Signal Transduction

Principles, Pathways, and Processes

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 Signal Transduction
Principles, Pathways, and Processes

EDITED BY

Lewis C. Cantley Tony Hunter

Harvard Medical School Salk Institute for Biological Studies

Richard Sever Jeremy Thorner

Cold Spring Harbor Laboratory University of California at Berkeley
Signal Transduction
Chapters online at cshperspectives.org and perspectivesinmedicine.org

All rights reserved

© 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Printed in the United States of America

Publisher John Inglis

Director of Editorial Development Jan Argentine
Project Manager Inez Sialiano
Permissions Coordinator Carol Brown
Production Editor Diane Schubach
Production Manager/Cover Designer Denise Weiss

Front cover artwork: Drawing by Nigel Hynes.

Library of Congress Cataloging-in-Publication Data

Signal transduction / edited by Lewis C. Cantley, Harvard Medical School, Tony Hunter, Salk Institute
for Biological Studies, Richard Sever, Cold Spring Harbor Laboratory, Jeremy Thorner, University of
California at Berkeley.
p. cm.
Summary: “This textbook provides a comprehensive view of signal transduction, covering both the
fundamental mechanisms involved and their roles in key biological processes. It first lays out the basic
principles of signal transduction, explaining how different receptors receive information and transmit it
via signaling proteins, ions, and second messengers. It then surveys the major signaling pathways that
operate in cells, before examining in detail how these function in processes such as cell growth and
division, cell movement, metabolism, development, reproduction, the nervous system, and immune
function”–Provided by publisher.
Includes bibliographical references and index.
ISBN 978-0-87969-901-7 (hardback)
ISBN 978-1-621822-30-1 (ebook)
1. Cellular signal transduction. 2. Developmental biology. 3. Pathology, Molecular. I. Cantley, Lewis,
editor of compilation. II. Hunter, Tony, 1943- editor of compilation. III. Sever, Richard, editor of
compilation. IV. Thorner, Jeremy W., editor of compilation.
QP517.C45S534 2013
571.7′4--dc23

2013043753

All World Wide Web addresses are accurate to the best of our knowledge at the time of printing.

Authorization to photocopy items for internal or personal use, or the internal or personal use of specific
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directly to the Copyright Clearance Center (CCC). Write or call CCC at 222 Rosewood Drive, Danvers,
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obtained at CCC Online at www.copyright.com.

For a complete catalog of all Cold Spring Harbor Laboratory Press publications, visit our website at
www.cshlpress.org.
This book is dedicated to the memory of Tony Pawson (1952–
2013). Tony was a giant in the field of signal transduction, who
established principles of protein–protein interactions that have
profoundly influenced our understanding of signal transduction.
His enduring legacy will be the discovery that the Src homology
2 (SH2) domain of one protein can selectively interact with a
tyrosine residue in a second protein, once it is phosphorylated in
response to an upstream signal. This type of inducible protein–
protein interaction can link intracellular signals generated in
response to various upstream stimuli to downstream signaling
events. This insight was the basis for his enormously influential
idea that eukaryotic signaling systems involve modular and
combinatorial interaction domains that propagate signals
throughout the cell.
Contents

Preface
Foreword
Edmond Fischer

SECTION I. GENERAL PRINCIPLES AND MECHANISMS

1 Signals and Receptors
Carl-Henrik Heldin, Benson Lu, Ron Evans, and J. Silvio Gutkind

2 Protein Regulation in Signal Transduction

Michael J. Lee and Michael B. Yaffe

3 Second Messengers
Alexandra C. Newton, Martin D. Bootman, and John D. Scott

4 Signaling Networks: Information Flow, Computation, and Decision

Making
Evren U. Azeloglu and Ravi Iyengar

SECTION II. SIGNALING PATHWAYS

MAP Kinase Pathways
Deborah K. Morrison

The PI3K-PKB/Akt Pathway

Brian A. Hemmings and David F. Restuccia

mTOR Signaling
Mathieu Laplante and David M. Sabatini

Calcium Signaling
Martin D. Bootman

The Cyclic AMP Pathway

Paolo Sassone-Corsi

Wnt Signaling
Roel Nusse

Hedgehog Signaling
Philip W. Ingham

Notch Signaling
Raphael Kopan

Signaling by the TGFβ Superfamily

Jeffrey L. Wrana

The JAK/STAT Pathway

Douglas A. Harrison

Toll-Like Receptor Signaling

Kian-Huat Lim and Louis M. Staudt

Immunoreceptor Signaling
Lawrence E. Samelson

Signaling by Nuclear Receptors

Richard Sever and Christopher K. Glass

The Hippo Pathway

 Kieran F. Harvey and Iswar K. Hariharan

SECTION III. SIGNALING PROCESSES

5 Signaling Pathways that Control Cell Proliferation
Robert J. Duronio and Yue Xiong

6 Signaling Pathways that Regulate Cell Division

Nicholas Rhind and Paul Russell

7 Signaling in Control of Cell Growth and Metabolism

Patrick S. Ward and Craig B. Thompson

8 Signaling Networks that Regulate Cell Migration

Peter Devreotes and Alan Rick Horwitz

9 Signaling Pathways in Cell Polarity

Luke Martin McCaffrey and Ian G. Macara

10 Signaling Mechanisms Controlling Cell Fate and Embryonic Patterning

Norbert Perrimon, Chrysoula Pitsouli, and Ben-Zion Shilo

11 Signaling by Sensory Receptors

David Julius and Jeremy Nathans

12 Synaptic Signaling in Learning and Memory

Mary B. Kennedy

13 Signaling in Muscle Contraction

Ivana Y. Kuo and Barbara E. Ehrlich

14 Organismal Carbohydrate and Lipid Homeostasis

D. Grahame Hardie

15 Signaling in Innate Immunity and Inflammation

Kim Newton and Vishva M. Dixit
16 Signaling in Lymphocyte Activation
Doreen Cantrell

17 Vertebrate Reproduction
Sally Kornbluth and Rafael Fissore

18 Cell Signaling and Stress Responses

Gökhan S. Hotamisligil and Roger J. Davis

19 Cell Death Signaling

Douglas R. Green and Fabien Llambi

20 Subversion of Cell Signaling by Pathogens

Neal M. Alto and Kim Orth

21 Signal Transduction in Cancer

Richard Sever and Joan S. Brugge

22 Outlook
Jeremy Thorner, Tony Hunter, Lewis C. Cantley, and Richard Sever

Index
Preface

S IGNAL TRANSDUCTION PROCESSES CAN BE VIEWED as the higher command

functions executed by cells on metabolic pathways (both catabolic and
biosynthetic), macromolecular machinery and organellar compartments that
allow an organism to maintain homeostasis and adjust cell number, cell
behavior, and organismal physiology appropriately in response to internal
cues and external stimuli. This book was conceived and organized as an
instructional resource to introduce advanced students, investigators new to
the field, and even researchers actively working in this general area to the
underlying foundations and basic mechanisms of signal transduction in
animal cells. Such a volume is needed because signaling impinges on every
aspect of molecular and cellular biology—from biochemistry and structural
biology to development and differentiation, endocrinology and systems
biology, pharmacology and neuroscience, and immunology and cancer
biology. Our objective is to explicate and illustrate the fundamental concepts,
principles, and processes involved in signaling quite comprehensively,
without necessarily being completely encyclopedic. We have taken a novel
approach to conveying this large body of information and making it
accessible, dividing the book up into distinct sections that describe principles,
pathways, and processes.
The first four principle chapters set the stage, presenting molecular
mechanisms and paradigms that are pertinent to all that follows. In Chapter 1,
Carl-Henrik Heldin, Benson Lu, Ronald Evans, and Silvio Gutkind discuss
signaling molecules and their receptors and downstream signaling events. In
Chapter 2, Michael Lee and Michael Yaffe introduce the central role of
proteins as transducers in signaling, describing the many ways by which
signaling can control protein level, function, activity, and location. In Chapter
3, Alexandra Newton, Martin Bootman, and John Scott discuss the nature,
generation, and action of intracellularly generated mediators (“second
messengers”). In Chapter 4, Evren Azeloglu and Ravi Iyengar consider the
circuit-like characteristics of signaling networks and systems, their emergent
properties, and mathematical models we can use to describe them.
There follows a series of 14 process chapters that cover the roles of
signaling in distinct biological processes and discuss how the general
principles described in the four principle chapters apply in a specific context.
Thus, the focus in these more specialized chapters is on the molecular basis
of a particular aspect of signaling, its logic and its physiological
consequences in biology, rather than a mere enumeration of pathway
components and their interactions. Nonetheless, familiarity with signaling
pathways used by cells is essential, and so separating the principles and
process chapters are a series of pathway diagrams with short accompanying
synopses written by other leaders in the field.
Different cell types possess a variety of mechanisms to sense and respond
to diverse stimuli. Dedicated receptor cells, for example, respond to physical
inputs from their surroundings, such as light, heat, and sound, as considered
in the chapter by David Julius and Jeremy Nathans. The information is
relayed via inorganic-ion-based electrical currents and release of and
response to amino acids (glutamate and glycine), amino-acid-derived
compounds, and other classes of substances that serve as neurotransmitters,
as discussed in the chapters by Mary Kennedy and by Ivana Kuo and Barbara
Ehrlich.
Cells respond to a plethora of other kinds of chemical signals, as disparate
as inorganic substances (including gases) and a host of other organic
molecules (from volatile substances to lipidic compounds to peptide
hormones, growth factors, and morphogens), as presented in Chapter 1 and in
the chapter by Norbert Perrimon, Chrysoula Pitsouli, and Ben-Zion Shilo. As
discussed in Chapter 3, in many cases, the encounter with such extracellular
ligands activates the production of second messengers, from
phosphoinositides to cyclic nucleotides to less familiar, newly discovered
metabolites. This allows amplification and spreading of the response by
affecting the level, localization, and activity of numerous proteins and other
cellular targets by mechanisms described in detail in Chapter 2. In addition to
responses to native extracellular signals and normal internal cues, the
specialized cells of our immune system must respond to attack by or
internalization of potentially dangerous prokaryotic, viral and fungal
pathogens, as reviewed in the chapters by Kim Newton and Vishva Dixit and
by Doreen Cantrell. Microbes, in turn, have evolved an armamentarium of
virulence factors and other effectors that they inject to specifically interdict
signaling by lymphocytes and other cells, which also provide useful tools for
experimentally interrogating signaling processes, as discussed in the chapter
by Neal Alto and Kim Orth.
It is especially important that cells and tissues stay acutely attuned to their
nutrient supply and adjust their metabolism accordingly. This aspect of
signaling is described in the chapters by Patrick Ward and Craig Thompson
and by Grahame Hardie. Cells also need to gauge their position in space and
time and alter their morphology and adjust their movements in response to
signals arising from cell–cell and cell–extracellular-matrix contacts, as
presented in the chapters by Luke McCaffrey and Ian Macara and by Peter
Devreotes and Rick Horowitz.
One reason for a cell to constantly gauge and integrate information about
its nutrient supply, its developmental state, its neighboring cells, and
demands of other tissues is to decide whether it should remain quiescent,
grow and divide, or enter a developmental pathway leading to production of a
highly specialized postmitotic cell type. The issue of how entry into the cell
division cycle is controlled by signaling pathways is discussed in detail in the
chapter by Robert Duronio and Yue Xiong. The internal, fail-safe signaling
mechanisms (checkpoints) that ensure the proper spatial and temporal order
of events in cell cycle progression, and act as delay timers to allow an
adequate hiatus for any necessary repairs, are considered in the chapter by
Nicholas Rhind and Paul Russell. When the normal signals that control the
decision of cells to divide are subverted, and the negative controls on cell
division are broken, malignant growth can occur. How defects in signaling lie
at the heart of the molecular basis of cancers is discussed in the chapter by
Richard Sever and Joan Brugge.
Concomitant with what may occur under optimal conditions, cells also
have to cope with decisions about how to manage their resources and
responses under more challenging and stressful conditions. Maybe the cell
can overcome the problems, but, if it suffers irreversible harm to the integrity
of its chromosomes, or to the functioning of a vital organelle, then alarm
signals are in place to try to prevent any rogue or damaged cell from
lingering. The signaling responses elicited by stressful conditions, and how
those responses promote cell survival, are examined in the chapter by
Gökhan Hotamisligil and Roger Davis. Conversely, how cells evoke and
respond to the signals that lead to their own demise is described in the
chapter by Douglas Green and Fabien Llambi.
Of course, most eukaryotes develop from multiplication of the single-
celled zygote formed by the union of two germ cells, and how signaling is
involved in gametogenesis and sexual reproduction is presented in the
chapter by Sally Kornbluth and Rafael Fissore.
At the end of the book, we present an Outlook that provides some
additional information and perspectives on recent developments (both
methodological and conceptual) that further set the stage for future advances
in the field of signal transduction. In it we discuss challenges and open
questions that we hope will help point the way forward.
We would like to express our gratitude to all the authors who took time
out of their busy schedules to contribute the fantastic chapters that make up
this book. We also want to express our deep gratitude to the many
investigators, too numerous to name individually here, who served as
anonymous referees to evaluate the accuracy and effectiveness of the contents
of this book. We would also like to thank Cell Signaling Technology, Inc.,
for financial support and for making available figures from which the
pathway diagrams shown in the book were derived and adapted. Finally, we
are indebted to Inez Sialiano, Diane Schubach, and Kathleen Bubbeo at Cold
Spring Harbor Laboratory Press for all their hard work helping to get the
book into print and online.

JEREMY THORNER
 RICHARD SEVER
TONY HUNTER
LEWIS C. CANTLEY
Foreword

T HIS TEXTBOOK ON Signal Transduction, edited by some of the foremost

experts in this area, presents an encyclopedic view of a field that
essentially did not exist 60 years ago. In those days, almost nothing was
known about the mechanism by which enzymes and physiological processes
were regulated, and terms such as “signaling” or “signal transduction” that
are so commonly used today would not have been understood.
First, although endocrinology was already well established as a discipline,
it remained purely at the phenomenological, mostly intact animal, level. The
action of hormones stopped at the cell membrane and what happened next
was totally unknown until Earl Sutherland and Ted Rall came along with
their stunning discovery of cAMP, which served as a second, intracellular
messenger for the action of epinephrine. Second, there was a fundamental
difference in the way science was conducted. At that time and, in fact, since
the days of Claude Bernard in the second half of the 19th century, one first
observed a physiological phenomenon and then tried to identify the factors or
enzymes involved. Whereas today, by and large, it is the other way around:
new proteins are first identified mostly through genome sequencing projects
and then, by overexpressing them or by knocking them in or out, one tries to
define their function. Finally, essentially nothing was known about enzyme
regulation. The prevailing idea was that they were regulated simply by the
rate at which they were synthesized and degraded. But in the late 1940s/early
1950s, people began to realize that this could not be the case, that this would
not work because protein synthesis and degradation are far too slow. Cells
had to have ways of modulating the activity of their enzymes once they had
been produced and liberated within the cells. They had to have the capability
of adapting to their environment, of satisfying their metabolic needs, almost
instantaneously in response to whatever internal or external demands are
placed upon them. And this is where cell signaling and signal transduction
came into play.
These fields did not originate from a single, explosive breakthrough or
discovery. They grew step-by-step through successive small advances in the
second half of the last century, originating perhaps with the finding that the
control of glycogen phosphorylase, an enzyme shown by the Coris to
catalyze the first step in the degradation of glycogen, occurs through a
phosphorylation–dephosphorylation reaction. Since then, reversible protein
phosphorylation has been found to be one of the most prevalent and versatile
means by which cellular processes are regulated, being involved in the
control of metabolism, gene expression, the immune response, cell
development and differentiation, and what not. In fact, it would be difficult to
find a physiological process that would not be, directly or indirectly,
regulated by this kind of mechanism. It is implicated in innumerable
hereditary diseases and pathological conditions, such as diabetes,
Alzheimer’s and Parkinson’s diseases, and myelogenous leukemia, in viral
diseases such as smallpox, and bacterial diseases such as cholera and plague.
Quantitatively, better than 99.9% of all these phosphorylation reactions
occur on serine and threonine. But one of the most exciting developments in
this field was the discovery, more than 30 years ago, that phosphorylation of
proteins on tyrosyl residues was intimately implicated in cell transformation
and oncogenesis, bringing into play a multitude of tyrosine kinases of cellular
or viral origin, or linked to growth factor receptors.
Although reversible protein phosphorylation seemed to be for many years
the main form of cellular regulation, a just as prevalent and far more complex
regulatory mechanism has since been uncovered—namely, ubiquitylation.
And it is very likely that other general regulatory systems might come to
light, such as reversible protein acetylation, methylation, and oxidoreduction
or the interaction of enzymes with their specific binding modules, anchors,
and chaperones.
These advances could not have been possible without the development of
sophisticated methodologies such as X-ray crystallography, nuclear magnetic
resonance, mass spectrometry, and cryo-electron microscopy for protein
structure determination and nanochemistry and the use of nanoparticles,
monoclonal antibodies, and genetically encoded fluorescent marker proteins
allowing one to monitor molecular processes without disrupting cell function.
Of course, the most spectacular advance occurred in genetic engineering
with the cloning, manipulation, expression, and sequencing of genes, without
which we would know essentially nothing about our genetic makeup or about
a variety of hereditary and viral diseases. With the pervasive presence of the
computer that allows one to display and analyze data and store and retrieve
them at the touch of a button, today’s investigators have at their disposal an
array of technologies absolutely undreamed of just a few years ago.
Finally, what are some of the main problems that remain to be solved in
signal transduction? Most of the major signaling pathways have probably
been elucidated, and the structure, properties, regulation, and physiological
function of the molecules involved have been well characterized. But these
molecules are only the words the cells use to perform their daily chores. We
know many of these words; we recognize probably bits and pieces of some of
the sentences they spell out to elicit a particular response. But we are only
just starting to understand the language the cell has to use to allow different
receptors or pathways to speak with one another to coordinate all the
reactions that take place. This communication often occurs through the
formation of large macromolecular complexes comprising anchoring and
scaffolding proteins and modules that link them to the cytoskeleton,
providing those systems with the specificity and selectivity they require;
however, how cells maintain and preserve the fidelity of signaling processes
remains poorly understood.
The problem is further complicated by the fact that during the several
billion years over which cells have evolved, they have had all the
opportunities in the world to put in place the vast array of secondary or
parallel pathways, shunts, compensatory mechanisms, feedback loops, and
fail-safe systems they need to regulate their growth and development, to
protect themselves against all sorts of adversity, and to program their own
death when the time comes. And we do not know the myriads of signals that
must exist to sort out all the reactions that take place.
Perhaps even more importantly, we do not understand the cross talk—the
interactivity that must exist among cells and how they communicate with one
another to synchronize their behavior in response to internal or external
signals. This cross talk, this sharing of information, is crucial for the
establishment of such sophisticated networks of communication as seen, for
instance, during embryonic development and organogenesis, in the immune
system, or in the infinitely more complex central nervous system, where a
thousand billion cells speak with one another through more than a million
billion synapses, leading ultimately to the generation of memory and thought
and consciousness. Solving these problems will be one of the major
challenges that will confront biologists in the years to come.
This textbook on signal transduction addresses most of these problems. It
is directed toward future practitioners of biology and medicine: advanced
graduate students, postdoctoral fellows, or researchers working in an
academic, biotechnological, or pharmaceutical environment. It will be of
enormous help to all those who would want to remain abreast of the field.

EDMOND FISCHER
University of Washington
SECTION I

GENERAL PRINCIPLES AND

MECHANISMS
CHAPTER 1

Signals and Receptors

Carl-Henrik Heldin1, Benson Lu2, Ron Evans2, and J. Silvio

Gutkind3
1Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, SE-75124
Uppsala, Sweden
2The Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, California 92037
3National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda,
Maryland 20892-4340
Correspondence: c-h.heldin@licr.uu.se

SUMMARY

Communication between cells in a multicellular organism occurs by the

production of ligands (proteins, peptides, fatty acids, steroids, gases, and
other low-molecular-weight compounds) that are either secreted by cells
or presented on their surface, and act on receptors on, or in, other target
cells. Such signals control cell growth, migration, survival, and
differentiation. Signaling receptors can be single-span plasma membrane
receptors associated with tyrosine or serine/threonine kinase activities,
proteins with seven transmembrane domains, or intracellular receptors.
Ligand-activated receptors convey signals into the cell by activating
signaling pathways that ultimately affect cytosolic machineries or
nuclear transcriptional programs or by directly translocating to the
nucleus to regulate transcription.
Outline
1 Introduction
2 Cell-surface receptors
3 The TNF receptor family
4 Cell adhesion receptors and mechanotransducers
5 Nuclear receptors
6 Functions of nuclear receptors
7 Gases
8 Concluding remarks
References

1 INTRODUCTION
Cells within multicellular organisms need to communicate with each other to
coordinate their growth, migration, survival, and differentiation. They do so
by direct cell–cell contact and secretion or release of molecules that bind to
and activate receptors on the surface of or inside target cells. Such factors can
stimulate the producer cell itself (autocrine stimulation), cells in the
immediate vicinity (paracrine stimulation), or cells in distant organs
(endocrine stimulation). The signaling induced within target cells is important
during embryonic development, as well as in the adult, where it controls cell
proliferation, differentiation, the response to infection, and numerous
organismal homeostatic mechanisms.
Many cell-surface receptors contain an extracellular ligand-binding
region, a single transmembrane segment, and an intracellular effector region,
which may or may not have an associated enzyme activity. Some receptors
contain multiple subunits that together form the ligand-binding site. Others,
including those encoded by the largest gene family in the human genome,
consist of a polypeptide that spans the cell membrane seven times. Finally,
there are receptors that are located intracellularly and are activated by ligands
that cross the cell membrane, such as steroid hormones. Below, we describe
the major families of ligands and receptors and the signal transduction
mechanisms they activate.

2 CELL-SURFACE RECEPTORS
2.1 Receptors with Associated Protein Kinase Activity
Several types of cell-surface receptors contain or are associated with kinase
activities that respond to the binding of a ligand. Perhaps best understood are
receptors with intrinsic protein tyrosine kinase domains. This receptor
tyrosine kinase (RTK) family has more than 50 human members (Lemmon
and Schlessinger 2010). RTKs have important roles in the regulation of
embryonic development, as well as in the regulation of tissue homeostasis in
the adult. Each has an extracellular, ligand-binding region, which consists of
different combinations of various domains, such as Ig-like, fibronectin type
III, and cysteine-rich domains. This is linked to a single transmembrane
segment and an intracellular region that includes a tyrosine kinase domain.
Based on their structural features, RTKs can be divided into 20 subfamilies
(Fig. 1), a well-studied example being the epidermal growth factor (EGF)
receptors (EGFRs).
Figure 1. Receptor tyrosine kinase (RTK) families. The 20 subfamilies of human RTKs and their
characteristic structural domains are shown. The individual members of each family are listed below.
(From Lemmon and Schlessinger 2010; adapted, with permission.)

Members of the cytokine receptor family in contrast lack intrinsic kinase

activity but associate with intracellular kinases. They have important roles in
the regulation of the immune system and also promote cell differentiation.
Cytokine receptors can be divided into two classes. The extracellular domains
of class I cytokine receptors contain cytokine receptor homology domains
(CHDs) consisting of two tandem fibronectin type III domains with a
characteristic WSXWS motif in the second (Liongue and Ward 2007). Based
on the number of CHDs and the presence of other types of domains, the class
I cytokine receptors can be divided into five groups (Fig. 2), the growth
hormone (GH) receptor being typical of the first group. Interferon receptors
are typical of the 12-member class II cytokine receptor family, which also
have extracellular regions based on tandem fibronectin domains but differ
from those of class I receptors (Fig. 2) (Renauld 2003). Both classes of
cytokine receptors have conserved box 1 and box 2 regions in their
intracellular regions, which bind to JAK family tyrosine kinases that are
activated upon ligand binding. The multisubunit antigen receptors on B cells
and T cells (Zikherman and Weiss 2009) and the Fc receptors present on
macrophages, mast cells, basophils, and other immune cells (Nimmerjahn
and Ravetch 2008) are also associated with intracellular tyrosine kinases;
activation of these receptors involves tyrosine phosphorylation by members
of the Src family, followed by docking and activation of SH2-domain-
containing Syk/Zap70 tyrosine kinases (see p. 125 [Samelson 2011]; Ch. 16
[Cantrell 2014]). Although receptors that have intrinsic tyrosine kinase
activity and those associated with tyrosine kinases are structurally different
and bind ligands of different kinds, the principles underlying their activation
and the intracellular signals they initiate are similar (see below).
Figure 2. Cytokine receptor families. The structural features of the five subfamilies of class I and class
II cytokine receptors are depicted. The characteristic cytokine homology domains (CHDs) with their
four cysteine residues (blue lines) and a WW motif (green line), as well as the box 1 and box 2 regions
(red bands), Ig-like domains, and FNIII domains are shown.

There is also a family of receptors that have intrinsic serine/threonine

kinase domains, and these respond to members of the transforming growth
factor β (TGFβ) family (see Moustakas and Heldin 2009; p. 113 [Wrana
2013]). The human genome has only 12 genes encoding receptors of this type
(Fig. 3). These receptors have rather small cysteine-rich extracellular
domains; their intracellular domains are most often also small and consist
mainly of the kinase domains. TGFβ receptors mediate signaling events
during embryonic development. Because they often inhibit cell growth, they
also exert a controlling function on the immune system and other tissues.
Figure 3. Serine/threonine kinase receptors. (A) The structural features of type I (TGFβRI) and type II
(TGFβRII) serine/threonine kinase receptors. (B) The different members of the type I and type II
receptor subfamilies and their evolutionary relations. Act, Activin; ALK, activin-receptor-like kinase.

2.2 Ligands
Each of these different receptor types responds to a subfamily of structurally
similar ligands. The ligands are normally small monomeric, dimeric, or
trimeric proteins, often derived by proteolytic processing from larger
precursors, some of which are transmembrane proteins. There is not a strict
specificity in ligand–receptor interactions within the families; normally each
ligand binds to more than one receptor, and each receptor binds more than
one ligand. Although it is rare that ligands for completely different types of
receptors bind to kinase-associated receptors, examples do exist.4

2.3 Activation by Dimerization

A common theme for activation of kinase-associated receptors is ligand-
induced receptor dimerization or oligomerization (Heldin 1995). The
juxtaposition of the intracellular kinase domains that occurs as a consequence
allows autophosphorylation in trans within the complex. For RTKs, the
autophosphorylation has two important consequences: it changes the
conformation of the kinase domains, leading to an increase in their kinase
activities; and it produces docking sites (phosphorylated sequences) for
intracellular signaling molecules containing SH2 or PTB domains (see Ch. 2
[Lee and Yaffe 2014]). The autophosphorylation that controls the kinase
activity occurs on residues in various regions of the receptors. Most RTKs are
phosphorylated in the activation loops of the kinases, that is, a flexible loop
of the carboxy-terminal domain, which can fold back and block the active site
of the kinase when unphosphorylated. Exceptions are Ret and the four
members of the EGFR family that can be activated without phosphorylation
in their activation loops (Lemmon and Schlessinger 2010). Several RTKs are
activated by phosphorylation in the juxtamembrane region, including MuSK,
Flt3, Kit, and Eph family members. Moreover, Tie2, platelet-derived growth
factor (PDGF) β receptor (PDGFRβ) and Ron have been shown to be
activated by phosphorylation of their carboxy-terminal tails. In each case,
autophosphorylation causes a change in conformation that opens up the
active site of the kinase. Interestingly, a phosphorylation-independent
mechanism for activation of EGFR kinase activity has been elucidated: the
carboxy-terminal lobe of one kinase domain makes contact with the amino-
terminal lobe of the other kinase domain in the dimer and thereby activates it
(Zhang et al. 2006). The juxtamembrane region of the receptor, which in
other RTKs has an inhibitory role, is needed to stabilize this dimeric
interaction (Jura et al. 2009; Red Brewer et al. 2009).
Different ligands use different mechanisms to cause receptor
dimerization. Thus, GH is a monomeric ligand that dimerizes its receptors by
inducing formation of an asymmetric complex containing one ligand and two
receptors (De Vos et al. 1992). EGF is also a monomeric factor, but each
molecule binds to a single receptor molecule, causing a conformational
change that promotes direct binding of two ligand-bound receptors (Fig. 4)
(Burgess et al. 2003). Similarly, dimeric ligands can induce symmetric 2:2
ligand–receptor complexes. In the case of nerve growth factor (NGF), the
ligand itself is solely responsible for the dimerization (Wehrman et al. 2007);
in the case of Kit and the PDGFRs, the ligand-induced receptor dimerization
is stabilized by direct interactions between the receptors (Yuzawa et al.
2007). In other cases, accessory molecules are needed to stabilize
dimerization; that is, heparin or heparan sulfate stabilizes fibroblast growth
factor (FGF)–induced receptor dimers by interacting with both FGF and
FGF-receptor subunits (Lemmon and Schlessinger 2010). Recent work on
EGFR has shown that its activation requires an interaction between the
amino-terminal parts of the transmembrane helices of the dimeric receptors,
which promotes an antiparallel interaction between the cytoplasmic
juxtamembrane segments and release of inhibition by the membrane
(Arkhipov et al. 2013; Endres et al. 2013).

Figure 4. Schematic illustration of different modes of dimerization of RTKs. (A) Some dimeric ligands,
such as nerve growth factor (NGF), bind to receptors in a symmetric manner, but the receptors do not
contact each other. (B) Other dimeric ligands, such as stem cell factor (SCF), also bind to RTKs in a
symmetric manner, but the receptor dimer is in addition stabilized by direct receptor–receptor
interactions. (C) In the case of fibroblast growth factor (FGF), a ternary complex involving the ligand,
the receptor, and heparin/heparin sulfate stabilizes the receptor dimer. (D) In the case of members of
the epidermal growth factor (EGF) receptor family such as ErbB, ligand binding induces a
conformational change in the extracellular domain of the receptor that promotes direct receptor–
receptor interactions. (From Lemmon and Schlessinger 2010; adapted, with permission.)
 In addition to homodimerization of receptors, heterodimerization also
often occurs. This is particularly common among cytokine receptors; a
ligand-specific receptor subunit often interacts with one or more common
subunits, such as gp130, βc, or γc, to form a heterodimer, heterotrimer, or
even more complicated multimer (Wang et al. 2009). Similarly, members of
different RTK subfamilies often form heterodimers. For instance, one
member of the EGFR subfamily, ErbB2, cannot bind to ligand itself, but acts
in heterodimers with other members of the family (Yarden and Sliwkowski
2001). In the PDGFR family, different dimeric isoforms of the ligand induce
formation of different dimeric complexes of PDGFα and PDGFβ receptors
(Heldin 1995). Because the downstream signaling pathways that are activated
to a large extent depend on the specific docking of SH2- or PTB-domain-
containing signaling molecules (see below and Ch. 2 [Lee and Yaffe 2014]),
differences in the autophosphorylation patterns of homodimeric versus
heterodimeric receptor complexes will give rise to different combinatorial
signals.
Serine/threonine kinase receptors present another variation on this theme.
These receptors are activated by ligand-induced assembly of two type I and
two type II receptors into heterotetrameric receptor complexes, in which
constitutively active type II receptors phosphorylate type I receptors in
serine- and glycine-rich sequences just upstream of the kinase domains. In
the TGFβ type I receptor, this causes a change in conformation that prevents
the inhibitory interaction of the receptor with the immunophilin FK506-
binding protein (FKBP12) and activates its kinase (Kang et al. 2009).
Note that certain receptors are present in dimeric complexes even in the
absence of ligand; examples include the erythropoietin cytokine receptor, and
the insulin and insulin-like growth factor-1 (IGF1) receptors, RTKs that
actually occur as disulfide-bonded dimers. Here, ligand binding induces
conformational changes that lead to activation of the receptor-associated
kinase. Importantly, there are indications that reorientation of the intracellular
regions of the receptors relative to each other in the dimer is important for
their activation (Jiang and Hunter 1999).

2.4 Signaling Downstream from Tyrosine-Kinase-Associated

 Receptors via SH2- or PTB-Domain-Containing
Molecules
An important aspect of signaling by tyrosine-kinase-associated receptors is
the formation of multiprotein signaling complexes (Pawson 2004). As
mentioned above, SH2- or PTB-domain-containing proteins bind to specific
phosphorylated tyrosine residues in the receptors themselves or in scaffolding
proteins associated with the receptors (see Ch. 2 [Lee and Yaffe 2014]).
Examples of the latter include the four members of the insulin receptor
substrate (IRS) family, which bind to insulin and IGF1 receptors; FRS2-
family protein molecules, which bind to FGF and NGF receptors; Gab
molecules, which bind to several RTKs; and SLP76 and LAT, which bind to
antigen receptors.
Some of the proteins that bind to these sequences contain intrinsic
enzymatic activities, for example, the tyrosine kinases of the Src family, the
tyrosine phosphatases of the SHP family, GTPase-activating proteins (GAPs)
that regulate Ras family GTPases, the ubiquitin ligase Cbl, and phospholipase
C (PLC) γ. Others are proteins that form stable complexes with enzymes, for
example, the regulatory p85 subunit of type 1 phosphoinositide 3-kinase
(PI3K), which forms a complex with the catalytic p110 subunit, and Grb2,
which forms a complex with SOS1, a guanine nucleotide exchange factor
(GEF) that stimulates Ras. Cytokine receptors and some RTKs also activate
STAT molecules, which translocate to the nucleus, where they act as
transcription factors (see p. 117 [Harrison 2012]). Finally, SH2-/SH3-
domain-containing adaptor molecules (e.g., Shc, Nck, and Crk) bind to
certain tyrosine phosphorylated sites in the receptors or associated molecules
and form bridges to other signaling molecules, including the enzymes
mentioned above.
Many of the signaling proteins that bind to the receptors also contain
domains that mediate interactions with other molecules. These include SH3
domains, which bind proline-rich motifs in proteins; PDZ domains, which
bind to the carboxy-terminal tails of proteins containing valine residues; and
pleckstrin homology (PH) domains, which bind lipids present in membranes.
Thus, large complexes of signaling proteins are transiently formed and
mediate signaling to cytoplasmic machinery (e.g., enzymes controlling the
cytoskeleton and cell migration), as well as to the nucleus, where
transcription is affected.
Note that the kinase domains of certain RTKs (e.g., ErbB3, CCK4,
EphB6, and Ryk) lack critical catalytic residues normally found in kinases
and thus have no, or very low, kinase activity. However, these receptors still
have important roles in signal transduction, acting as phosphorylatable
scaffolds in heterodimers with other receptors.
There are also indications that the EGFRs can translocate to the nucleus to
regulate transcription (Lin et al. 2001). However, the functional significance
of this needs to be further clarified.

2.5 Downstream Signaling by Serine/Threonine Kinase

Receptors
Serine/threonine kinase receptors activate members of the Smad transcription
factor family (p. 113 [Wrana 2013]); similar to STATs, these molecules
oligomerize after phosphorylation and activation and then translocate to the
nucleus, where they induce transcription. In addition, serine/threonine kinase
receptors can activate non-Smad pathways. The TGFβ type I receptor, for
example, can autophosphorylate on tyrosine residues, which bind to the
adaptor Shc, leading to activation of Ras and the ERK1/2 mitogen-activated
protein kinase (MAPK) pathway (see p. 81 [Morrison 2012]). Moreover, the
p38 MAPK pathway is activated via binding of the ubiquitin ligase TRAF6 to
the TGFβ type I receptor (Kang et al. 2009). In addition to activating the type
I receptor, the TGFβ type II receptor also contributes to signaling by
phosphorylating the polarity complex protein PAR6 (Ozdamar et al. 2005;
Ch. 9 [McCaffrey and Macara 2012]).

2.6 Interaction with Coreceptors

Several RTKs form complexes with nonkinase receptors, such as the
hyaluronan receptor CD44 and integrins, which bind to extracellular matrix
molecules. Such interactions can modulate their signaling. Similarly, the
heparan sulfate proteoglycan agrin activates MuSK receptors by binding to
low-density lipoprotein (LDL)-receptor-related protein 4 (LRP4); thus, LRP4
acts as a coreceptor for MuSK (Kim et al. 2008). Another example is glial-
ceU-derived neurotrophic factor (GDNF) receptor α, a
glycosylphosphatidylinositol (GPI)-anchored molecule needed for GDNF to
bind to and activate Ret receptors (Schlee et al. 2006).

2.7 Feedback and Amplification Mechanisms

Signaling via kinase-associated receptors is controlled by multiple feedback
mechanisms to ensure an appropriate level. Thus, many tyrosine kinase and
cytokine receptors bind protein tyrosine phosphatases (PTPs), including
SHP1 and SHP2, that contain SH2 domains. Through dephosphorylation of
specific tyrosine residues, PTPs modulate signaling quantitatively as well as
qualitatively (Lemmon and Schlessinger 2010).
Another example is activation of the small G protein Ras, which occurs
by docking of the GEF SOS1 in complex with the adaptor Grb2 (see Ch. 2
[Lee and Yaffe 2014]). PDGFRβ and possibly other receptors simultaneously
bind the Ras-GAP molecules via another phosphorylated tyrosine residue,
which counteracts Ras activation by promoting hydrolysis of bound GTP.
In addition, another downstream target of receptor signaling, protein
kinase C (PKC), is involved in feedback control of certain RTKs, including
EGFR, the insulin receptor, Met, and Kit. Activation of PLCγ leads to
production of diacylglycerol (DAG) and increases in intracellular calcium
levels, which activate the classical isoforms of PKC (see Ch. 3 [Newton et al.
2014]). Phosphorylation of the receptors by PKC subsequently inhibits the
kinase activity of the receptors.
After the induction of immediate-early genes encoding various effector
molecules by signaling to the nucleus, RTKs induce the expression of
delayed-early genes, many of which encode proteins that suppress signaling.
Examples include NAB2, which binds to and inhibits EGFR; FOSL1, which
binds to and inhibits AP1 transcription factors; Id2, which inhibits the TCF
transcription complex; DUSPs, which dephosphorylate and inactivate
MAPKs; and ZFP36, which recognizes AU-motifs in the 3′ ends of mRNA
molecules and causes their degradation (Amit et al. 2007). Similarly, TGFβ
receptors and cytokine receptors induce Smad7 (Moustakas and Heldin 2009)
and SOCS proteins (Yoshimura et al. 2003), respectively, which exert
negative-feedback control by promoting ubiquitin-dependent degradation of
receptors by targeting receptor-containing vesicles to lysosomes (see below).

2.8 Endocytosis of Kinase-Associated Receptors

After ligand binding, kinase-associated receptors are internalized into the cell
via clathrin-dependent or clathrin-independent pathways (Zwang and Yarden
2009). Whereas internalization of RTKs is induced by ligand binding,
internalization of serine/threonine receptors is constitutive and independent of
ligand binding. Internalization has both positive and negative effects on
signaling. Thus, when present in endosomes, the receptors are still active; in
some cases, internalization is even necessary for the receptors to interact with
signaling molecules on intracellular endosomes (Miaczynska et al. 2004).
Examples include TGFβ receptors, which need to be internalized to interact
with Smad molecules presented to the receptors by SARA and endofin
proteins. These proteins reside on endosomes, their FYVE domains binding
to phosphatidylinositol 3-phosphate (PI3P), a phospholipid that is enriched in
endosomal membranes (Tsukazaki et al. 1998). Signaling is interrupted when
the pH of the endosomes becomes so low that the ligand dissociates; at this
stage, receptors are dephosphorylated and recycle back to the cell surface.
Alternatively, the receptors are recognized by components of the endosomal
sorting complex required for transport (ESCRT) machinery, which facilitates
translocation to multivesicular bodies and degradation in lysosomes (Raiborg
and Stenmark 2009). The latter route is promoted by ubiquitylation of the
receptors by Cbl or other ubiquitin ligases.

2.9 Kinase-Associated Receptors and Disease

Because tyrosine-kinase-associated receptors often stimulate cell proliferation
and survival, overactivity of these receptors is linked to the development of
cancer and other diseases characterized by excess cell proliferation, such as
inflammatory and fibrotic conditions. There are several examples of gain-of-
function mutations in RTKs that occur in malignancies (Lengyel et al. 2007;
Ch. 21 [Sever and Brugge 2014]). Point mutations in Kit and PDGFRα have
been found in gastric intestinal stromal tumors, and the kinase domains of
PDGFRs and FGFRs occur as constitutively active cytoplasmic fusion
proteins in several rare leukemias. Moreover, the ERBB2 gene is amplified in
∼20% of breast cancers, and a mutated version of the EGFR gene is amplified
in ∼30% of glioblastoma cases.
Because overactivity of these receptors is common in malignancies,
several antagonists have been developed, including inhibitory antibodies,
ligand traps, and low-molecular-weight kinase inhibitors. Several of these are
now used routinely in the clinic or are undergoing clinical trials.
The serine/threonine kinase receptors often relay growth inhibitory and
apoptotic signals and therefore have tumor-suppressive effects. Thus, loss-of-
function mutations of TGFβ type I and type II receptors have been observed
in some cancers (e.g., colorectal carcinomas).

2.10 Receptors Activated by Proteolytic Cleavage

The highly conserved Notch family of receptors consists of four members,
which have important roles during embryonic development and tissue
renewal (Kopan and Ilagan 2009). The Notch receptors are single-pass
transmembrane proteins with large extracellular domains containing multiple
EGF-like repeats. They are cleaved extracellularly by furin-like proteases
during transit to the plasma membrane to create a heterodimer held together
by noncovalent interactions (p. 109 [Kopan 2012]).
There are five canonical Notch ligands (Delta-ligand-like 1, 3, and 4, and
Jagged 1 and 2). These are also single-pass transmembrane proteins with
extracellular EGF-like regions and other domains present on neighboring
cells. The Notch receptor is normally triggered upon cell–cell contact. Notch
activation involves a series of proteolytic events. Ligand binding induces
cleavage of Notch by ADAM metalloproteases at a site about 12 amino acid
residues outside the transmembrane domain. Removal of the extracellular
domain allows an intramembrane cleavage by γ-secretase, which causes
release of the intracellular domain (ICD) of Notch. Because the ICD has a
nuclear localization sequence, it translocates to the nucleus, where it, together
with the DNA-binding protein RBPjκ/CBF-1 and certain coactivators and
corepressors (together referred to as CSL), regulates transcription of target
genes via its transactivation domain. Because the Notch ligands are
membrane-associated, signaling may also be induced in the ligand-bearing
cells after binding to Notch.
Some RTK receptors (Ni et al. 2001) and the type I TGFβ receptor (Mu et
al. 2011) are also subjected to sequential cleavage by metalloproteases and γ-
secretase. The intracellular region of the receptors can then translocate to the
nucleus to regulate transcription. Meanwhile, the extracellular portion of the
receptor is liberated and can negatively regulate signaling by acting as a
decoy for ligand (Ancot et al. 2009). In addition, some RTKs are classified as
“dependence receptors.” When they are not occupied by ligand, they can be
cleaved by caspases (a group of proteases that control apoptosis) to generate
fragments with apoptotic activity. For example, fragments of EGFR, ErbB2,
Ret, Met, TrkC, ALK, and EphA4 have apoptotic effects, which contrast with
the normal antiapoptotic effects of the full-length receptors stimulated by
their ligands (Ancot et al. 2009).

2.11 G-Protein-Coupled Receptors

Approximately 2% of all genes in the human genome encode G-protein-

coupled receptors (GPCRs), which represent the largest family of cell-surface
molecules involved in signal transmission. They are so called because their
signals are transduced by heterotrimeric G proteins; members of the GPCR
family regulate a wide range of key physiological functions, including
neurotransmission, blood pressure, cardiac activity, vascular integrity,
hemostasis after tissue injury, glucose and lipid metabolism, sensory
perception, regulation of endocrine and exocrine gland function, immune
responses, multiple developmental processes, and stem cell function and
maintenance (Pierce et al. 2002; Dorsam and Gutkind 2007). Reflecting this
remarkable multiplicity of activities, GPCR dysregulation contributes to
some of the most prevalent human diseases. Indeed, GPCRs are the direct or
indirect target of >50% of all available medicines (Flower 1999; Pierce et al.
2002).
2.12 A Shared Heptahelical Structure Transduces Signals
Initiated by Highly Diverse Molecular Agonists

GPCRs are characterized by the presence of an extracellular amino terminus,

an intracellular carboxy-terminal tail, and a shared structural core composed
of seven transmembrane α helices that weave in and out of the membrane,
thus forming three intracellular and three extracellular loops (Fig. 5) (Pierce
et al. 2002). They are regulated by a diverse array of agonists, as small as
photons, ions, nucleotides, amino acids, biogenic amines, bioactive lipids,
and glucose metabolites, and as large as chemokines, glycoproteins, and
proteases. They can be grouped into three classes and subfamilies of these
according to the structural features involved in ligand–receptor recognition
and subsequent stimulation (Fig. 5).
Figure 5. GPCRs use distinct structural features for ligand recognition. GPCRs have been classified
based on their sequence similarities and ligand-binding properties. In class A GPCRs, the largest group,
the ligand-binding site is deep within the transmembrane domains in subfamily 1. It involves
interactions with the amino terminus, the extracellular loops, and the transmembrane domains in
subfamily 2. The ligand-binding site is in the long extracellular domain in subfamily 3. Class B GPCRs
are activated by high-molecular-weight hormones, which bind to the ligand-binding site within the long
amino-terminal region, as well as some of the extracellular loops. Class C GPCRs are characterized by
a very long amino terminus that shares some sequence similarity with periplasmic bacterial proteins;
activation involves obligatory dimerization. The Frizzled family of receptors contains the Frizzled and
“Smoothened” subfamilies, which are structurally distinct and have complex mechanisms of agonist
activation. Wnt binds to and activates Frizzled through an interaction with a cysteine-rich amino-
terminal region, whereas low-density lipoprotein-receptor-related protein 5 (LRP5) or LRP6 (single-
transmembrane-span proteins) acts as a coreceptor (Lim and Nusse 2013). When Hedgehog binds to
Patched, a negative regulatory effect of Patched on Smoothened activity is relieved, and Smoothened
regulates both G-protein-dependent and -independent signals (p. 107 [Ingham 2012]).

In class A GPCRs, the ligand-binding site is deep within the

transmembrane domains in subfamily 1, which includes most receptors for
small molecules, such as neurotransmitters, lipid mediators, and odorants.
Subfamily 2 members are activated by protein ligands, such as chemokines
and the tethered ligand resulting from thrombin-mediated cleavage of the
protease-activated receptor 1 (PAR1) receptor. Subfamily 3 members have a
very long extracellular domain, which binds to leuteinizing hormone (LH;
also known as lutropin), thyroid-stimulating hormone (TSH), and follicle-
stimulating hormone (FSH) (Ch. 17 [Kornbluth and Fissore 2014]). This
subfamily also includes LGR5, LGR6, and LGR7, which are GPCR-like
receptors involved in adult stem cell specification and function (Hsu et al.
2000; Leushacke and Barker 2011). The class B GPCRs are activated by
high-molecular-weight hormones (e.g., glucagon, secretin, and vasoactive
intestinal peptide [VIP]), whereas class C GPCRs include metabotropic
glutamate receptors, calcium-sensing receptors, γ-amino butyric acid
(GABA) B receptors, and receptors for taste compounds. Although many
class A and class B GPCRs can form homo- and heterodimers (Terrillon and
Bouvier 2004), dimer formation is obligatory for class C GPCRs (Kniazeff et
al. 2011). The Frizzled family of receptors comprises the “Frizzled” and
“Smoothened” subfamilies, which are structurally distinct and have complex
mechanisms of agonist activation (p. 107 [Ingham 2012]; Lim and Nusse
2013).

2.13 GPCR Activation, Trafficking, and G-Protein-Coupling

Specificity

The heterotrimeric G proteins that relay signals from GPCRs are associated
with the underside of the plasma membrane and are composed of an α subunit
and a βγ dimer. Agonist-activated GPCRs act as GEFs that catalyze the
exchange of GDP bound to the α subunit for GTP, causing release of Gβγ
(Ch. 2 [Lee and Yaffe 2014]). A single ligand-bound GPCR can activate
several G proteins, providing the first layer of signal amplification. The GTP-
bound Gα subunits and Gβγ subunits can then promote the activation of a
variety of downstream effectors, stimulating a network of signaling events
that is highly dependent on the G-protein-coupling specificity of each
receptor (Fig. 6). The human G-protein α subunits are encoded by 16 distinct
genes and can be divided into four subfamilies: Gαs (Gαs and Gαolf), Gαi
(Gαt, Gαgust, Gαi1-3, Gαo, and Gαz), Gαq (Gαq, Gα11, Gα14, and Gα15/16), and
Gα12 (Gα12 and Gα13) (Fig. 6) (Cabrera-Vera et al. 2003). A single GPCR
can couple to either one or more than one family of Gα subunits. Five
different β subunits and 12 γ subunits that form functional βγ dimers have
been described.

Figure 6. Regulation of classical second messenger systems and Ras and Rho GTPases by GPCRs.
Agonist-activated GPCRs promote the dissociation of GDP bound to the α subunit pf heterotrimeric G
proteins and its replacement by GTP. Gα and Gβγ subunits can then activate numerous downstream
effectors. The 16 human G protein α subunits can be divided into the four subfamilies shown, and a
single GPCR can couple to one or more families of Gα subunits. Downstream effectors regulated by
their targets include a variety of second messenger systems, as well as members of the Ras and Rho
families of small GTP-binding proteins, which, in turn, control the activity of multiple MAPKs,
including ERK, JNK, p38, and ERK5. G-protein-dependent activation of these by GPCRs and β-
arrestin-mediated G-protein-independent activation of ERK and JNK can have multiple effects in the
cytosol. MAPKs also translocate to the nucleus, where they regulate gene expression. Activation of the
PI3K–Akt and mTOR pathways plays a central role in the regulation of cell metabolism, migration,
growth, and survival by GPCRs.

Studies of the structural perturbations caused by ligand binding to class A

GPCRs reveal that agonist binding causes a contraction of a ligand-binding
pocket located within the transmembrane α-helical regions. In particular,
upon ligand binding, the transmembrane (TM) α helix 6 moves outward from
the center of the transmembrane bundle, loses contact with TM3, and moves
closer to TM5. The consequent conformational changes in the
intracytoplasmic loops lead to the formation of a new pocket between TM3,
TM5, and TM6, which binds to the carboxyl terminus of the Gα subunits,
leading to G-protein activation by promoting the release of GDP and its
exchange for GTP (Kobilka 2011). Ligands that bind and stabilize the
receptors in conformations other than this fully activated state act as partial
agonists, and they provoke a more limited G-protein activation and hence a
restricted response. Ligands that stabilize the inactive conformation of the
GPCR act as classical antagonists or inverse agonists. There is now a great
interest in the development of novel drugs that act as allosteric modulators by
binding at a site distinct from that to which the natural GPCR ligands bind.
This new generation of pharmacological agents changes the receptor
conformation, thereby modifying the affinity and/or efficacy of the
endogenous ligands (May et al. 2007). Note that the nature of the agonist
binding can impact receptor conformation, thus biasing the choice of G
protein and hence the signaling output.

2.14 Regulation of Classical Second Messenger Systems by G

Proteins and Their Linked GPCRs

Many of the immediate actions of GPCRs involve the rapid generation of

second messengers (Fig. 6). Gαs stimulates adenylyl cyclases, which
increases the cytosolic levels of cAMP, whereas Gαi inhibits adenylyl
cyclases and hence decreases cAMP levels as a result of tonic
phosphodiesterase activity. The different adenylyl cyclase isoforms appear to
be distinctly regulated by Gαs and Gαi, as well as by Gβγ subunits,
intracellular calcium, and PKCs (Taussig and Gilman 1995). Thus, the effects
of different GPCR agonists on the intracellular levels of cAMP are highly
dependent on the adenylyl cyclases expressed in a given cell type. Gαt and
Gαgust (also known as transducin and gustducin, respectively) activate
phosphodiesterases in the visual system and gustative papillae, respectively,
thus decreasing the cytoplasmic levels of cGMP by converting it to GMP
(Cabrera-Vera et al. 2003). Members of the Gαq family bind to and activate
phospholipase Cβ, which cleaves phosphatidylinositol 4,5-bisphosphate
(PIP2) into DAG and inositol 1,4,5-trisphosphate (IP3). The latter causes an
increase in cytosolic calcium levels by promoting the release of calcium from
intracellular stores and also subsequent calcium influx into the cells, whereas
DAG activates PKC (Hubbard and Hepler 2006; see Ch. 3 [Newton et al.
2014] and Ch. 11 [Julius and Nathans 2012] and p. 99 [Sassone-Corsi 2012]).
The released Gβγ dimers can independently activate many signaling
molecules, including PLCβ, adenylyl cyclases, and ion channels (particularly
the GIRK family of potassium channels). Although, in principle, the
activation of any G protein by GPCRs should result in the release of Gβγ
dimers and hence activation of their downstream targets, Gi and Go are often
the most highly expressed G proteins and represent the most frequent source
of free Gβγ complexes.
The targets of diffusible second messengers activated by G proteins
include a large number of ion channels, calcium-sensitive enzymes, and
kinases such as PKA, PKC, cGMP-dependent kinase (PKG), and calcium-
/calmodulin-dependent kinases (CaMKs), which are stimulated by cAMP,
calcium/DAG, cGMP, and calcium, respectively. Heterotrimeric G proteins
can also regulate other effectors, including signaling molecules that activate
kinase cascades and small GTPases.

2.15 GPCRs Regulate a Network of Ras- and Rho-Related

GTPases, MAPK Cascades, and PI3K-Regulated
Signaling Circuits

GPCRs stimulate pathways that control cell migration, survival, and growth
in part by activating MAPKs, a group of highly related proline-targeted
serine/threonine kinases that link multiple cell-surface receptors to
transcription factors. MAPKs include ERK1/2, JNK1-3, p38α-δ, and ERK5
MAPKs (Gutkind 1998; p. 81 [Morrison 2012]).
The small GTPase Ras, tyrosine kinases, PI3Ks, PKCs, and arrestins can
act downstream from GPCRs to promote the activation of ERK1/2 in a cell-
specific fashion (for review, see Gutkind 2000). The JNK cascade is activated
downstream from the small G proteins Rac, Rho, and Cdc42 (Coso et al.
1995). Indeed, Rac and Cdc42 can mediate signaling of Gβγ dimers and
Gα12, Gα13, Gαq, and Gαi to JNK (Gutkind 2000; Yamauchi et al. 2000).
Many GPCRs coupled to Gi activate Rac and JNK through the direct
interaction of Gβγ subunits with the P-REX1/2 family of Rac-GEFs (Welch
et al. 2002; Rosenfeldt et al. 2004). Similarly, Gαq activates Rho GTPases,
and hence JNK, through direct interaction with p63-RhoGEF and Trio (Lutz
et al. 2007). Gα12 and Gα13 bind to and act on three GEFs—p115-RhoGEF,
PDZ-RhoGEF, and LARG—to promote the activation of Rho downstream
from GPCRs (Hart et al. 1998; Fukuhara et al. 2001). Additional GEFs can
also contribute to this network. How GPCRs activate p38 and ERK5 is much
less clear, but, in general, these MAPKs are activated primarily by Gαq,
Gα12/13, and Gβγ dimers (Gutkind 2000).
Although human cancer-associated viruses express constitutively active
viral GPCRs, emerging data from deep sequencing studies have revealed that
a large fraction of human malignancies harbour mutations in GPCRs and G-
protein α subunits (O’Hayre et al. 2013). This has increased the interest in the
molecular mechanisms by which G proteins and GPCRs control normal and
cancer cell growth. Recent findings suggest that although GPCRs can
stimulate multiple diffusible-second-messenger-generating systems, their
ability to promote normal and aberrant cell proliferation often relies on the
persistent activation of Rho GTPases and MAPK cascades based on the direct
interaction of Gα subunits with RhoGEFs. The MAPKs regulate the activity
of nuclear transcription factors and coactivators, such as Jun, Fos, and YAP
(Yu et al. 2012; Vaqué et al. 2013).
Activation of the PI3K–Akt and mTOR pathways plays a central role in
cell metabolism, migration, growth, and survival (Zoncu et al. 2011). PI3K
generates 3′-phosphorylated inositol phosphates that participate in activation
of the kinase Akt and mTOR, which relay downstream signals (p. 87
[Hemmings and Restuccia 2012] and p. 91 [Laplante and Sabatini 2012]).
PI3Kγ shows restricted tissue distribution and is activated specifically by
GPCRs by the direct interaction of its catalytic (p110γ) and regulatory (p101)
subunits with Gβγ subunits (Lopez-Ilasaca et al. 1997). PI3Kγ is involved in
the chemokine-induced migration of leukocytes and plays significant roles in
innate immunity (Costa et al. 2011). In cells lacking PI3Kγ expression,
GPCRs can use PI3Kβ to stimulate phosphatidylinositol-(3,4,5)-tris-
phosphate (PIP3) synthesis (Ciraolo et al. 2008).

2.16 GPCR-Interacting Proteins in Receptor

Compartmentalization, Trafficking, and G-Protein-
Independent Signaling

GPCRs interact with a diverse array of proteins, which regulate

compartmentalization to plasma membrane microdomains, endocytosis,
trafficking between intracellular compartments and the plasma membrane,
and G-protein-independent signaling (see below). These include receptor-
activity-modifying proteins (RAMPs), GPCR-associated sorting proteins
(GASPs), Homer, β-arrestins, arrestin-domain-containing proteins
(ARRDCs), and DEP-domain proteins (Ballon et al. 2006; Magalhaes et al.
2012).
RAMPs are single-transmembrane-span proteins that associate with some
class C GPCRs, such as the calcitonin receptor and calcitonin-like receptor.
RAMPs facilitate the glycosylation of calcitonin family receptors in the
endoplasmic reticulum, thereby facilitating their expression at the cell
surface, and remain associated at the plasma membrane, where RAMPs
contribute to ligand binding and receptor signaling (Bouschet et al. 2005).
GASPs interact with the carboxy-terminal tail of many GPCRs and are
primarily involved in their postendocytic sorting (Whistler et al. 2002).
Homer 1a-c, Homer 2, and Homer 3 represent a class of proteins that harbor
Enabled/VASP homology (EVH)–like domains and bind to metabotropic
glutamate receptors (mGluRs) through a carboxy-terminal polyproline
sequence (PPXXFP) (Bockaert and Pin 1999).
GPCRs can also associate with molecules containing protein–protein
interaction domains, such as DEP, PDZ, WW, SH2, and SH3 domains (Ch. 2
[Lee and Yaffe 2014]), as well as polyproline-containing regions (Bockaert
and Pin 1999; Brzostowski and Kimmel 2001). These interactions facilitate
the localization of GPCR-initiated signaling to specific cellular structures or
membrane microdomains, including the neuronal synapse, and also determine
the signaling output by favoring the activation of a subset of GPCR targets by
increasing their local accumulation in the vicinity of the GPCR.
β-Arrestins were initially described as adaptor proteins promoting the
endocytosis of activated GPCRs (see below) but are now believed to scaffold
a wide variety of signaling complexes (Luttrell and Gesty-Palmer 2010;
Rajagopal et al. 2010). In particular (Andreeva et al. 2007), they can interact
with Src family kinases as well as multiple serine/threonine kinases, small
GTPases and their GEFs, E3 ubiquitin ligases, phosphodiesterases, and
transcription factors (Luttrell and Gesty-Palmer 2010; Rajagopal et al. 2010).
β-Arrestins can act downstream from GPCRs within endocytic vesicles in a
pathway leading to the activation of ERK1/2 and JNK, particularly in
response to β-arrestin-biased agonists for some GPCRs, thus initiating
intracellular signaling independently of the activation of heterotrimeric G
proteins (Rajagopal et al. 2010). Interestingly, β-arrestins can form
multimeric signaling complexes with ERK1/2 and JNK that are retained in
the cytosol, thus restricting the nuclear translocation of these MAPKs, which
instead act on cytosolic substrates (Fig. 6) (Rajagopal et al. 2010). Besides
the best-studied β-arrestins, a family of α-arrestins that are conserved from
budding yeast to humans has recently received increased attention because of
their potential role in GPCR trafficking and degradation (Nabhan et al. 2010).

2.17 GPCR-Independent Activation of G Proteins

Heterotrimeric G proteins can be also activated in a GPCR-independent

fashion by a family of proteins known as activators of G-protein-mediated
signaling (AGS proteins) (Blumer et al. 2007). These proteins substitute for
GPCRs by promoting nucleotide exchange on Gα subunits (e.g., AGS1), or
can regulate the physical interaction and localization of G-protein subunits
without affecting nucleotide exchange (e.g., AGS3, also known as LNG or
PINS proteins) (Blumer et al. 2007). The latter play an important conserved
role in cell polarity and polarized cell division and share the presence of a
GoLoco motif by which they bind to Gα subunits to prevent nucleotide
exchange (Willard et al. 2004).

2.18 GPCR Signal Termination

Considerable attention has focused on mechanisms of termination of GPCR

signaling, because persistent activation occurs in many diseases (Pierce et al.
2002). This desensitization is highly regulated and occurs through several
well-understood mechanisms, including GPCR-targeted kinases known as
GPCR kinases (GRKs), and more general second-messenger-regulated
kinases, such as PKC and PKA. PKC and PKA phosphorylation uncouples
receptors from their respective G proteins, presumably by phosphorylating G-
protein-interaction sites or by recruiting arrestins (Benovic et al. 1985),
thereby forming a negative-feedback loop. Activation of PKA and PKC can
also result in the heterologous desensitization of multiple GPCRs within a
cell (Chuang et al. 1996). In contrast, GRKs phosphorylate only the activated
or agonist-occupied form of the receptor, primarily in the carboxy-terminal
tail, which then binds arrestin dimers that can prevent G-protein interaction
and promote the removal of the receptor from the cell surface by endocytosis
(Shenoy and Lefkowitz 2011). Internalized receptors can be recycled back to
the plasma membrane or degraded in lysosomes, a process influenced by the
ability of ligand-bound receptors to interact with ubiquitin ligases and a
complex repertoire of sorting molecules (Hanyaloglu and von Zastrow 2008).
Concomitantly, a family of regulators of GPCR signaling (RGS) molecules
act as GAPs on GTP-bound G-protein α subunits, accelerating GTP
hydrolysis and hence signal termination (Berman and Gilman 1998).
Signaling and inactivation are intertwined, because molecules such as
arrestins can also regulate GPCR signaling specificity and/or localization
(Shenoy and Lefkowitz 2011), and many G-protein targets include RGS
domains or act as GAPs, thus acting as direct effectors that concomitantly
limit the duration of G-protein signaling.

2.19 GPCR Signal Integration

GPCRs are best known for their ability to control the activity of adenylyl
cyclases, phosphodiesterases, phospholipases, ion channels, and ion
transporters. The rapid regulation of these classical diffusible-second-
messenger-generating systems and their direct molecular targets is now
believed to represent a subset of the extensive repertoire of molecular
mechanisms deployed by GPCRs in physiological and pathological contexts.
Our recently gained understanding of GPCR signaling circuitries, including
GEFs, Ras and Rho GTPases, MAPKs, PI3Ks, and their numerous
downstream cytosolic and nuclear targets, provide a more global view of the
general systems by which these receptors exert their numerous physiological
and pathological roles. Indeed, the final biological outcome of GPCR
activation most likely results from the integration of the network of GPCR-
initiated biochemical responses in each cellular context. A new, systems-level
understanding may provide a molecular framework for the development of
novel approaches for therapeutic intervention in some of the most prevalent
human diseases.

3 THE TNF RECEPTOR FAMILY

3.1 TNF Isoforms and TNF Receptors
Tumor necrosis factor (TNF) belongs to a 19-member family of structurally
related factors that bind to the 29 members of the TNF receptor (TNFR)
family. Most TNF and TNFR members are expressed in the immune system,
where they regulate defense against infection (see Ch. 15 [Newton and Dixit
2012]); however, some members are expressed elsewhere, regulating
hematopoiesis and morphogenesis. Perturbation of signaling by TNFRs is
implicated in several diseases, including tumorigenesis, bone resorption,
rheumatoid arthritis, and diabetes (Aggarwal 2003; Croft 2009).
Members of the TNFR family have an amino-terminal extracellular region
consisting of one to four cysteine-rich domains, each of which contains three
conserved intrachain disulfide bridges. These are linked to a single
transmembrane segment and a cytoplasmic region that lacks enzymatic
activity (Locksley et al. 2001). Several receptors contain binding motifs for
members of the TRAF family of ubiquitin ligases in their intracellular
regions, and eight of them contain death domains (DDs), which are involved
in apoptotic signaling (Fig. 7). Some instead lack intracellular and/or
transmembrane regions and act as decoy receptors.
Figure 7. The tumor necrosis factor (TNF) receptor family. The structural features of the members of
the TNF receptor family (left) and their ligands (right) are shown. Cysteine-rich domains, death
domains, and interaction motifs for various members of the TRAF family are indicated. Cleavage sites
for various proteases involved in processing of the ligands are also shown. (From Aggarwal 2003;
adapted, with permission.)

The TNF family ligands are also transmembrane proteins but have
extracellular carboxyl termini. Intriguingly, these can be shed following
cleavage by proteases, and some inhibit the effects of the membrane-bound
ligand (Suda et al. 1997). The ligands are characterized by a conserved TNF
homology domain that mediates receptor binding. An exception is nerve
growth factor (NGF), which in addition to binding to an RTK also binds to
p75, a member of the TNFR family. Several ligands of this family bind to
more than one receptor (Fig. 7).

3.2 Activation of TNFRs

Most TNF family ligands are noncovalent trimers that form symmetric 3:3
complexes with their receptors. Homomeric as well as heteromeric receptor
complexes have been described (Schneider et al. 1997). Preformed receptor
oligomers exist and TNFR1 and TNFR2 have a pre-ligand-binding assembly
domain (PLAD) that is required for the assembly of TNFR complexes (Chan
et al. 2000). Whereas juxtaposition of the receptors is important for
activation, trimerization itself appears not to be necessary because there are
examples of agonistic bivalent monoclonal antibodies directed against the
extracellular domain, and because one of the ligands, NGF, is a dimer.

3.3 Signaling via TNFRs

The receptors that have DDs, including TNFR1, Fas (also known as CD95),
death receptor (DR) 3, DR4, DR5, DR6, EDAR, and the p75 NGF receptor,
induce apoptosis and necrosis of cells (Moquin and Chan 2010; Ch. 15
[Newton and Dixit 2012]; Ch. 19 [Green and Llambi 2014]). Ligand-induced
receptor activation leads to the formation of complexes referred to as death-
inducing signaling complexes (DISCs), in which the receptor DDs interact
with DDs in adaptor molecules such as TRADD and FADD. The protease
procaspase 8 is also recruited to the DISC, triggering a caspase cascade that
results in apoptosis.
In addition to apoptosis, TNFRs control survival and differentiation.
Many of their effects are mediated by the TRAF family of ubiquitin ligases,
which stimulate NF-κB, PI3K, and JNK and p38 MAPK pathways (Faustman
and Davis 2010). Activation of NF-κB is of central importance and occurs
downstream from all members of the TNFR family, except the decoy
receptors. Moreover, certain members of the TNFR family stimulate cell
proliferation, for example, by activation of the ERK1/2 MAPK.

3.4 TNFRs and Disease

Overactivity of members of the TNFR family is seen in immune-related
diseases, and encouraging results have been obtained using inhibitory anti-
TNF antibodies or ligand traps in the treatment of Crohn’s disease (Suenaert
et al. 2002) and rheumatoid arthritis (Feldmann and Maini 2001). Similarly,
blocking signaling via the receptors OX40, 4-1BB, CD27, and DR3 is
effective in various immune diseases (Croft 2009). Promising attempts have
also been made to induce apoptosis of cancer cells by treatment with agonists
of TNFR1, Fas, and TRAIL receptors (Fox et al. 2010).

4 CELL ADHESION RECEPTORS AND

MECHANOTRANSDUCERS
4.1 Mechanosensing Signaling through Integrins
Cells deploy multiple signaling mechanisms to sense the biophysical
properties of their surroundings and communicate with their neighboring
cells. Integrins are perhaps the best known cell-surface receptors involved in
these essential processes. They function primarily in cell adhesion to the
extracellular matrix (ECM) or to other cells, the latter through a repertoire of
cell-surface ligands involved in cell–cell interactions. Integrins accumulate at
cell–ECM and cell–cell contact points and orchestrate the local assembly of
multimolecular structures known as adhesion complexes, often referred to as
focal adhesions (FAs) or adhesomes (Hynes 2002; Geiger and Yamada
2011).
Integrins provide a link between the extracellular environment and the
intracellular cytoskeleton, and their dynamic engagement at cell–ECM and
cell–cell adhesions results in the rapid activation of multiple intracellular
signaling circuits, a process known as outside-in signaling (Hynes 2002;
Miranti and Brugge 2002). In turn, the adhesive properties of integrins and
hence the strength of the interactions with the ECM and other cells are
regulated by a variety of signaling pathways, which impinge on the
interaction of integrins with a key cytoskeletal molecule, talin (see below);
this process is known as inside-out signaling (Hynes 2002). Integrins can be
regulated in this way by signals from growth factors acting on RTKs and
GPCRs, as well as by inflammatory cytokines and bioactive lipids (Hynes
2002; Miranti and Brugge 2002). The latter is of particular importance for
immune cells and platelets, in which integrins are maintained in an inactive
state that enables cells to circulate in the bloodstream without interacting with
endothelial cells in the blood vessel wall, but quickly deploy their adhesive
properties in response to immune cell activation and the coagulation cascade
(Moser et al. 2009). For example, cytokine-induced activation of the
leukocyte integrin LFA1 (also known as αLβ2) leads to its rapid binding to
intercellular adhesion molecule (ICAM)-1, an adhesion molecule expressed
on the surface of activated endothelial cells, thereby stabilizing cell–cell
interactions and facilitating the transmigration of circulating leukocytes
through the endothelial cell layer into damaged tissues (Moser et al. 2009).
The integrins also provide important survival signals, because many cells
undergo anoikis, a form of programmed cell death, upon detaching from their
surrounding ECM (Frisch and Screaton 2001). Overall, integrin-mediated cell
adhesion controls cell migration, survival, growth, and differentiation,
whereas at the organismal level, integrins play fundamental roles in tissue
morphogenesis during development, and in the immune response, hemostasis,
and tissue maintenance and repair (Hynes 2002; Miranti and Brugge 2002;
Ch. 8 [Devreotes and Horwitz 2013]).
Each integrin is composed of a heterodimer of two transmembrane
subunits (α and β). In humans, there are 18 α subunits (Fig. 8), which
associate with eight different β subunits to form at least 24 heterodimeric
complexes displaying distinct ligand-binding specificities and signaling
capacities. Some of these dimers are widely expressed, whereas others show
more restricted distributions and can therefore have more specific biological
functions. Integrins typically have large extracellular regions, which interact
with cell-surface ligands and with specific sequence motifs in ECM proteins,
such as the tripeptide RGD motif originally described in fibronectin (Geiger
et al. 2001; Hynes 2002). Some integrins are promiscuous and can bind to
multiple ligands, for example, the ECM components vitronectin, fibrinogen,
fibronectin, laminin, collagen, and tenascin. Other integrins show a more
restricted binding pattern or bind to other cell adhesion receptors—for
example, ICAMs, vascular cell adhesion molecule (VCAM), and E-cadherin
(see Table 1). Most integrins possess a relatively short intracellular
cytoplasmic domain (40–70 amino acids), with the exception of integrin β4,
which has a long cytoplasmic domain.

Figure 8. The integrin family of cell adhesion receptors. Integrins are composed of a heterodimer of
two transmembrane α and β subunits. The 18 α subunits and eight β subunits can form at least 24
heterodimeric complexes displaying distinct binding specificity and signaling capacity. (Adapted from
Hynes 2002.)

Table 1. Ligands for α–β integrin heterodimers

Integrin Representative ligands
α1β1 Collagen, laminin
α2β1 Collagen, laminin
α3β1 Laminin, fibronectin
α4β1 VCAM-1, fibronectin, thrombospondin
α4β7 VCAM-1, fibronectin, MAdCAM-1
α5β1 Fibronectin, neural adhesion molecule L1
α6β1 Laminin
α6β4 Laminin
α7β1 Laminin
α8β1 Osteopontin, fibronectin, vitronectin, tenascin
α9β1 Tenascin, osteopontin, VCAM-1
α10β1 Collagen
α11β1 Collagen
αEβ1 E-cadherin
αLβ2 ICAM-1, ICAM-2, ICAM-3
αMβ2 ICAM-1, ICAM-2, ICAM-4, fibrinogen
αXβ2 ICAM-1, fibrinogen
αDβ2 ICAM-3, VCAM-1
αIIbβ3 Fibrinogen, fibronectin, vitronectin, thrombospondin, von Willebrand factor
αVβ3 Vitronectin, fibronectin, fibrinogen, osteopontin
αVβ5 Vitronectin
αVβ6 Vitronectin, fibronectin
αVβ8 Fibronectin, laminin, collagen, vitronectin
α6β4 Laminin
α4β7 VCAM-1, fibronectin

The conformation of the integrin extracellular domains changes

dramatically upon cell–ECM and cell–cell adhesions (Shattil et al. 2010).
This results in the separation of the intracellular cytoplasmic tails, which lack
enzymatic activity but instead act as platforms for the assembly of multimeric
protein complexes that are involved in signal transduction (Shattil et al. 2010;
Wehrle-Haller 2012) and link integrins to the cytoskeleton. The direct
binding of talin to the cytoplasmic tails of most activated integrin β subunits
is one of the earliest events after integrins bind to the ECM. This causes the
local accumulation of PIP2 upon recruitment of the lipid kinase
phosphatidylinositol 4-phosphate 5-kinase via its interaction with talin and
the subsequent recruitment of vinculin to the nascent adhesions (Legate and
Fassler 2009; Moser et al. 2009; Shattil et al. 2010). This helps stabilize FAs,
because integrin β subunits interact weakly with actin through talin in the
absence of vinculin. Vinculin and talin also bind to α-actinin, which has a
high affinity for actin and hence helps strengthen the interactions between β
integrins and actin filaments. Other molecules linking the β subunits to actin
include filamin, kindlins, migfilin, and integrin-linked kinase (ILK), a
pseudokinase that bridges β integrins and parvin, another actin-binding
protein (Hynes 2002; Legate et al. 2009; Geiger and Yamada 2011). ILK
stabilizes the FA by retaining integrins in a clustered state and by reinforcing
the connection with the cytoskeleton, whereas kindlins partner with talins in
integrin inside-out signaling (Legate and Fassler 2009; Moser et al. 2009).
Tensin is recruited to FAs and helps to establish additional connections
between integrins and actin fibers. Ultimately, these multiple links between
integrins and actin promote localized actin polymerization at sites of cell–
ECM and cell–cell contact. This allows rapid assembly and disassembly of
adhesion contacts and dynamic control of the cellular cytoskeleton during
cell migration (Huttenlocher and Horwitz 2011; Ch. 8 [Devreotes and
Horwitz 2013]).
FAs (Geiger and Yamada 2011) contain many cytoskeletal, adaptor, and
signaling proteins. Among them, multiple nonreceptor tyrosine kinases,
including focal adhesion kinase (FAK), the related proline-rich tyrosine
kinase 2 (Pyk2), Src, and Src-family kinases act both as signal transducers
and as scaffolds (Fig. 9) (Miranti and Brugge 2002; Geiger and Yamada
2011). Activation of these tyrosine kinases upon integrin ligation results in
their autophosphorylation and cross-phosphorylation at several tyrosine
residues, creating binding sites for multiple signaling proteins. These include
the adaptor protein Grb2, which leads to recruitment of SOS (a GEF for Ras)
and the consequent activation of Ras and the MAPK ERK1/2. In the case of
FAK, the phosphotyrosines recruit Src, Grb2, and the p85 subunit of PI3K
via their SH2 domains (Miranti and Brugge 2002; Legate et al. 2009; Shattil
et al. 2010). The latter generates PIP3 at the plasma membrane, which recruits
ILK, Akt, and other proteins to the FA complex (Miranti and Brugge 2002;
Shattil et al. 2010; Ch. 2 [Lee and Yaffe 2014]). Akt relays prosurvival and
growth-promoting signals. A direct substrate of FAK, paxillin, acts as a
multidomain adaptor protein that forms a scaffold organizing a variety of
signaling molecules, including Src, ILK, Crk, and vinculin (Miranti and
Brugge 2002; Geiger and Yamada 2011). Crk is an adaptor protein that binds
to tyrosine-phosphorylated paxillin or another adaptor protein, p130Cas, and
can then use its SH3 domain to recruit multiple additional proteins. These
include the GEF DOCK180, which activates the small GTPase Rac1 (Miranti
and Brugge 2002; Legate et al. 2009). The Rac/Cdc42-GEF α-Pix is also
activated upon integrin ligation by binding to phosphorylated paxillin and by
the local accumulation of PIP3. Moreover, Src or FAK can phosphorylate and
activate Vav, a Rac GEF that is recruited to the newly formed adhesive
structures (Miranti and Brugge 2002; Legate et al. 2009). This results in the
rapid remodeling of the actin cytoskeleton by Rho GTPases and their
downstream effectors and the relay of the signals to the nucleus by JNK and
p38 (Miyamoto et al. 1995; Miranti and Brugge 2002; p. 81 [Morrison
2012]).

Figure 9. Integrin-based cell adhesion and signaling. Integrin engagement at cell-matrix adhesions or
interaction with a repertoire of cell-surface ligands results in the rapid assembly of a multifunctional
protein network (Geiger and Yamada 2011) containing many cytoskeletal, adaptor, and signaling
proteins. This contributes to cell adhesion and activates multiple signaling events. The adhesive
properties of integrins are, in turn, regulated by a variety of signaling pathways; this is known as inside-
out signaling.

The assembled protein network thus both contributes to adhesion to the

ECM and other surrounding cells and orchestrates signaling events that
enable the cells to respond appropriately to mechanical cues. In addition to
outside-in and inside-out signaling, integrin activation can also induce the
rapid clustering of multiple RTKs, such as EGFR, PDGFR, and FGFR
(Miyamoto et al. 1996; Geiger and Yamada 2011). This enhances signaling in
response to cell adhesion. Finally, note that rather than being rigid
intracellular structures, most FAs are dynamic (Ch. 8 [Devreotes and Horwitz
2013]), and regulation of their multiple components and adhesive properties
by mitogens and chemoattractants, for example, is essential for the rapid
dissolution of preexisting adhesions and the establishment of new contact
sites during cell migration.

4.2 Signaling by Cell Adhesion Molecules (CAMs)

The formation of adhesive structures between adjacent cells, including
adherens junctions and tight junctions, contributes to the establishment of cell
polarity, differentiation, and survival and consequently key morphogenetic
processes involved in embryonic development, control of tissue homeostasis,
and tissue repair in adults. A large family of cell adhesion molecules (CAMs)
provides mechanical adhesion among cells, while initiating signaling via
networks that control cellular behavior in response to the microenvironment.
In general, cadherin molecules mediate cell–cell adhesion at adherens
junctions, claudins contribute to the formation of tight junctions, and
immunoglobulin-like CAMs (Ig-CAMs) accumulate throughout the
intercellular boundary (Cavallaro and Dejana 2011). Other CAMs, known as
nectins, can participate in both adherens and tight junctions (Takai et al.
2008).
The cadherins are calcium-dependent, homophilic, cell–cell adhesion
molecules expressed in nearly all cells in solid tissues. These molecules
participate in cell–cell recognition, and only cells expressing the same type of
cadherins may adhere to each other (Nose et al. 1988). The “classical”
cadherins were originally named based on the tissue in which they are most
prominently expressed, for example, E-cadherin in epithelial cells, VE-
cadherin in vascular endothelial cells, and N-cadherin in nervous system and
mesenchymal cells (Gumbiner 2005). These cadherins are single-pass
transmembrane proteins that form a core adhesion complex in which a
cadherin dimer binds through its extracellular region to another cadherin
dimer on an adjacent cell in a calcium-dependent manner. The cadherin
intracellular region is anchored in the plasma membrane and linked to the
cytoskeleton through a family of proteins known as catenins (Gumbiner
2005). β-Catenin interacts with the distal part of the cadherin cytoplasmic
tail, and p120 catenin interacts with a more proximal region (Gumbiner
2005). α-Catenin binds primarily to cadherin-associated β-catenin and
provides a physical link to the actin cytoskeleton, by binding actin filaments
either directly or indirectly through other actin-binding proteins, such as
vinculin, α-actinin, and formins (Kobielak and Fuchs 2004; Mege et al.
2006). p120 regulates cell movement through its ability to control both cell
adhesion by governing the availability of cadherins at the plasma membrane
and actin cytoskeleton organization by regulating Rho GTPases (Anastasiadis
and Reynolds 2001; Grosheva et al. 2001; Yanagisawa and Anastasiadis
2006). p120 can also directly affect gene expression by repressing
transcription by scaffolding a nuclear complex including the gene silencer
Kaiso (Daniel and Reynolds 1999).
The formation of cadherin-dependent adhesions contributes to growth
inhibition upon cell–cell contact. This has often been associated with
inhibition of the canonical Wnt pathway by cadherins, which retain β-catenin
at the plasma membrane and therefore limit the pool of free β-catenin
available for nuclear signaling (Nelson and Nusse 2004). Recent evidence
suggests that cadherin engagement can also activate the Hippo pathway,
which results in the cytoplasmic retention of the transcriptional coactivator
YAP that is necessary for cell growth (Kim et al. 2011). In epithelial cells, E-
cadherin acts as a tumor and metastasis suppressor. Its expression and
function are down-regulated or altered in many human cancers, and its
reexpression decreases both the proliferative and invasive capacity of tumor
cells (Vleminckx et al. 1991; Thiery and Sleeman 2006). However, the
engagement of cadherins in newly formed cell contacts promotes cell
proliferation and survival through the activation of MAPKs, PI3K, and Rho
GTPases (Pece et al. 1999; Pece and Gutkind 2000; Braga and Yap 2005).
This process often involves the engagement of growth factor receptors such
as EGFR, VEGFR2, FGFR, and PDGFR, promoting their ligand-independent
clustering and activation and prolonging the activation by ligands by
enhancing receptor recycling and limiting their degradation (Carmeliet et al.
1999; Pece and Gutkind 2000; Suyama et al. 2002; Cavallaro and Dejana
2011).
Cadherins can also control multiple cellular processes by associating with
Src-family kinases, G proteins of the Gα12/13 family, and phosphatases, such
as density-enhanced phosphatase (RPTPη), the tyrosine phosphatase SHP2,
and vascular endothelial protein tyrosine phosphatase (VE-PTP) (Cavallaro
and Dejana 2011). Cadherins thus contribute to cell–cell recognition and
adhesion while promoting cell survival and restricting cell motility/growth by
regulating intracellular signaling at the plasma membrane. In some cases, the
cadherin intracytoplasmic tail can translocate to the nucleus and regulate
transcription after shedding of the ectodomain by cell-surface matrix and
disintegrin family proteases and cleavage of the carboxy-terminal tail by
intracellular proteases, such as γ-secretase (Cavallaro and Dejana 2011),
which is reminiscent of Notch signaling.
Tight junctions involve numerous adhesion molecules, including
occludin, junctional adhesion molecules (JAMs), and the claudin family of
tetraspan transmembrane proteins, as well as intracellular adaptors, such as
ZO1 and ZO2 (Tsukita et al. 2001). Claudins are the major adhesive proteins
at tight junctions; whether they play a direct role in cell signaling is not clear
yet.
Ig-CAMs are cell-surface glycoproteins that accumulate at the cell–cell
boundary, and their homophilic interactions contribute to cell–cell
recognition and adhesion in a calcium-independent fashion (Loers and
Schachner 2007; Cavallaro and Dejana 2011). Although most Ig-CAMs have
a transmembrane region and a cytoplasmic tail, some associate with the cell
surface via a GPI anchor. In the former case, the cytoplasmic tail of Ig-CAMs
can interact with cytoskeletal proteins such as actin, ankyrins, and spectrin
and can also initiate signal transduction (Cavallaro and Dejana 2011). For
example, the formation of NCAM-based adhesions results in the activation of
a kinase cascade including CaMKIIα, the Src-family kinase Fyn, and FAK,
and this promotes neurite outgrowth and neuronal survival (Bodrikov et al.
2008). NCAM also stimulates PKCβII by recruiting it to the membrane
through the formation of a signalling complex involving a protein known as
growth-associated protein 43 (GAP43) (Korshunova and Mosevitsky 2010).
In common with cadherins, NCAM can interact with multiple growth factor
receptors, including FGFR, regulating their signaling capacity.
Signaling by CAMs—either direct or indirect actions engaging and
prolonging the activity of RTKs—is likely to play a key role during the
formation of cell–cell contacts, particularly during embryonic development,
morphogenesis, and tissue repair (Pece and Gutkind 2000; Dumstrei et al.
2002; Andl et al. 2003; Fedor-Chaiken et al. 2003). This may accelerate the
growth rate without the need for elevated local levels of growth factors. The
stabilization of cell–cell contacts may subsequently reduce signaling by
ligand-activated RTKs by sequestering them in CAM-containing clusters,
while favoring their ligand-independent activation of survival pathways, such
as PI3K–Akt signaling (Pece and Gutkind 2000; Qian et al. 2004).
Concomitantly, CAMs may restrict cell and organ overgrowth by limiting the
availability of free β-catenin and YAP (Nelson and Nusse 2004; Kim et al.
2011). In these cases, CAMs may help prevent tumor formation while
generating survival and differentiation signals. The microenvironment and
RTK signaling networks can, in turn, modulate the localization, expression,
and stability of CAMs and the proteins that they associate with, thus
regulating their adhesive properties and signaling capacity. Conversely,
CAMs can regulate RTK signaling, acting as rheostats governing the
intensity and duration of their signals in response to environmental cues such
as cell density, tissue architecture, and polarity.

5 NUCLEAR RECEPTORS
Nuclear receptors (NRs) comprise a large superfamily of intracellular
transcription factors that can effect gene expression changes in response to a
wide variety of lipophilic ligands (p. 129 [Sever and Glass 2013]). In this
respect, they differ from most other receptors in that they do not reside in the
plasma membrane, which many of their ligands can cross. Typical NR
ligands include steroids, vitamins, dietary lipids, cholesterol metabolites, and
xenobiotics. Ligands for 24 NRs have been identified; the remaining family
members are considered orphan receptors (Mangelsdorf et al. 1995). There
are 48 NRs in humans (49 in mice and 18 in Drosophila). NRs are believed
to be only one of two transcription factor families that are metazoan specific
(King-Jones et al. 2005; Degnan et al. 2009). Because many are endocrine
hormone receptors, this suggests a potentially critical role in the evolution of
animal physiology. Hormonal NRs are typically classified by the type of
ligands to which they bind, whereas orphans have various different
abbreviations, indicating, for example, similarity to known receptors (e.g.,
estrogen-related receptor, ERR). Binding of their cognate ligands, in the most
simplistic scenario, either changes the cellular localization of the NR or its
interaction with repressive and activating cofactors in the nucleus. What
distinguishes NRs from other genres of receptors is the ability to mediate
transcription without intermediate signaling cascades. Instead, they directly
bind to target genes. Nuclear–cytoplasmic cycling of some NRs allows them
to have nongenomic effects and also to be targets of cytoplasmic signaling
cascades (Wehling 1997).

5.1 Structure and Mechanisms of Receptor Activation

Nuclear receptors generally contain five functional domains. The A/B region
at the amino terminus is divergent and in some NRs contains a ligand-
independent transcriptional activation function domain (AF1). The highly
conserved DNA-binding domain (DBD) is located in the central C domain.
The carboxyl terminus is the E region, which contains the ligand-binding
domain (LBD). A flexible hinge D region links the DBD and LBD (Fig. 10).
The DBD contains tetracysteine (C4) zinc fingers that are unique to NRs and
define membership in the superfamily. The DBD typically targets the NR to a
specific DNA element termed a hormone-response element (HRE). The LBD
is composed of 12 α helices and mediates ligand recognition, dimerization,
interaction with coactivators/corepressors, and ligand-dependent
transcriptional activation. The last helix, helix 12, within the LBD is the AF2
domain, which enables NRs to interact with short LxxLL motifs in
coactivators or corepressors. These are termed the NR box or CoRNR box,
respectively (Hu and Lazar 1999). Whereas AF1 domains vary greatly among
all NRs and have a propensity to stay disordered, AF2 domains have similar
structures (Warnmark et al. 2003).

Figure 10. General structure and binding of nuclear receptors. (A) Domain organization of a typical
nuclear receptor. (B) Three modes of signal transduction: as monomers, heterodimers, and homodimers.
(From Sonoda et al. 2008: modified, with permission, © Elsevier.)

5.2 Nuclear Receptor Classification

NRs can be classified based on their DNA-binding mechanism (Fig. 10) or
their ligand (Table 2). On the basis of their mode of actions, nuclear receptors
are classified into four types. Type I and type III NRs are normally
sequestered in the cytoplasm by heat shock proteins. Ligand binding to type I
NRs dissociates heat shock proteins and results in nuclear enrichment. Type I
NRs bind to DNA as homodimers, and the response elements they recognize
are inverted repeats. Type II receptors, unlike type I receptors, are normally
enriched in the nucleus and are bound to DNA even in the absence of ligand.
They generally bind to DNA as heterodimers with the retinoid X receptor
(RXR). In the absence of ligand, type II receptors repress transcription via
association with corepressors. In the presence of ligand, corepressors are
dissociated, and coactivators including histone-modifying enzymes are
recruited by the type II receptors for gene activation. Examples of type II
receptors include the thyroid hormone receptor (TR) and retinoic acid
receptor (RAR). Type IV receptors bind as monomers and recognize half-site
response elements.

Table 2. The nuclear receptor superfamily

Steroid receptors
GR Glucococorticoid
MR Mineralocorticoid
AR Androgens
PR Progesterone
TRα, TRβ Thyroid hormone
ERα, ERβ Estrogen
Vitamin receptors
VDR Vitamin D
RARα, RARβ, RARγ Retinoic acid
Fatty acids and derivatives
PPARα, PPARβ/δ, PPARγ Fatty acids
LXRα, LXRβ Oxysterol
FXR Bile acids
RXRα, RXRβ, RXRγ Rexinoids
Xenobiotic receptors
CAR Androstane metabolites
PXR Pregnane derivatives
Adopted orphan receptors
HNF4α, HNF4γ Fatty acids
REV-ERBα, REV-ERBβ Heme
RORα, RORβ, RORγ Cholesterol, retinoic acid
SF1, LRH1 Phosphatidylinositols
ERRα, ERRβ, ERRγ Estrogen
Orphan receptors
SHP
DAX1
TLX
PNR
GCNF
TR2, 4
NR4A1, NR4A2, NR4A3
COUP-TFα, COUP-TFβ, COUP-TFγ
5.3 Three Modes of Binding to Promoter Regions Influence
Transcription
Hormone-responsive NRs are sequestered in the cytoplasm or are bound to
HREs in repressive complexes that include chromatin modifiers such as
histone deacetylases (HDACs), nuclear corepressors (N-CoRs), and SMRT
proteins (for “silencing mediator of retinoid and thyroid receptors”) (Perissi
et al. 2010). Ligand binding typically promotes nuclear translocation and/or
recruitment of coactivators that displace corepressors to initiate transcription
(Rosenfeld et al. 2006). Coactivators that mediate NR function include
PGC1, SIRT1, p160, and p300 proteins (Sonoda et al. 2008).
Mechanistically, NRs can mediate transcription (either repression or
activation) in the following manner.

5.3.1 Homodimers
Type I and type III receptors bind to DNA as homodimers when bound to
their cognate ligands. Type I receptors include all steroid receptors, and their
response elements typically consist of two hexameric inverted (palindromic)
repeat half-sites, for example, AGAACA (the glucocorticoid response
element, GRE) or AGAACA (the estrogen response element, ERE). These
sequences can mediate either activation or repression in response to hormone.
The DBDs of steroid receptors are highly similar. Thus, the glucocorticoid
receptor (GR), the mineralocorticoid receptor (MR), the androgen receptor
(AR), and the progesterone receptor (PR) all bind to overlapping response
elements. Type III receptors are similar to type I receptors except that they
recognize direct repeats instead of inverted repeats.

5.3.2 Heterodimers
RXR forms heterodimers with various nonsteroidal members of the NR
superfamily that do not bind to HREs efficiently by themselves. Depending
on the type of NR, HREs have specific conformations. Unlike the type I
receptors mentioned above, type II receptors form retinoid-acid-related-
receptor (RXR)-containing heterodimers that recognize direct repeats of
AGGTCA or similar sequences separated by specific spacing (Umesono et al.
1991). The RAR- and peroxisome proliferator-activated-receptor-γ (PPARγ)-
containing dimers RAR–RXR and PPARγ-RXR bind to a direct repeat (DR-
1), whereas the vitamin D3 receptor (VDR)-, TR-, and RAR-containing
dimers VDR–RXR, TR–RXR, and RAR–RXR bind to DR-3, DR-4, and DR-
5, respectively (Evans 2005).

5.3.3 Monomers
Type IV NRs bind to just one AGGTCA site as monomers in a ligand-
independent manner. These receptors use an extended DBD. There are
currently three known types of monomer-binding receptors, which are
defined by unique sequences 5′ to the consensus site. REV-ERBs tend to bind
to A(A/T)CTAGGTCA sequences, whereas ERRs bind to TNAAGGTCA
sequences.

6 FUNCTIONS OF NUCLEAR RECEPTORS

6.1 Steroid and Thyroid Hormone Receptors
Steroid and thyroid receptors maintain homeostasis by acting as sensors for
circulating hormones from the adrenals, ovaries, testes, and thyroid. GR, MR,
AR, and PR are closely related and bind common DNA sequences. Following
ligand treatment, these receptors translocate from the cytoplasm to the
nucleus and bind to palindromes of the GR half-site AGAACA as
homodimers. All other members of the superfamily, including ER and TR,
bind to response elements that contain variations of the ER half-site
AGGTCA.

6.2 Nonsteroidal Hormonal Receptors

Instead of acting as homodimers, all nonsteroidal hormone receptors,
including TR, use RXR as a partner to form heterodimers. VDR and RAR are
activated by calcitriol (vitamin D) and retinoic acid. RXR is conserved in
invertebrates and may be an ancient member of the NR family. RXRs (α, β,
and γ) are activated by the novel retinoid isomer 9-cis retinoic acid and other
synthetic agents (rexinoids) (Mangelsdorf et al. 1992). RXR forms three
different types of dimers. Other than the nonpermissive heterodimers
mentioned above (e.g., RAR and VDR), which cannot be activated by the
RXR ligand, RXR can form homodimers and permissive heterodimers (e.g.,
with PPAR and liver X receptor, LXR) that can be activated by RXR-ligand
binding.

6.3 Receptors for Fatty Acids and Derivatives

The PPAR subfamily includes PPARα, PPARδ, and PPARγ. Each of the
PPARs binds to common response elements as heterodimers with RXR, using
fatty acid ligands, which function as metabolic sensors. PPARα is highly
expressed in the liver and is the target of the fibrate class of drugs used to
control hyperlipidemia (Issemann and Green 1990). PPARδ is universally
expressed and promotes fatty-acid oxidation. PPARγ is required for
adipogenesis, promotes fat storage, and has been widely studied for its role in
the metabolic syndrome (Barak et al. 1999). LXRα and LXRβ and farnesoid
X receptor (FXR) are sensors for cholesterol metabolites such as oxysterols
and bile acids, respectively. Both LXRs and FXR form heterodimers with
RXR. LXRs are important for maintaining cholesterol homeostasis, whereas
FXR acts as the primary bile acid sensor by repressing expression of 7α-
hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid production,
through up-regulation of two orphan NRs, SHP and LRH1, upon bile acid
stimulation (Lu et al. 2001). SHP and LRH1 form inactivating heterodimers
to repress transcription of CYP7A1, thus turning off bile acid production.

6.4 Xenobiotic Receptors

The xenobiotic receptors include the constitutive active/androstane receptor
(CAR) and pregnane X receptor (PXR). Both CAR and PXR form
heterodimers with RXR and bind promiscuously to different response
elements (Lehmann et al. 1998; Sueyoshi et al. 1999). Their role is to induce
expression of enzymes such as CYP3A that promote the metabolism of
exogenous compounds (xenobiotics). Unlike typical NRs, PXR has a
promiscuous LBD pocket that binds a wide variety of natural and synthetic
compounds. Activation of xenobiotic receptors is not always beneficial to the
host. For example, upon exposure to high doses of acetaminophen (a
common component of pain killers and cocaine), CAR can mediate
production of toxic by-products through up-regulation of three CYP enzymes
that lead to acute hepatotoxicity (Zhang et al. 2002).

6.5 Adopted Orphan Receptors

Discovery of natural or synthetic ligands for several orphan NRs has led to
their classification as “adopted” orphan receptors (e.g., REV-ERBα and
REV-ERBβ). REV-ERBs are repressors that compete with retinoid-related
orphan receptors (RORα, RORβ, and RORγ) for binding to (A/T)6RGGTCA
HREs (Forman et al. 1994). The REV-ERB ligand is neither a lipid nor a
hormone but, rather, a single heme molecule (Reinking et al. 2005). Many
heme-containing proteins also bind to diatomic gases, and REV-ERBs are
also responsive to NO and CO (see below). RORs are proposed to bind
cholesterol and retinoid acid, although whether this is important in
antagonizing REV-ERBs is not clear (Kallen et al. 2002; Stehlin-Gaon et al.
2003). NR5A subfamily members SF1 (NR5A1) and LRH1 (NR5A2) were
originally thought to be constitutively active. Recent analysis, however,
reveals that these receptors can bind to phosphatidylinositols, including PIP2
and PIP3 (Ch. 3 [Newton et al. 2014]). They may therefore link transcription
and phospholipid signaling (Krylova et al. 2005).

6.6 True Orphan Receptors

True orphans are constitutive activators or repressors that do not bind ligands.
The NUR subfamily (NR4A1, NR4A2, and NR4A3) binds to DNA as
monomers, homodimers, and heterodimers with RXR (or another NUR).
NURs have no known ligands, and their activity is controlled by their
expression levels. ERRα, ERRβ, and ERRγ interfere with ER and AR
signaling. ERRs are activated by coactivators such as PGC1α, an important
metabolic regulator (Ch. 14 [Hardie 2012]). Germ-cell nuclear receptor
(GCNF) is an unusual member of the NR superfamily that binds to a DR-0
motif and may function as a transcriptional repressor in stem cells (Fuhrmann
et al. 2001).
NRs thus represent an important metazoan strategy for regulating gene
expression programs by integrating different intracellular and extracellular
signals. Moreover, cross talk between NRs and other signal transduction
pathways adds an additional layer of complexity to their already diverse
functions.

7 GASES
The ability to sense and adapt to changes in levels of diatomic gases (O2, NO,
and CO) is crucial for an organism’s survival. Like steroids, gases are able to
cross the plasma membrane and bind to intracellular targets. Signal
transduction via gases is predominantly mediated by heme-containing
proteins. Binding of the gas to the sensor proteins via the heme moiety can
affect their activity or stability. In the case of oxygen, the source of the gas is
the environment. In contrast, nitric oxide and carbon monoxide can be
generated in neighboring cells and function as bona fide paracrine signals
(through covalent and noncovalent binding).

7.1 Signal Transduction via Oxygen

Levels of oxygen inside mammalian cells are mainly detected by hypoxia-
inducible factor (HIF) (Wang et al. 1995). HIF is a heterodimer comprising a
regulatory α subunit and a DNA-binding β subunit. At high oxygen levels,
the HIFα subunit is hydroxylated by prolyl hydroxylase domain (PHD)
proteins and targeted for proteosomal degradation by interacting with the von
Hippel–Lindau (VHL) protein, which is the targeting subunit of a cullin
RING ligase (CRL) 2 E3 ubiquitin ligase. The PHD proteins are
dioxygenases that catalyze the incorporation of both atoms of molecular
oxygen into their substrates. At low oxygen levels, HIFα degradation is
inhibited and HIFα accumulates inside the cell. It associates with HIFβ,
which binds to DNA elements termed hypoxia responsive elements (HREs)
that have the core sequence RCGTG. HIF targets include erythropoietin,
which stimulates red blood cell production; vascular endothelial factor
(VEGF), which stimulates angiogenesis (growth of new blood vessels); and
other enzymes in relevant pathways, including glycolytic enzymes.

7.2 Signal Transduction via Nitric Oxide

Nitric oxide (NO) was first discovered as an endothelial-derived relaxing
factor (EDRF) and subsequently found to be a powerful mediator in other
biological processes, including platelet aggregation, synaptic transmission,
and inflammatory responses (Moncada et al. 1991). Vascular NO is a key
regulator of endothelial function and stimulates blood vessel dilation,
prevents platelet aggregation and adhesion, and inhibits proliferation of
vascular smooth muscle cells. NO is produced by NO synthase (NOS) from
the amino acid L-arginine and detected by the heme-containing enzyme
soluble guanylyl cyclase (sGC). Synthesis of NO from arginine involves two
mono-oxygenation steps and requires one O2, one NADPH, and
tetrahydrobiopterin (BH4).
The electronic structure of NO makes it an excellent ligand for heme,
allowing it to bind to sGC heme at a low and nontoxic concentration. sGC is
a heterodimer composed of α1 and β1 subunits. H105 in the β1 amino
terminus is the heme ligand (Wedel et al. 1994). In a two-step process, NO
first binds to the sGC heme moiety, forming a 6-coordinate complex. In the
second step, the heme-H105 bound breaks forming a 5-coordinate complex.
The second step triggers a conformational change in the sGC catalytic
domain, leading to its activation. When activated by NO, sGC converts GTP
to cGMP, which then acts as a second messenger (Ch. 3 [Newton et al.
2014]). Besides sGC, NO can also interact with other heme-containing
enzymes, including methionine synthase, cytochrome c oxidase, cytochrome
P450, hemoglobin, and ferritin (Moncada and Palmer 1991; Brown and
Cooper 1994; Danishpajooh et al. 2001). REV-ERBα and REV-ERBβ have
also been shown to be able to bind NO through their heme-containing LBDs,
which results in inhibition of the ability of REV-ERB to repress transcription
(Pardee et al. 2009).
In S-nitrosylation, NO covalently links to the thiol side chain of cysteine
residues in target proteins and modifies their activities. An example of NO-
mediated S-nitrosylation is the modification of β-catenin in the adherens
junction to increase endothelial permeability (Thibeault et al. 2010).

7.3 Signal Transduction via Carbon Monoxide

Endogenous carbon monoxide is generated by heme oxygenase (HO), an
enzyme that degrades heme into iron, biliverdin, and CO in aging red blood
cells and is induced by many cellular stressors and toxins, including UV,
hydrogen peroxide, heavy metals, and arsinite. Like NO, CO has affinity for
heme-containing proteins and activates sGC by binding to its heme motif,
which stimulates similar biological processes, including intestinal smooth
muscle relaxation and neural transmission (Rodgers 1999). In olfactory
neurons, CO acts as a neurotransmitter via sGC to produce cGMPs (Verma et
al. 1993). It can also bind to REV-ERBs, although at a lower affinity. In
addition, CO can bind to other circadian-rhythm-regulating transcription
factors, such as neuronal PAS domain protein 2 (NPAS2), which controls
circadian gene expression. Upon exposure to CO, NPAS2 cannot form
heterodimers with its partner Bmal (Dioum et al. 2002).
Gases are thus not only cell stress indicators, but also important
endogenous signaling molecules. Their levels are also crucial and often
disrupted in pathological processes, including cardiovascular disease,
cancers, and CNS diseases. Future work on diatomic gas signaling thus has
the potential lead to the development of entirely new classes of therapeutic
agents.

8 CONCLUDING REMARKS
Signaling mechanisms controlling cell growth, migration, survival, and
differentiation involve a plethora of different types of ligands, including
proteins, peptides, and certain lipids, that bind to receptors at the cell surface,
and steroids and gases that can pass across the plasma membrane and bind to
intracellular receptors. Recent work has provided immense insights into how
receptors are activated after ligand binding, how they initiate signaling inside
the cell, and how signaling is terminated. A striking feature is that regulated
dimerization or oligomerization is involved in the control of many plasma
membrane receptors and intracellular signaling molecules. Moreover, signal
transduction often occurs by the reversible formation of complexes and
specific translocations of signaling molecules, rather than by random
diffusion events. Another striking feature is that many different types of
posttranslational modifications of proteins are involved in the regulation of
the activity, stability, and subcellular localization of signaling molecules.
Furthermore, different signaling inputs converge on a few well-conserved
intracellular pathways, several of which link to transcriptional regulation of
gene programs.
Whereas the initial phase of signal transduction often involves
amplification mechanisms, such as inhibition of phosphatases in conjunction
with activation of tyrosine kinases, subsequent phases contain different
negative-feedback and termination mechanisms, acting at many different
levels. There is also extensive cross talk between different signaling
pathways. An important question that remains to be answered is exactly how
specificity of signaling is achieved. Moreover, most of our knowledge about
receptor signal transduction mechanisms comes from studies of cultured
cells; much more work is needed before we understand signal transduction at
the organismal level.

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4The Ryk and Ror families of RTK, for example, bind members of the Wnt family. Ryk has a Wnt
inhibitory factor-1 (WIF1) domain, and the two Ror receptors have cysteine-rich domains related to a
domain in the Frizzled family of serpentine receptors to which ligands of the Wnt family bind (van
Amerongen et al. 2008).

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005900
CHAPTER 2

Protein Regulation in Signal

Transduction

Michael J. Lee and Michael B. Yaffe

David H. Koch Institute for Integrative Cancer Research at MIT, Department
of Biology and Department of Biological Engineering, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139
Correspondence: myaffe@mit.edu

SUMMARY

Cells must respond to a diverse, complex, and ever-changing mix of

signals, using a fairly limited set of parts. Changes in protein level,
protein localization, protein activity, and protein–protein interactions are
critical aspects of signal transduction, allowing cells to respond highly
specifically to a nearly limitless set of cues and also to vary the
sensitivity, duration, and dynamics of the response. Signal-dependent
changes in levels of gene expression and protein synthesis play an
important role in regulation of protein levels, whereas posttranslational
modifications of proteins regulate their degradation, localization, and
functional interactions. Protein ubiquitylation, for example, can direct
proteins to the proteasome for degradation or provide a signal that
regulates their interactions and/or location within the cell. Similarly,
protein phosphorylation by specific kinases is a key mechanism for
augmenting protein activity and relaying signals to other proteins that
 possess domains that recognize the phosphorylated residues.

Outline
1 Introduction
2 Posttranslational modifications and the regulation of protein activity
3 Regulation of protein–protein interaction
4 Regulation of protein location
5 Regulation of protein production
6 Protein degradation
7 Concluding remarks: What does the future hold?
References

1 INTRODUCTION
Signal transduction processes are, in many respects, protein-driven events.
One common way to describe these circuits involves designating different
proteins, or modular domains within an individual protein, as “readers,”
“writers,” and “erasers.” In this model, catalytic domains that add specific
posttranslational modifications, such as kinases and acetyltransferases, are
called writers because they leave behind a physical mark on the proteins they
act on. Conversely, phosphatases, deacetylases, and other enzymes that
remove these modifications are examples of erasers. In addition to writers
and erasers, there are also domains that bind to specific posttranslationally
modified or unmodified sequences of amino acids. These types of domains
read the sequences produced by writers and erasers and are therefore called
readers. These readers can be involved in protein–protein interactions or
interactions between two parts of the same protein. Other readers can bind
directly to specific phospho- and neutral lipids, or to specific ions, such as
calcium, rather than to amino acid sequences. The targets of these readers,
writers, and erasers are often short amino acid sequences (typically three to
15 amino acids in length) called motifs. Using these modular parts, cells
dynamically encode information in response to environmental, chemical, or
developmental stimuli, to transduce the signal (see Table 1).

Table 1. Examples of modular domain readers of motifs and protein/lipid

posttranslational modifications
Modular
protein
domain Posttranslation modification
reader recognized (if any) Example motifs Biological processes
SH2 domain Phosphotyrosine pYxxφ Growth factor signaling
SH3 domain None, usually recognizes proline- RxxPxxP, Signaling scaffolds
rich motifs PxxPxR/K
14–3–3 Phosphoserine/phosphothreonine RSx(pS/pT)xP, Chaperone/scaffold,
Rxx(pS/pT)xP subcellular localization
FHA domain Phosphothreonine pTxxφ, pTxxD DNA damage response,
gene expression, transport
BRCT Phosphoserine/phosphothreonine (pS/pT)xxφ DNA damage response
domain
Polo-box Phosphoserine/phosphothreonine S(pS/pT)P/X Mitotic control by Polo-like
domain kinases
WW domain Most do not recognize any PTMS; a PPxY, PPLP, Gene transcription, proline
few recognize PPR (pS/pT)P isomerization and cell-
phosphoserine/phosphothreonine cycle control,
development
Bromo Acetyl-lysine – Gene expression, chromatin
domain structure, and remodeling
Chromo Methyl-lysine – Chromatin structure and
domain remodeling
Tudor Methyl-lysine – Chromatin structure and
domain remodeling, DNA damage
response
EVH1 None, usually recognizes Pro-rich FPXφP, PPxxF Actin cytoskeleton control,
domain motifs neurotransmission
PX domain Phosphatidyl-3-phosphate, -3,4- (Not applicable) Membrane targeting and
bisphosphate, and -3,4,5- trafficking, endosomal
trisphosphate sorting
PH domain Phosphatidyl-4,5-bisphosphate, -3,4- (Not applicable) Membrane trafficking,
bisphosphate, and -3,4,5- cytoskeletal control
trisphosphate
FYVE Phosphatidyl-3-phosphate (Not applicable) Vacuolar protein sorting,
domain endosomal sorting
Additional motifs beyond those shown are also recognized. (–) No distinct motif has emerged.
(Not applicable) Lipid posttranslational modifications rather than protein sequence motifs.
pY, Phosphotyrosine; pS, phosphoserine; pT, phosphothreonine; F, hydrophobic amino acids.

For some cellular processes, the mere presence of a protein, monitored,

for example, by the levels of its RNA transcript, is sufficient to provide
insight into the current state of the cell. In contrast, this provides relatively
limited information about the current state of a signaling network, primarily
because an amazing amount of regulation occurs at the protein level, via
control of the readers, writers, and erasers. Moreover, this occurs
dynamically, reversibly, and sometimes very quickly. Below, we focus on the
basic mechanisms of regulation that occur at the level of the protein,
specifically focusing on how signal transduction can control protein number,
protein localization, protein activity, and protein–protein interactions.

2 POSTTRANSLATIONAL MODIFICATIONS AND THE

REGULATION OF PROTEIN ACTIVITY
Many signaling proteins have intrinsic enzymatic activities. For example,
they may contain catalytic domains capable of phosphorylating or
dephosphorylating proteins, lipids, or sugars (kinases) or hydrolyzing specific
lipids or phospholipids (lipases and phospholipases, respectively). In addition
to performing such covalent posttranslational modifications, signaling
proteins may also stimulate the exchange or hydrolysis of nucleotides in
nucleotide-binding proteins such as small G proteins and heterotrimeric G
proteins by acting as guanine-nucleotide exchange factors (GEFs) or GTPase-
activating proteins (GAPs), respectively. Other types of catalytic domains
participate in the formation of cyclic nucleotides such as cyclic AMP (cAMP)
or cyclic GMP (cGMP) (Ch. 3 [Newton et al. 2014]), or synthesize specific
types of gas molecules that are involved in signaling, such as nitric oxide,
superoxide, and other reactive oxygen and nitrogen species.
The activity of these signaling molecules must be tightly controlled so
that the products of the reactions that they catalyze—phospholipids,
phosphoproteins, gases, activated or inactivated G proteins—are only
produced at the appropriate time and place. Some of this control occurs
through regulation of protein levels or protein localization, but much of it
involves regulation of protein activity by posttranslational modification of the
signaling molecule itself, intramolecular interactions between different
domains within the protein, and/or intermolecular interactions with other
proteins. In each case, this may involve a phenomenon known as allostery, in
which the site where the control is exerted (e.g., where the domains interact)
is different from the site where catalysis occurs. Transmission of information
via a conformational change allows the allosteric site to control the protein’s
catalytic activity, even though the two may be far removed in physical space
(Fig. 1). Key examples in signaling include binding of calcium to calmodulin
to activate calcium/calmodulin-dependent protein kinases (CaM kinases), and
binding of cyclic nucleotides to activate protein kinase A (PKA) and protein
kinase G (PKG) (see Ch. 3 [Newton et al. 2014]).

Figure 1. Allosteric modulation of protein activity. (A) Cartoon example of protein allostery. In this
example, an allosteric-regulation site exists in a region of the protein that is spatially distinct from the
active site. Modulation of the allosteric site (through posttranslational modification or the binding of a
ligand, cofactor, or protein) causes changes in the active site. Importantly, these changes can be either
activating or inhibiting. (B) Allosteric regulation of dihydrofolate reductase (DHFR). (Left panels) Two
surface views of the DHFR enzyme, highlighting the active site (bound to folate), cofactor binding site
(bound to NADPH), and residues involved in allosteric communication between the two sites (shown in
blue). (Middle panels) A view of a slice through the protein core. (Right panels) A cartoon
representation of the slice mappings shown in B. (Figure generously supplied by Rama Ranganathan
and Cell Press.)

2.1 Posttranslational Modifications

Proteins can be exquisitely regulated by relatively small covalent changes to
their basic chemical structure. These posttranslational modifications can
profoundly alter a protein’s activity, localization, stability, and/or binding
partners, and therefore constitute the “front line” of many signaling systems
within the cell. More than 350 different posttranslational modifications have
been discovered, many of which are reversible.
One of the first types of protein posttranslational modifications to be
identified was phosphorylation. Although phosphorus was first noted as a
trace element in purified egg white by Gerrit Mulder in 1835 (Mulder would
subsequently invent the term protein to describe such materials), it was not
until 1906 that an isolated cleavage fragment from a single protein, vitellin,
was shown to contain a constant composition of ∼10% phosphorus (Levene
and Alsberg 1906). Subsequent studies in the 1930s revealed that the
hydrolysis products of vitellin included serinephosphoric acid, indicating that
the phosphorus was part of a covalent modification of the protein’s
constituent amino acids (Lipmann and Levene 1932). In the 1950s, George
Burnett and Eugene Kennedy identified an enzymatic activity in
mitochondrial lysates that was capable of transferring radioactive phosphate
to exogenous substrates (Burnett and Kennedy 1954), but it was not until the
pioneering work of Edwin Krebs and Edmond Fischer that protein
phosphorylation was shown to reversibly regulate a key biological process
(Krebs and Fischer 1956)—in this case, controlling whether glucose
molecules are stored as polymers of glycogen or glycogen molecules are
broken down to supply the body with glucose.
Using kinases as writers, phosphate groups are added by formation of
esters with amino acids whose side chains contain alcohols. In mammals, the
vast majority of protein phosphorylation occurs on serine residues (∼85%),
with a lesser amount on threonine (∼15%) and only a tiny fraction on tyrosine
residues (∼0.4%). In addition to being relatively rare, tyrosine
phosphorylation is restricted to higher eukaryotes, having evolved just before
the origin of the metazoan lineage. Nevertheless, tyrosine phosphorylation
seems to be particularly important in signaling and is frequently dysregulated
in cancer. A handful of other residues can also be phosphorylated, including
histidine, aspartic acid, arginine, and lysine. These modifications are typically
labile and difficult to purify/study, but it is becoming increasingly clear that
histidine phosphorylation, for example, is relatively common and an
important player in many signaling systems.
Protein phosphorylation controls the functions of proteins through a
variety of different molecular mechanisms. At physiological pH, the addition
of a phosphate group adds about 1.5 electrostatic units of negative charge. In
some cases, this negative charge can drive the formation of ionic bonds with
positively charged residues, such as lysine and arginine, in other parts of the
protein. In enzymes, these new bonds can shift the positions of α helices and
loops in the protein to make the enzyme more or less active. For example,
many protein kinases themselves are phosphorylated on residues in a region
called the activation loop. Ionic interactions and hydrogen bonds involving
these phosphorylated residues to nearby arginine and lysine residues lead to a
dramatic rearrangement of the protein that opens up the binding site for ATP
and substrates, while simultaneously reorienting the catalytic residues into a
conformation that allows the protein kinase to transfer a phosphate group
from ATP to a serine, threonine, or tyrosine residue on the substrate (Fig. 2).
Because protein kinases often both catalyze the phosphorylation of proteins
and are themselves substrates for other, upstream protein kinases, they can be
linked together into pathways where one protein kinase phosphorylates
another, which then phosphorylates a third, which then phosphorylates some
other type of protein. This creates a signal amplifier in which the activity of
the first kinase is magnified by the catalytic activities of the other kinases
downstream from it in the pathway and is a particularly prominent type of
signaling circuit used by mitogen-activated protein kinases (MAPKs) (see p.
81 [Morrison 2012]). Phosphorylation is negatively regulated by a class of
erasers called phosphatases, which are grouped into three main families based
on sequence and structural similarity: phosphoprotein phosphatases, protein
phosphatase metal-ion-dependent phosphatases, and protein tyrosine
phosphatases. Each family is also further divided into subfamilies based on
mechanisms of catalysis and substrate specificity.

Figure 2. Mechanism of kinase activation. (A) Conformational changes in protein kinases upon
phosphorylation enhance their catalytic ability. The structure of ERK2, an MAPK, is shown in its
inactive nonphosphorylated state and its active phosphorylated state. The carboxy-terminal lobes of the
kinase in both states (brown and red, respectively) have been superimposed. Note that following
phosphorylation of the activation loop, there is a marked rotation and reorientation of the amino-
terminal lobe (yellow and purple, respectively), bringing key catalytic residues, including those present
in a critical α helix, αC, into position, converting the kinase into an active state that can now
phosphorylate downstream substrates. (B) Close-up of phosphorylation-induced conformational
changes in the activation loop. Two key residues in the activation loop of MAPKs, a threonine and a
tyrosine residue, separated by a singe amino acid (i.e., a TXY motif) can interact with a network of
surrounding arginine residues only when they are in their phosphorylated states. These interactions not
only shift the positions of the threonine and tyrosine residues themselves (curved arrows), but drag the
entire activation loop into a new conformation that communicates with the rest of the protein to move
the entire amino-terminal lobe relative to the carboxy-terminal lobe, as shown in A.

Besides changing the enzymatic activity of a protein, phosphorylation can

also disrupt the interactions between two or more proteins or cause two
proteins to interact (see below), often changing the subcellular location of the
phosphorylated protein.
A second common reversible posttranslational modification of proteins is
acetylation. In this case, the positively charged ε amino groups on lysine
residues are converted into neutral amides by the addition of acetate. This
loss of positive charge prevents the acetylated lysines from making
electrostatic interactions with phosphate groups and is therefore a prominent
posttranslational modification found on DNA-binding histones. Because the
DNA backbone is built from esters of phosphates and sugars, acetylation
weakens binding of the histones in nucleosomes to DNA, allowing other
DNA-binding proteins, such as transcription factors and RNA polymerase, to
bind instead. This often results in changes in chromatin structure and
transcriptional activity. Acetylation is also common in enzymes involved in
metabolism (Wang et al. 2010) and probably functions, at least in part, by
changing the activity of the enzyme, in a similar way to protein
phosphorylation. Like phosphorylation, protein acetylation can also drive two
proteins to bind to each other if one of the proteins has a domain that
specifically recognizes acetyl-lysine (e.g., a bromo domain), or it can
specifically enhance the dynamics of recruitment of other proteins to the
acetylated protein relative to the unmodified form. For example, acetylation
of the DNA damage kinase ATM by the acetyltransferase Tip60 (also known
as Kat5) promotes recruitment of ATM to sites of DNA damage and ATM-
dependent signaling. Similarly, microtubules containing acetylated tubulin
are better able to recruit molecular motors that drive vesicular trafficking
within the cell (Perdiz et al. 2011). As in the case of phosphorylation,
acetylation is balanced by erasers, called deacetylases. Historically, histone
proteins were thought to be the main targets of acetylation. Acetyl
transferases and deacetylases are therefore commonly referred to as histone
acetyltransferases (HATs) and histone deacetylases (HDACs), respectively.
However, because neither of these classes of enzyme is specific for histone
proteins, the terms lysine acetyltranferase (KAT) and lysine deacetylase
(KDAC) are more appropriate.
Lysine and arginine residues can also be modified by methylation and/or
demethylation (Fig. 3). Here, the amino acid side-chain nitrogen atoms have
one or more of their hydrogen atoms replaced with methyl groups. A single
lysine residue can contain one, two, or three methyl groups, whereas an
arginine residue can contain one or two methyl groups distributed in different
ways among the three side-chain guanidino nitrogens. Lysine methylation,
like acetylation, can weaken interactions between histones and DNA (but can
also lead to repression of transcription, depending on which histone lysine
residue is methylated) and is therefore a major mechanism for the epigenetic
control of gene expression. In addition, both lysine and arginine methylation
can drive direct protein–protein interactions when the methylated
lysine/arginine residues on one protein are recognized by modular domains of
the Royal superfamily (e.g., Tudor, chromo, MBT, PWWP, and plant Agenet
domains) on the other protein (Maurer-Stroh et al. 2003). For example, in
response to DNA damage, kinases such as ATM and ATR phosphorylate a
host of substrates to initiate cell-cycle arrest, DNA repair, and potentially cell
death if the damage is too severe. One of these substrates is the
methyltransferase MMSET (also known as NSD2 or WHSC1).
Phosphorylated MMSET is recruited to sites of DNA damage, where it
methylates histone H4 on lysine residue K20. Methylated H4K20 recruits the
DNA repair protein 53BP1 through a Tudor domain in 53BP1, facilitating
DNA repair (Pei et al. 2011).
Figure 3. Examples of protein posttranslational modifications and modular protein-binding domains
that recognize these modified amino acids. (A) Structures of common amino acid posttranslational
modifications. The parent amino acid structure is shown in black; the modification is shown in red. (B)
Cartoon representations of modular binding domains. α Helices are shown in cyan; β-strands are shown
in purple; loops are shown in orange. SH2 domains recognize peptides containing phosphotyrosine,
FHA domains recognize peptides containing phosphothreonine, Bromo domains recognize peptides
containing acetyl-lysine, and tandem Tudor domains recognize dimethylarginine. In the examples
shown, the SH2 domain is from Src kinase, the FHA domain is from Chk2, the bromo domain is from
Brd4, and the tandem Tudor domains are from SND1.

Other types of posttranslational modifications include glycosylation,

nitrosylation, and nitration. Glycosylation occurs when sugar residues are
covalently attached to the amide nitrogens of asparagine (N-linked
glycosylation) or to the hydroxyl groups of serine or threonine residues (O-
linked glycosylation), usually as branched chains, in secreted proteins or the
extracellular regions of transmembrane proteins. In most cases, these
modifications help the protein to fold correctly or facilitate its transit and
secretion, or insertion into the cell membrane; however, the addition of a
single residue of a particular amino sugar—N-acetylglucosamine—to serine
and threonine residues of cytoplasmic proteins may function in some cases by
preventing those same residues from being phosphorylated (Dias et al. 2012).
Protein nitrosylation involves the covalent incorporation of nitric oxide
into the thiol side chain of cysteine residues within proteins, whereas protein
nitration involves the incorporation of nitric oxide and/or its reactive nitrogen
species onto the ring –OH group of tyrosine residues to generate
nitrotyrosine. Three isoforms of nitric oxide synthase (NOS), the enzymes
that produce NO, are known, all of which appear to participate in protein
nitrosylation and nitration. Although less well understood than protein
phosphorylation, both S-nitrosylation and O-nitration can also regulate
protein structure, catalytic activity, stability, localization, and protein–protein
interactions. Protein nitration appears to occur primarily as a consequence of
oxidative stress and is believed to affect tissue homeostasis (Radi 2013). In
contrast, protein thiol nitrosylation is emerging as a prominent mechanism for
regulating signal transduction pathways, particularly those within the
cardiovascular system (Lima et al. 2010). Although the best-known role for
NO in controlling vasodilation is through the generation of cGMP by
activation of guanylyl cyclase (Ch. 3 [Newton et al. 2014]), many of the
effects of NO are mediated by S-nitrosylation. For example, the chemokine
SDF1 induces cell migration and angiogenesis by activating endothelial
NOS, which S-nitrosylates and inactivates the MAPK phosphatase MKP7 to
enhance downstream signaling. One of the most intriguing targets of protein
nitrosylation is small G proteins of the Ras superfamily. Nitrosylation of a
specific cysteine residue seems to facilitate their conversion from an inactive
to an active form (see below) (Foster et al. 2009). Additional posttranslational
modifications include ubiquitylation and lipidation (discussed in greater
detail below).

2.2 Noncovalent Regulation of Protein Activity

Signals are not only transmitted in the form of protein posttranslational
modifications. Ions, various lipids, and nucleotides can be produced or
relocalized to function as second messengers that transmit a signal (Ch. 3
[Newton et al. 2014]). Another major class of signaling enzymes is guanine-
nucleotide-binding proteins, also called G proteins, a large family of
signaling proteins that control a wide array of cellular functions, including
motility, hormone responses, sensory perception, and neurotransmission. G
proteins function as molecular switches. Their activity is regulated by the
intrinsic ability to bind and hydrolyze GTP to GDP.
In the basic GTPase cycle, G proteins exist in the “off” state bound to
GDP (Fig. 4). G proteins have high affinity for GDP (and GTP); thus, the
dissociation rates are very low. In addition, the nucleotide-free (i.e., empty)
form of the protein is unstable, such that removal of GDP requires assistance
from a guanine-nucleotide exchange factor (GEF). After dissociation of GDP,
the empty G protein will favor binding to GTP because of the 10:1 ratio of
GTP to GDP within the cell (Bos et al. 2007). The extra phosphate on GTP
induces a conformational change in three switch regions near the nucleotide-
binding pocket of the G protein, allowing substrate recognition. Signaling is
promoted when G proteins are in the active state through binding to motifs in
other proteins that specifically recognize the GTP-bound conformation of the
G protein. Although the GTP-bound G protein is generally the active state,
capable of transducing downstream signals, in some systems, GDP-bound G
proteins transduce the active signal, particularly in plants (Temple and Jones
2007). G proteins have a slow intrinsic GTPase activity (kcat = 101–10−3 min
−1) that results in GTP hydrolysis and returns the protein to the “inactive”

GDP-bound state; however, the rate of GTP hydrolysis can be dramatically

enhanced (by a factor of 103–106) by interaction with GTPase-activating
proteins (GAPs). Another important class of regulators is guanine-nucleotide
dissociation inhibitors (GDIs). These bind to and stabilize the inactive GDP-
bound state, maintaining the G protein in the inactive conformation. Thus,
coordinated actions of GEFs, GAPs, and GDIs are critical factors in
determining the amplitude, dynamics, and duration of G-protein-transduced
signals. The two major classes of G proteins are small G proteins and
heterotrimeric G proteins.

Figure 4. The GTPase cycle. G proteins can be small (Ras-like) or large (heterotrimeric). Depicted here
is the nucleotide cycle for heterotrimeric G proteins, but the reactions are conceptually the same for
small GTPases. G-protein-coupled receptors (GPCRs) bind extracellular ligands, and transmit signals to
intracellular G proteins. The ligand-bound receptor functions as a guanine-nucleotide exchange factor
(GEF), causing the Gα subunit to exchange GDP for GTP. GTP-bound Gα no longer interacts with the
Gβγ dimer, and both entities are free to interact with downstream effector proteins. Gα controls the
duration of the signal because it is a GTPase, whose activity can be stimulated by GTPase-activating
proteins (GAPs) such as RGS proteins.

2.2.1 Small G Proteins

Small G proteins are ∼20–25 kDa in size and consist of a single monomeric
subunit that has nucleotide binding and GTPase activities. More than 100
small G proteins exist in humans. The prototypic small G protein is Ras; thus,
this class of proteins is sometimes referred to as the Ras superfamily. At least
10 distinct subfamilies exist within the Ras superfamily. Members within a
single subfamily generally share similar sequence, structure, and functions
(Wennerberg 2005). For example, members of the Rho subfamily (which
include RhoA, Rac1, and Cdc42) are generally involved in cytoskeletal
dynamics and cell morphology, whereas members of the Arf subfamily
control vesicular transport. Targets for small G proteins are often themselves
signaling proteins, which creates signaling cascades. For example, Ras
activates the kinase Raf, and Rho activates the kinase ROCK.
The characteristics, mechanisms of action, and regulation of GEFs, GAPs,
and GDIs differ between small G proteins and heterotrimeric G proteins.
GEFs for small G proteins differ in structure and domain architecture for each
of the Ras subfamilies, but their mechanisms of action generally involve
interaction with the so-called switch regions of the GTPases and the
coordination of a magnesium ion within the nucleotide-binding pocket
(Sprang 1997). Small G proteins typically have only marginal GTPase
activity (often 100-fold to 1000-fold slower than the Gα subunits of
heterotrimeric G proteins). GAPs for this class of protein contain a catalytic
“arginine finger” region that inserts into the binding pocket, greatly
increasing the rate of hydrolysis (Kötting et al. 2008). GDIs generally
stabilize the inactive state, but may also alter membrane association or
stabilize the GTP-bound state. Members of the Ras subfamily—particularly
H-Ras, K-Ras, and N-Ras—are important in many types of cancer because
they control cell proliferation (see Ch. 21 [Sever and Brugge 2014]). Two
constitutively active mutations are commonly seen in various cancers,
including lung, colon, and pancreas. Mutation of the phosphate-binding loop
glycine (G12) to valine or aspartic acid results in loss of sensitivity to GAPs,
resulting in a prolonged activation period and a higher basal level of
signaling. Mutation of the catalytic glutamine residue (Q61), which normally
coordinates a water molecule that attacks the β–γ bond in GTP to a leucine,
results in an enzymatically inactive protein because the enzymatic transition
state cannot be stabilized (Privé et al. 1992).

2.2.2 Heterotrimeric G Proteins

The second class of G proteins is heterotrimeric G proteins, which function
downstream from G-protein-coupled receptors (GPCRs) (Hepler and Gilman
1992; Ch. 1 [Heldin et al. 2014]). The GTPase in this system is the Gα
subunit. Roughly 20 different Gα subunits exist in humans, each of which
contains two main domains: a GTPase domain that has sequence and
structural similarity to Ras (also called the Ras-like domain) and an α-helical
domain that can be posttranslationally modified and contributes to GTPase
regulation (Dohlman and Jones 2012). The other components of the
heterotrimer are the Gβ and Gγ subunits, which generally exist as an obligate
dimer. In the basal state, Gα and Gβγ form a tripartite complex in which Gα
is bound to GDP; however, the ligand-bound GPCR induces a conformational
change in Gα, resulting in GDP-for-GTP exchange. In the GTP-bound state,
Gα and Gβγ dissociate, and each is able to transduce a signal to downstream
effector proteins. The GEFs for heterotrimeric G proteins are typically the
GPCRs themselves (Weis and Kobilka 2008). Some non-GPCR GEFs have
been identified, including Ric8a, Arr4, and various AGS family proteins, but
their roles are still unclear. Gα subunits have GTP hydrolysis rates about 100
times higher than the Ras superfamily, but GAPs targeting them nevertheless
exist. These proteins each contain a regulator of G-protein-signaling (RGS)
domain. Some RGS proteins, like Sst2 in yeast or RGS2 and RGS4 in
mammals, are relatively simple, essentially containing only an RGS box;
others are more complex, containing numerous functional domains
(Siderovski and Willard 2005). Unlike GAPs for small G proteins, RGS
proteins function by interacting with the switch regions on Gα, and
stabilizing the transition state between GDP- and GTP-bound forms (Tesmer
et al. 1997).

2.2.3 The GPCR GEF Reaction

It has been estimated that 30%–50% of all drugs target GPCRs or some
aspect of GPCR-mediated signal transduction (Wise et al. 2002). Despite this,
our understanding of how GPCRs activate G proteins remains incomplete.
Unlike GEFs for small GTPases, GPCRs do not make contact with the switch
regions on Gα. Furthermore, other than the mobile switch regions, the Gα
subunit was not thought to undergo large conformational changes upon
activation. Recent high-resolution crystal structures of a GPCR–
heterotrimeric G-protein-ternary complex, however, indicate that binding of
ligand to the GPCR may induce a major displacement of the all-helical
domain of Gα relative to the Ras-like GTPase domain during catalysis
(Chung et al. 2011; Rasmussen et al. 2011). These structures suggest a
mechanism by which binding of ligand to a GPCR induces nucleotide
exchange.

3 REGULATION OF PROTEIN–PROTEIN
INTERACTION

3.1 Protein Interaction Domains

What exactly is a protein domain, and what kind of amino acid sequences do
reader domains involved in protein–protein interactions read? A domain is a
segment of a protein, generally 50–400 amino acids in length, that folds
independently into a stable three-dimensional structure and is capable of
some type of independent function. Most signaling proteins are built of
multiple domains. The sequences that connect the domains together are
usually short and less structured parts of the protein. More than 1000 modular
protein domains have been characterized using bioinformatics (Letunic et al.
2011).
A classic example of modular protein reader domains is SH2 domains,
which bind to phosphotyrosine-containing sequences. Another example is
SH3 domains, which bind to proline-containing sequences. Other domains,
such as PTB domains, HYB domains, and some C2 domains, can also bind to
phosphotyrosine-containing sequence motifs, whereas WW, EVH1, and GYF
domains, like SH3 domains, bind to short proline-rich sequences.
A wide variety of domains recognize other posttranslational
modifications. Domains such as 14–3–3, FHA domains, tandem BRCT
domains, MH2 domains and Polo-box domains, for example, bind to short
phosphoserine- and/or phosphothreonine-containing motifs (Yaffe and
Smerdon 2004). Bromo domains recognize specific acetyl-lysine-containing
sequences, and chromodomains, Tudor domains, MBT domains, and PWWP
domains bind to sequences that contain methyl-lysine and methylarginine.
Other domains, such as CUE, PAZ, UBA, and UEV domains, can bind to
ubiquitin or specific types of ubiquitin chains. The ability of these domains to
distinguish unmodified proteins from proteins containing these different
types of posttranslational modifications ensures that protein–protein
interactions occur only when one of the two proteins has been appropriately
marked and modified by a writer. This use of readers allows protein–protein
interactions and the assembly of multiprotein signaling machines, or even the
activity of a single protein, to be precisely controlled by the actions of writers
and erasers in response to a signal. For domains like SH3 domains that do not
bind to modified sequences, their reader functions are typically regulated by
conformational changes in other parts of the protein that modulate access of
the domain to binding partners.
Reader domains are often found within proteins that also contain writer or
eraser domains. Frequently, these interact with each other or with sequences
that lie outside the domain, and this can have important functional
consequences. This is perhaps best illustrated by the tyrosine kinase Src. Src
contains a kinase writer domain, together with SH2 and SH3 reader domains.
In the inactive state, the SH2 domain of Src is bound to a carboxy-terminal
tyrosine residue that has been phosphorylated, while its SH3 domain is bound
to a proline-containing linker region between the SH2 domain and the kinase
domain. In this conformation, the SH2 and SH3 domains rest against the back
surface of the kinase domain, holding it in a catalytically inactive form.
Dephosphorylation of the carboxy-terminal tyrosine in Src by phosphatases
such as SHP1 or SHP2, or displacement of the SH2 and SH3 domains
through competitive binding to phosphotyrosine sequences (such as those
found in platelet-derived growth factor receptor [PDGFR]) or focal adhesion
kinase (FAK) and polyproline sequences (such as those found in the arrestin-
bound β2 adrenergic receptor, NEF, or Sin) then releases the kinase domain
to promote activation. Together with phosphorylation of a tyrosine residue in
the activation loop, this results in full catalytic activity (Fig. 5).
Figure 5. A multistep mechanism for maximal Src activation. (A) In the inactive state, Src is folded up
as a consequence of multiple interactions between the reader domains and motifs in Src itself. The Src
SH2 domain is bound to a phosphotyrosine residue (Y527) in the carboxyl terminus, while the SH3
domain binds to a polyproline-type helix in the linker that connects the SH2 domain to the kinase
domain. (B) The kinase opens up into an active conformation when a ligand such as a growth factor
receptor or an adaptor protein engages the SH2 and SH3 domains directly, usually accompanied by
dephosphorylation of the Y527 site to prevent intramolecular reassociation into the closed form. (C)
Autophosphorylation of Y416 in the activation loop of Src, or phosphoprylation of this site by another
kinase, results in maximal activity. (From Xu et al. 1999; adapted, with permission, © Elsevier.)

A similar intramolecular interaction between reader and writer domains

keeps the mitotic kinase Polo-like kinase 1 (Plk1) inactive until a certain level
of cyclin-dependent kinase (CDK) activity has been reached (Ch. 6 [Rhind
and Russell 2012]). Plk1 contains an amino-terminal kinase writer domain
and a carboxy-terminal Polo-box reader domain that fold back against each
other in interphase cells to keep the protein inactive (Lowery et al. 2005).
When cells approach mitosis, CDKs phosphorylate substrates such as
Cdc25C and Wee1 to generate phosphoserine/threonine motifs that can bind
directly to the Polo-box domain, prying it away from the kinase domain.
Again, together with phosphorylation of a threonine residue in the activation
loop, this drives Plk1 into the fully active form required for cells to complete
mitosis.
For many domains, portions that are not directly involved in motif
recognition can also be instrumental in stabilizing protein–protein
interactions. These types of domain–domain interactions are often important
in assembly of multisubunit signaling complexes. For example, non-ligand-
binding portions of the SH2 domain in phospholipase Cγ (PLCγ) are used to
stabilize interactions with fibroblast growth factor 1 (FGFR1). Intriguingly,
evolution has led to preferential cosegregation of particular protein domains
within individual proteins (Jin and Pawson 2012). For example, SH2 domains
and SH3 domains frequently co-occur, as do PX domains and SH3 domains.
Presumably this is because specific combinations of domains have already
mastered the molecular origami required for both productive interactions and
allosteric control over additional domains with which they coassociate (see
Table 1 for common domains, their partners, and motifs).

3.2 Motifs
For readers, writers, and erasers to function together to form signaling
networks, they must operate on common short amino acid sequence motifs in
their targets. Most such motifs contain ∼15 amino acid residues and include
particular amino acids that confer specificity to the writers and readers. For
example, CDKs and MAPKs (writers) preferentially phosphorylate S-P and
T-P motifs, whereas Polo-box domains (readers) preferentially recognize
motifs with the consensus S-pS/pT-P (where pS and pT are phosphorylated
serine and threonine). Similarly, many tyrosine kinases (writers)
phosphorylate tyrosine residues that are surrounded by acidic amino acid
residues, whereas SH2 domains (readers) prefer bind to phosphotyrosine-
containing sequences that contain specific patterns of hydrophobic or small
amino acid residues carboxy terminal to the phosphotyrosine. Some domains
can bind multiple sequence motifs or even bind ligands in multiple
orientations, as is the case for SH3 domains and SUMO-SIM domains.
This type of limited overlap between the sequences that the writer
domains generate and those that the reader domains recognize gives rise to
the specificity we observe in signaling networks. That is, only a small
fraction of the targets of any particular writer are then recognized by a
particular reader. As mentioned above, not all motifs require posttranslational
modification (e.g., proline-rich sequences are recognized by SH3 domains,
WW domains, and EVH1 domains, among others), which leads to
competition between a motif and multiple readers, depending on localization
and timing. In contrast, some types of domains appear to recognize specific
combinations of posttranslational modifications. Some bromo domains, for
example, recognize specific phosphoacetyl-methyl motif combinations on
histones (Filippakopoulos et al. 2012), whereas 14–3–3 proteins are able to
recognize both phosphorylated and phosphoacetylated histone sequences
(Macdonald et al. 2005). A similar effect can be achieved by adjacent
protein–protein interaction domains that bind to different posttranslational
modifications. These domains or domain combinations function as readers of
a more complex code, essentially creating “AND gates,” which can be used
to dictate greater specificity and control, or functioning as integrators of
signaling from multiple pathways.
Because most motifs are defined by the primary structure of a protein
rather than a complicated 3D arrangement of noncontiguous elements, it is a
relatively straightforward process to go motif hunting using protein
sequences and bioinformatics search tools (Obenauer et al. 2003; Obenauer
and Yaffe 2004). The small, “portable” nature of these motifs means that they
have frequently moved around within the sequences of evolutionarily related
proteins. Thus, a motif can sometimes be seen in one part of a protein
sequence in a human protein and in another part of the sequence in a yeast
ortholog.

4 REGULATION OF PROTEIN LOCATION

Signaling processes occur in the context of the multicompartment, 3D
environment of the cell. Information transfer from the cell exterior, across
lipid membranes, and through/within the cytosol must be tightly regulated in
both space and time, and a key component to achieving specificity is the
dynamic regulation of protein localization.

4.1 Compartmentalization
A common theme in signaling is modulating compartmentalization of
signaling proteins within the cell. This can dictate access to substrates or
environmental conditions that promote activation. Subcellular compartments,
particularly organelles and cytoskeletal structures, can be very dynamic. In
these cases, signaling can promote the formation of these compartments. The
spatial segregation that organelles provide represents an important layer of
regulation, allowing similar proteins to execute different functions in
different environments. For example, mitotic kinases like Plk1, Aurora B,
and Never in mitosis kinase 2 (Nek2) achieve substrate specificity through
nonoverlapping subcellular localization, despite sharing partially overlapping
substrate selection motifs (Alexander et al. 2011).
Such compartmentalization can be achieved in different ways. Some
proteins have short sequences (also called signal peptides) that function as
localization signals, allowing transport to a particular organelle. The best
recognized of these are nuclear localization signals (NLSs), which typically
feature one or more short sequences of positively charged lysine or arginine
residues (Hung and Link 2011). Similar signals have been identified for
many other organelles, including mitochondria, lysosomes, and peroxisomes.
For proteins with localization sequences, regulated localization can be
achieved through posttranslational modifications or conformational changes
that mask or unmask a signal peptide. Type I nuclear receptors, for example,
are typically retained in the cytoplasm in an inactive complex with HSP90
(see p. 129 [Sever and Glass 2013]; Ch. 1 [Heldin et al. 2014]). Ligand
binding induces a conformational change, dissociation of HSP90,
homodimerization, and active transport into the nucleus, promoting DNA
binding and gene expression. Phosphorylation of the MAPK ERK on an SPS
motif within the kinase-insert region results in recognition by the nuclear
transport receptor importin β7 (Chuderland et al. 2008). Following nuclear
import, ERK then phosphorylates nuclear substrates, including transcription
factors Fos, Myc, and Elk1 (see p. 81 [Morrison 2012]). For proteins without
localization sequences, protein–protein interactions or association with lipid
membranes can localize them to specific subcellular regions.

4.2 Membrane Localization

Attachment to various cellular membranes can function as an anchor that
restricts a protein or its activation to certain subcellular areas. Another benefit
is that signaling is more efficient in two dimensions, because the local
concentration near the membrane is greatly increased. Some estimates have
suggested that membrane association increases the local protein concentration
as much as 1000-fold (McLaughlin and Aderem 1995). Membrane-localized
proteins can be either transmembrane proteins embedded in the membrane or
peripheral membrane proteins that attach to membranes through protein–lipid
interactions. Because they connect two physically separated environments,
transmembrane proteins are well positioned to function as pumps, ion
channels, or cell-surface receptors (Ch. 1 [Heldin et al. 2014]). Peripheral
membrane proteins, which may interact with transmembrane receptors,
typically function as signaling intermediaries. The most common method of
membrane attachment for these proteins is through the covalent attachment of
lipid groups (e.g., glycophosphatidylinositol [GPI] anchors and other lipid
chains) (Casey 1995; Nadolski and Linder 2007) and reversible recruitment
to the plasma membrane is a common method of signal regulation. Upon
activation of certain receptor tyrosine kinases, for example, phosphoinositide
3-kinase (PI3K) is recruited to the receptor via interactions between the SH2
domains of the p85 subunit of PI3K and phosphorylated tyrosine residues on
the activated receptor. Active PI3K then phosphorylates phosphatidylinositol
4,5-bisphosphate (PIP2) to create phosphatidylinositol-3,4,5-trisphosphate
(PIP3). Downstream effectors like the kinase Akt then are recruited to the
membrane through a PH-domain-dependent interaction with PIP3. Once at
the membrane, Akt can then be phosphorylated and activated by its activating
kinase PDK1, which also has a PIP3-binding PH domain. Similar
mechanisms in which subcellular localization and activation are intrinsically
coupled are also used for activation of Ras family GTPases. These involve
recruitment of activation factors such as GEFs to the plasma membrane.

4.3 Lipid Modification

Membrane attachment can be induced by protein lipidation, owing to the
hydrophobicity of the modifying lipid. Many types of lipid modification
exist, each unique in its regulation, chemical properties, and mechanism of
linkage to proteins (Fig. 6). Myristoylation is the cotranslational addition of a
saturated 14-carbon acyl chain to an amino-terminal glycine. The myristoyl
moiety has a relatively low level of hydrophobicity. Thus, myristoylated
proteins, such as Src-family kinases and some G proteins, typically achieve
membrane attachment by using myristoylation in concert with other lipid
modifications or nearby polybasic amino-acid stretches. Myristoylation can
allow proteins to attach to membranes, but, in some cases, it alters protein
conformation or protein–protein interaction (Boutin 1997). A myristoyl group
added to the C subunit of PKA, for example, can insert into a lipid a bilayer,
promoting membrane localization, or alternatively, fold into a hydrophobic
pocket of the enzyme, allowing regulation of the protein–lipid
conformational state by phosphorylation. Another common modification is
prenylation, the posttranslational addition of a 15-carbon farnesyl or 20-
carbon geranylgeranyl chain attached to a carboxy-terminal cysteine. The
enzymes responsible for prenylation have been identified, and inhibitors of
these enzymes have received much attention as anticancer agents, owing to
the importance of prenylation of GTPases in the Ras family (Resh 2012).

Figure 6. Common forms of protein lipidation. The table highlights four lipid moieties that are
commonly used to modify proteins posttranslationally or (in the case of myristoylation)
cotranslationally. These modifications differ in terms of their consensus sequences, position of the
modification, hydrophobicity, and mechanism of regulation.

Another example is palmitoylation, the addition of a 16-carbon-chain

fatty acid to cysteine residues through a thioester bond. Protein subcellular
localization is often dictated by palmitoylation, through the localization of the
palmitoyl acyltransferase (PAT). For example, proteins with only a myristoyl
or prenyl group are thought to transiently interact with membranes, sampling
many membrane surfaces within the cell. However, palmitoylation of a singly
lipid-modified protein induces stable membrane attachment. Thus, through
limited spatial expression of the PATs, palmitoylation can dictate protein
subcellular localization. For example, the Ras family member H-Ras can
localize to the plasma membrane or Golgi apparatus, having unique signaling
roles at each location. H-Ras subcellular localization is dictated by
palmitoylation on one of two carboxy-terminal cysteine residues (Roy et al.
2005). Furthermore, unlike myristoylation or prenylation, palmitoylation is a
reversible process and can be dynamically regulated during signal
transduction.

4.4 Membrane Microdomains and Spatially Restricted

Signaling
Proteins and lipids in the plasma membrane are thought to be distributed
heterogeneously, forming membrane microdomains (Maxfield 2002). This
may help sequester low-abundance molecules, increasing their local
concentrations or facilitating the formation of signaling machines. Examples
of such microdomains include invaginations of the plasma membrane called
caveolae and lipid rafts, cholesterol- and sphingolipid-rich portions of the
membrane that potentially function as organizing centers for
compartmentalized signaling. GPI-linked proteins are preferentially localized
to such microdomains, which may play an important role in many signaling
processes. Membrane microdomains can be used to promote spatially
restricted signaling, such as those seen in the immunological synapse (the
interface between an antigen-presenting cell and effector T cell) (see Ch. 16
[Cantrell 2014]).
Spatially regulated signal transduction is a well-established phenomenon,
but an emerging paradigm is the positive and negative regulation of plasma-
membrane-associated signals at intracellular locations. For example, H-Ras,
well known to transduce signals from the plasma membrane, also exists at
Golgi bodies, where it transmits a unique signal (Bivona et al. 2003). In
addition, a growing body of evidence supports a positive role for the
endocytic pathway in signal transduction (Zastrow and Sorkin 2007; Murphy
et al. 2009). Numerous signals transmitted from endosomal locations have
been identified, including those coupled to RTK- (Di Guglielmo et al. 1994)
and GPCR-coupled signals (Lefkowitz and Shenoy 2005; Slessareva et al.
2006).
Signals can also be spatially restricted through binding to protein
scaffolds. The co-occurrence of multiple motifs on a single molecule can lead
to the formation of signaling scaffolds and signaling hubs. If a single protein
contains multiple motifs and each is recognized by a different protein, then
these will all converge. Such an arrangement is used, for example, to
organize MAPK signaling in yeast and mammalian cells (Elion 2001;
Engström et al. 2010; p. 81 [Morrison 2012]). By using scaffolds to bring two
or more proteins together, cells overcome the diffusion problem and no
longer require the proteins to find each other in the crowded interior of the
cell. As is the case for membrane attachment, the tethering of proteins to
scaffolds greatly increases the effective concentration of signaling proteins. If
the different proteins that are recruited to a multimotif protein do not
communicate directly with each other, but instead act on other proteins, then
the multimotif protein serves as a hub, where multiple signaling events
converge in time and space. Such an arrangement is used, for example, by A-
kinase-anchoring proteins (AKAPs) to coordinate signaling through PKA
pathways (Ch. 3 [Newton et al. 2014]), and by the cytoplasmic tails of
growth factor receptors to coordinate signaling by Ras, PI3K, PKC, and
MAPKs (Ch. 1 [Heldin et al. 2014]).

5 REGULATION OF PROTEIN PRODUCTION

At its most basic level, signaling is controlled by the relative stoichiometry of
positive and negative signaling intermediaries, which are frequently proteins.
Any process that changes the balance of these proteins can, in turn, modulate
the signal. One basic and widely used strategy is to change how much of the
signaling intermediate—whether a ligand, second messenger, or protein—is
available to transmit information. Cells use an incredibly diverse array of
methods to regulate protein levels—so many, in fact, that an in-depth review
of this topic would be a textbook unto itself. Here, we therefore focus on
basic ways cells control signaling by modulating the rate of protein synthesis
or protein degradation. The processes that can be targeted include
transcription, RNA stability, RNA splicing, and translation. Of these,
transcriptional regulation is probably the best studied and most commonly
recognized target of signal transduction pathways.

5.1 Transcriptional Control

The output of many signaling pathways is transcription of genes that encode
proteins necessary for the desired cellular response. The regulation can occur
by modulating nuclear localization of transcription factors, coactivators, and
other regulatory proteins by modulating the DNA-binding capabilities of
these proteins or by posttranslationally modifying histones to modulate
chromatin architecture. A good example is the transcriptional control of the
tumor suppressor p53, which is a transcription factor, and its downstream
targets in response to DNA damage signals (Brown et al. 2009; Ch. 5
[Duronio and Xiong 2013]). In unstressed cells, p53 expression remains low,
owing to a low level of transcription and constitutive ubiquitin-dependent
degradation of p53 promoted by an E3 ligase protein called MDM2 (Momand
et al. 1992). In response to various cellular stressors, including ionizing
radiation, UV, and other forms of genotoxic damage, the transcription of the
p53 gene is dramatically increased, and the p53 protein is phosphorylated on
one or more of its many phosphorylation sites. There are two important
consequences of this phosphorylation event. The first is increased levels of
p53 protein due to loss of interaction with MDM2, an E3 ubiquitin ligase that
drives ubiquitin-mediated degradation (see below) of p53 in unstimulated
cells. Second, as a result of MDM2 dissociation, p53 undergoes a
conformational change that allows it to interact with DNA and drive
transcription of new p53 target genes. Among the genes activated by p53 are
those that control cell-cycle arrest, DNA repair, and apoptotic cell death, and
even the p53 gene itself (Bieging and Attardi 2012).

5.2 RNA Stability and miRNAs

Signal transduction can also involve modulation of mRNA processing or
stability. Several splicing factors are phosphorylated in response to signaling,
and these events may control alternative splicing of pre-mRNAs to give
different mRNA isoforms (Lynch 2007). Cells can regulate mRNA stability
by modulating mRNA maturation (via 5′ capping or 3′ polyadenylation) or
interactions between RNA-binding proteins and mRNA transcripts (Wu and
Brewer 2012). An emerging area of research is the control of signaling by
cellular micro-RNAs (miRNAs) and other noncoding RNAs, such as long
noncoding RNAs (lnc-RNAs). miRNAs are short (∼21–23 nucleotide)
untranslated RNAs (Ambros 2001) that typically induce degradation of target
RNAs or block mRNA translation through sequence-specific base-pairing
interactions. They are important regulators of normal developmental timing
(Ambros 2011). miRNAs like Lin-4/mir-125 regulate the temporal transitions
between pluripotent and differentiated states of numerous stem cell
populations.
miRNAs are also common regulators of the dynamics, duration, and
sensitivity of signaling processes. Many signaling pathways—including the
Wnt, Notch, Hedgehog, and p53 pathways, for example—achieve specificity
and sensitivity through active repression (i.e., basal repression or default
repression). In this context, miRNAs target core pathway components or their
transcriptional targets, maintaining cells in the inactive state, causing the
system to require greater levels of stimulus for activation and also sharpening
the response to activating stimuli. For example, miR-125 targets many
components of p53 signaling, and loss of miR-125 causes spontaneous p53
activation.

5.3 Translational Control

Translation is also a target of signal transduction pathways. For example, the
mammalian target of rapamycin (mTOR) and related pathways are now well
known to control protein translation in response to nutrient, growth factor,
and amino acid signals (Laplante and Sabatini 2012). A critical target
involved in the regulation of translation by the mTORC1 complex is the
eukaryotic translation initiation factor 4E (eIF4E)–binding protein 1 (4E-
BP1) (Hara et al. 1997). 4E-BP1 inhibits cap-dependent translation by
binding to eIF4E, the initiation factor that recognizes the 5′ cap on mRNA
molecules. Phosphorylation of 4E-BP1 at T37 or T46 is thought to prime 4E-
BP1 for subsequent phosphorylation at S65 and T70, leading to loss of
interaction with eIF4E (Gingras et al. 1999), which results in a general
increase in protein translation. The mTORC1 complex also regulates the
activity of p70 S6 kinase 1 (S6K1), which has many targets whose
phosphorylation activates translation (Pullen and Thomas 1997). Key among
these is the S6 subunit of the 40S ribosome, which when phosphorylated by
S6K1 promotes increased translation of mRNA transcripts containing an
oligopyrimidine sequence in their 5′ untranslated region (5′-UTR).
Additionally, translation of specific mRNAs can be controlled through
feedback signaling at the level of translation initiation. For example, iron
homeostasis is maintained in part by regulating the translation of proteins
involved in iron import. In the presence of high concentrations of
intracellular iron, the transferrin receptor mRNA is repressed by iron-induced
binding of iron-response-element-binding protein (IREBP) to a 5′-UTR
element in the transferrin receptor mRNA.

6 PROTEIN DEGRADATION
Every protein has a baseline level of expression resulting from the
equilibrium between its synthesis and breakdown (see Box 1). A control
mechanism widely used in signal transduction is regulation of the rate of
protein degradation. Many methods exist for regulated degradation of
proteins, including lysosomal and ER-mediated degradation (Ciechanover
2012), but the mechanism that seems to be most important in cell signaling is
ubiquitin-dependent proteasomal degradation (Hershko and Ciechanover
1998). Ubiquitin is a small 76-amino-acid protein that can be conjugated
through its carboxyl terminus to lysine residues on target proteins or to other
ubiquitin molecules to form ubiquitin chains that serve as a marker for
various cellular functions. Through diversity in chain length or the orientation
of chain attachment, ubiquitin can regulate protein trafficking and protein–
protein interactions, or function as a signaling scaffold (Muratani and Tansey
2003; Kirkin and Dikic 2007; Walczak et al. 2012).

BOX 1. PROTEIN LEVELS DESCRIBED MATHEMATICALLY

Protein levels are best described using a deceptively simple equation:

where [P] is the protein concentration, k1 is the synthesis rate, and k2 is

the degradation rate. Obviously, k1 and k2 reflect complex processes that
are themselves regulated by signaling events at multiple levels. At
steady state, when [P] is constant, d[P]/dt = 0 and [P] = k1/k2. Therefore,
to increase [P], we need either to increase the synthesis rate or to
decrease the degradation rate. The two key questions from a biologist’s
point of view are these: (1) what will the new level be? and (2) how fast
will the changes happen—that is, how long until the new steady-state
level is reached? Consider the case when the synthesis of a protein
ceases entirely—that is, k1 = 0. Because the protein is now subject only
to degradation, the new steady-state level will be 0—that is, [P] = 0/k2—
but the rate of decline is given by k2; that is, t1/2 = ln 2/k2. The somewhat
surprising thing is that the same is true if we change the synthesis rate to
some value other than zero. For example, if we double the synthesis rate,
the new steady-state level will be twice the old steady-state level ([Pss] =
k1/k2 → 2k1/k2), but the time it takes to reach the new steady-state level
will be determined not by the change in synthesis rate but by the
degradation rate, k2. Thus, big changes in synthesis rate only cause rapid
changes in steady-state protein levels if the degradation rate is fast, that
is, if the protein is short lived. Similarly, minor changes in degradation
rates are manifest with kinetics that depend not on the degradation rate
but the synthesis rates—that is, rapid changes in degradation rate only
manifest in changes in steady-state protein levels if the synthesis rate is
fast.
 Perhaps its major role, however, is to form polyubiquitin chains that target
proteins for destruction by the proteasome. Recognition by the proteasome
requires at least four ubiquitin monomers linked to each other through amide
bonds involving the carboxyl terminus of one ubiquitin molecule and K48 on
another ubiquitin molecule. Although chains of four monomers are required,
longer chains are typical, presumably increasing the efficiency of
proteasomal recognition. Ubiquitin chains are formed through a cascade of
enzymatic processes performed by a ubiquitin-activating protein (E1), a
ubiquitin-conjugating protein (E2), and a ubiquitin ligase (E3) (Fig. 7). The
chains target proteins to the proteasome, a large protein complex containing
multiple proteolytic enzymes, where ubiquitylated proteins are subsequently
degraded and their parts recycled by the cell. A classic example of this
process in action is the regulated expression of the cyclin proteins (Ch. 6
[Rhind and Russell 2012]), whose oscillating levels allow the periodic
activation of cyclin-dependent kinases (CDKs) that regulates progression
through the cell cycle (Malumbres and Barbacid 2005). These oscillations are
achieved primarily by increased degradation of cyclins by ubiquitin-
dependent proteasomal degradation, coupled with increased cyclin synthesis
at different points in the cell cycle (Sudakin et al. 1995).
Figure 7. Protein ubiquitylation. (A) Structure of the ubiquitin monomer, highlighting the amino and
carboxyl termini, as well as key lysine residues. (B) Schematic depiction of the posttranslational
modification of substrate proteins with ubiquitin. Ubiquitin is added to proteins through a three-step
enzymatic reaction featuring a ubiquitin-activating enzyme (E1), a ubiquin-conjugating enzyme (E2),
and a ubiquitin ligase (E3). Subsequent rounds of ubiquitylation can result in the formation of ubiquitin
chains. (C) Ubiquitin chain diversity. Ubiquitin contains seven lysine residues, each of which can be
used as the anchorage point for subsequent ubiquitin monomers, in homotypic (same linkage
throughout chain), heterotypic (mixed chains), or branched fashion (multiple ubiquitin monomers
conjugated to a single ubiquitin). In addition, the amino-terminal methionine on a ubiquitin monomer
linked to a substrate protein can be linked to the carboxy-terminal end of another ubiquin monomer
(linear chains). Although all of these forms have been shown to exist in cells, physiological roles for
many of these chains are still being elucidated.
 Other types of polyubiquitin chains, in which the individual ubiquitin
molecules are connected through amide bonds involving residues in ubiquitin
other than K48 can also be added to proteins (Fig. 7) (Ikeda and Dikic 2008).
Seven such chain types can be formed, in which the lysine side-chain amino
group in one of the seven lysine residues present in ubiquitin is linked to the
carboxy-terminal di-glycine motif of the next ubiquitin molecule through an
isopeptide bond. Prominent examples of these other ubiquitin chains in
signaling include the use of K63-linked chains to control protein–protein
interaction rather than protein degradation. In the IKK/NF-κB pathway (see
p. 121 [Lim and Staudt 2013]), for example, stimulation of the ubiquitin
ligase activity of TRAF6, results in K63-linked polyubiquitylation of
substrate proteins like NEMO and TRAF6 itself. K63-ubiquitylated TRAF6
binds to the NZF reader domain in TAB2/TAB3, which then recruits and
activates TAK1, which, in turn, phosphorylates and activates IKK (Chen
2005).
In addition, an eighth chain type exists, linear ubiquitin chains, in which
peptide bonds are formed between the amino-terminal methionine of a
ubiquitin monomer and the carboxy-terminal carboxy group of another
ubiquitin monomer. Linear ubiquitin chains are important for generating
protein-binding surfaces and promote the activation of inflammation and
stress-signaling pathways, such as those downstream from the tumor necrosis
factor (TNF) receptor (Walczak et al. 2012). Furthermore, proteins can also
be modified by the attachment of ubiquitin-like proteins, such as SUMO (for
small ubiquitin-like molecule) and Nedd8 (for neural precursor cell expressed
developmentally down-regulated 8). Like K63 and linear ubiquitin chains,
these molecules are thought to control protein–protein interactions rather than
regulate protein degradation.

7 CONCLUDING REMARKS: WHAT DOES THE

FUTURE HOLD
Modulation of proteins represents the frontline response of most signaling
systems. Many proteins are exquisitely sensitive to even very small
posttranslational chemical modifications, such as phosphorylation,
methylation, and the other posttranslational modifications highlighted here.
These modifications can create new protein–protein binding surfaces or alter
the conformation or localization of the target protein (which can, in turn,
offer new protein–protein interaction possibilities). Furthermore, these
modifications can dramatically alter the abundance of a protein in both
positive (as in the case of signaling driving transcription) and negative
directions (as in the case of ubiquitin-mediated degradation).
An equally important question regarding posttranslational changes that
modulate signal transduction is not “what” or “how,” but rather “why.” Why
have these signaling systems used so much regulation at the protein level?
Why are these complex regulatory mechanisms preferred? Many benefits
have been proposed (and some validated) for the types of regulatory
complexity that are seen in signaling biology, including signaling speed,
robustness, reversibility, accuracy, and/or sensitivity that may be required for
certain biological responses. The greatest benefit of these designs, however,
is their diversity. Signaling processes were selected not to maximize
efficiency but rather to maximize possibility. The modular organization of
signaling components allows them to be imported into new biological
contexts, where they may create new biological functions. This may be best
illustrated by the roles of protein ubiquitylation in inactivating signals in
some biological contexts and activating signals in others. Thus, using the tool
kit of protein modules that function as readers, writers, and/or erasers, along
with changes in protein abundance and location, signaling systems have
evolved the potential to use a very limited list of parts to respond to a nearly
limitless set of cues.
Although our knowledge of protein regulation in signal transduction is
broad, it is important to stress that it is only deep at the detailed molecular
level in a limited number of areas. Much remains to be learned, particularly
with respect to how protein regulatory mechanisms work together at the
systems level, and how protein regulatory behavior can be captured in the
language of mathematics (Ch. 4 [Azeloglu and Iyengar 2014]). In addition,
we can be sure that additional types of posttranslational modifications, new
modular binding domains, and new mechanisms of protein regulation will
emerge.
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005918
CHAPTER 3

Second Messengers

Alexandra C. Newton1, Martin D. Bootman2, and John D.

Scott3
1Department of Pharmacology, University of California at San Diego, La Jolla, California 92093
2Department of Life, Health and Chemical Sciences, The Open University, Walton Hall, Milton
Keynes MK7 6AA, United Kingdom
3Department of Pharmacology, Howard Hughes Medical Institute, University of Washington School of
Medicine, Seattle, Washington 98195
Correspondence: anewton@ucsd.edu

SUMMARY

Second messengers are small molecules and ions that relay signals
received by cell-surface receptors to effector proteins. They include a
wide variety of chemical species and have diverse properties that allow
them to signal within membranes (e.g., hydrophobic molecules such as
lipids and lipid derivatives), within the cytosol (e.g., polar molecules
such as nucleotides and ions), or between the two (e.g., gases and free
radicals). Second messengers are typically present at low concentrations
in resting cells and can be rapidly produced or released when cells are
stimulated. The levels of second messengers are exquisitely controlled
temporally and spatially, and, during signaling, enzymatic reactions or
opening of ion channels ensure that they are highly amplified. These
messengers then diffuse rapidly from the source and bind to target
proteins to alter their properties (activity, localization, stability, etc.) to
 propagate signaling.

Outline
1 Introduction
2 Cyclic nucleotides
3 Lipid and lipid-derived second messengers
4 Ions as intracellular messengers
5 Concluding remarks
References

1 INTRODUCTION
Signals received by receptors at the cell surface or, in some cases, within the
cell are often relayed throughout the cell via generation of small, rapidly
diffusing molecules referred to as second messengers. These second
messengers broadcast the initial signal (the “first message”) that occurs when
a ligand binds to a specific cellular receptor (see Ch. 1 [Heldin et al. 2014]);
ligand binding alters the protein conformation of the receptor such that it
stimulates nearby effector proteins that catalyze the production or, in the case
of ions, release or influx of the second messenger. The second messenger
then diffuses rapidly to protein targets elsewhere within the cell, altering the
activities as a response to the new information received by the receptor.
Three classic second messenger pathways are illustrated in Figure 1: (1)
activation of adenylyl cyclase by G-protein-coupled receptors (GPCRs) to
generate the cyclic nucleotide second messenger 3′-5′-cyclic adenosine
monophosphate (cAMP); (2) stimulation of phosphoinositide 3-kinase (PI3K)
by growth factor receptors to generate the lipid second messenger
phosphatidylinositol 3,4,5-trisphosphate (PIP3); and (3) activation of
phospholipase C by GPCRs to generate the two second messengers
membrane-bound messenger diacylglycerol (DAG) and soluble messenger
inositol 1,4,5-trisphosphate (IP3), which binds to receptors on subcellular
organelles to release calcium into the cytosol. The activation of multiple
effector pathways by a single plasma membrane receptor and the production
of multiple second messengers by each effector can generate a high degree of
amplification in signal transduction, and stimulate diverse, pleiotropic,
responses depending on the cell type.

Figure 1. Second messengers disseminate information received from cell-surface receptors. Indicated
are three examples of a receptor activating an effector to produce a second messenger that modulates
the activity of a target. On the right, binding of agonists to a GPCR (the receptor) can activate adenylyl
cyclase (the effector) to produce cAMP (the second messenger) to activate protein kinase A (PKA; the
target). On the left, binding of growth factors to a receptor tyrosine kinase (RTK; the receptor) can
activate PI3K (the effector) to generate PIP3 (the second messenger), which activates Akt (the target).
In the center, binding of ligands to a GPCR (receptor) activates phospholipase C (PLC; the effector) to
clear the phospholipid PIP2, to generate two second messengers, DAG and IP3, which activate protein
kinase C (PKC; the target) and release calcium from intracellular stores, respectively.

Second messengers fall into four major classes: cyclic nucleotides, such
as cAMP and other soluble molecules that signal within the cytosol; lipid
messengers that signal within cell membranes; ions that signal within and
between cellular compartments; and gases and free radicals that can signal
throughout the cell and even to neighboring cells. Second messengers from
each of these classes bind to specific protein targets, altering their activity to
relay downstream signals. In many cases, these targets are enzymes whose
catalytic activity is modified by direct binding of the second messengers. The
activation of multiple target enzymes by a single second messenger molecule
further amplifies the signal.
Second messengers are not only produced in response to extracellular
stimuli, but also in response to stimuli from within the cell. Moreover, their
levels are exquisitely controlled by various homeostatic mechanisms to
ensure precision in cell signaling. Indeed, dysregulation of the second
messenger output in response to a particular agonist can result in cell/organ
dysfunction and disease. For example, chronic exposure to cAMP in the heart
results in an uncontrolled and asynchronous growth of cardiac muscle cells
called pathological hypertrophy. This early stage heart disease presents as a
thickening of the heart muscle (myocardium), a decrease in size of the
chamber of the heart, and changes in contractility. Because such prolonged
exposure to second messengers has deleterious effects, specific enzymes,
channels, and buffering proteins exist to rapidly remove second messengers,
either by metabolizing them or sequestering them away from target
molecules.

2 CYCLIC NUCLEOTIDES
2.1 cAMP and a Major Target, PKA
The “fight or flight” mechanism, more accurately referred to as the adrenal
response, prepares the body for situations of extreme stress. Release of the
hormone epinephrine, also known as adrenaline, from the adrenal gland into
the blood rapidly triggers vital cellular and physiological reactions that
prepare the body for intense physical activity. One of the most important
effects is the breakdown of glycogen into glucose to fuel muscles (Sutherland
1972).
Earl Sutherland first showed that epinephrine bound to cell-surface
receptors stimulates membrane-associated adenylyl cyclases to synthesize the
chemical messenger cAMP. Martin Rodbell and Alfred Gillman then
discovered that G-protein subunits are the intermediates that shuttle between
receptors and a family of eight adenylyl cyclase isoforms in the plasma
membrane. Each G protein is a trimer consisting of Gα, Gβ, and Gγ subunits.
The Gα subunits in the G proteins Gs and Gi are distinct and provide the
specificity for activation and inhibition of adenylyl cyclase, respectively. Gs
and Gi can thus couple binding of ligands to GPCRs with either activation or
inhibition of adenylyl cyclase, depending on the receptor type. This increases
or reduces production of cAMP, which diffuses from the membrane into the
cell. Edwin Krebs and Edmund Fischer later found that a principal task of
cAMP is to stimulate protein phosphorylation (Fischer and Krebs 1955),
ultimately showing cAMP-dependent protein kinase (also known as protein
kinase A, PKA) is responsible (Krebs 1993; Gilman 1995).
PKA is the major target for cAMP (see Fig. 2) (p. 99 [Sassone-Corsi
2012]). It is a heterotetramer consisting of two regulatory (R) subunits that
maintain two catalytic (C) subunits in an inhibited state. When cAMP levels
are low, the PKA holoenzyme is dormant; however, when cAMP levels are
elevated, two molecules bind in a highly cooperative manner to each R
subunit, causing a conformational change that releases the active C subunits
(Taylor et al. 2012). In humans, four genes encode R subunits (RIα, RIβ,
RIIα, and RIIβ) and two genes encode C subunits (Cα and Cβ), combinations
of which are expressed in most, if not all, tissues. The C subunits of PKA
phosphorylate serine or threonine residues on target substrates, typically
within the sequence RRxS/TΦ, in which Φ is an aliphatic hydrophobic
residue or an aromatic residue (Kemp et al. 1976). Around 300–500 distinct
intracellular proteins can be phosphorylated by PKA in a typical cell. These
include glycogen phosphorylase as part of the fight-or-flight mechanism.
Active phosphorylase catalyzes the production of glucose 1-phosphate, a
metabolic intermediate that is ultimately converted into other modified sugars
that are used in various aspects of cellular catabolism and ATP production
(see Ch. 14 [Hardie 2012]). On its release from the PKA holoenzyme, the C
subunit of PKA can diffuse into the nucleus, where it phosphorylates
transcription factors such as the cAMP-response-element-binding protein.
cAMP can thereby ultimately influence transcriptional activation and
reprogramming of the cell.

Figure 2. (A) cAMP is the archetypical second messenger. Its levels increase rapidly following
receptor-mediated activation of adenylyl cyclase (AC), which catalyzes the conversion of adenosine
monophosphate (AMP) to cAMP. This small second messenger activates PKA at specific cellular
locations as a result of anchoring of PKA to A-kinase-anchoring proteins (AKAPs). In addition, cAMP
activates EPACs (exchange proteins directly activated by cAMP). (B) AC activity is controlled by the
opposing actions of the Gs and Gi proteins. The cAMP produced by AC activates PKA by binding to
its R subunit. This releases the C subunit. The signal can be terminated by the action of
phosphodiesterase (PDE) enzymes.

Additional layers of regulation ensure that PKA phosphorylates the

correct proteins at the right time. For example, prostaglandin E1 and
epinephrine both produce similar increases in cAMP and PKA activity in the
heart, but only epinephrine increases glycogen phosphorylase activity. This is
achieved in part through attachment of PKA to subcellular structures via
interactions between R subunits and A-kinase-anchoring proteins (AKAPs)
(Wong and Scott 2004). Anchoring of PKA to subcellular structures by
AKAPs is a means to limit the range of action of PKA and avert the
indiscriminate transmission of these responses throughout the cell.
A typical cell expresses 10 to 15 different AKAPs. These proteins all
contain an amphipathic-helix motif that binds to a docking and dimerization
domain formed by the R subunits of PKA and targeting domains that tether
the AKAPs to intracellular membranes or organelles. AKAPs promote
signaling efficacy by placing PKA near preferred substrates and insulating
different anchored PKA complexes from one another (Scott and Pawson
2009). Most AKAPs also organize other signaling proteins, such as GPCRs,
GTPases, protein phosphatases, phosphodiesterase (PDE), and other protein
kinases.

2.2 3′-5′-Cyclic Guanosine Monophosphate and cGMP-

Dependent Protein Kinase
3′-5′-cyclic guanosine monophosphate (cGMP) is another cyclic nucleotide
that serves as a second messenger. cGMP is often synthesized by receptor
guanylyl cyclases, which have their catalytic domains close to the inner face
of the plasma membrane and are stimulated by small peptide factors that bind
to their extracellular domains. It can also be produced by soluble cytoplasmic
guanylyl cyclases, which are stimulated by nitric oxide. This latter signaling
pathway is involved in a number of important physiological processes,
including smooth muscle relaxation and neurotransmission (see Ch. 1 [Heldin
et al. 2014]). cGMP targets cGMP-dependent protein kinase (PKG), a
dimeric enzyme that has an R and C domain within the same polypeptide
chain. When cGMP levels are low, PKG is dormant; however, when cGMP
levels are elevated, two molecules bind to each R domain in the dimer,
exposing the active catalytic domains. There are two PKG isozymes: the type
I enzyme is soluble and predominantly cytoplasmic, whereas the type II
enzyme is particulate and is attached to a variety of biological membranes.
PKG phosphorylates peptide sites with the general consensus—RRXS/TX.
Hence, PKA and PKG have overlapping substrate specificities. However, a
major difference is that PKG expression is restricted to the brain, lungs, and
vascular tissue, where this enzyme controls important physiological
processes, including the regulation of smooth muscle relaxation, platelet
function in the blood, cell division, and aspects of nucleic acid synthesis.
PKG causes relaxation of vascular smooth-muscle cells, for example, by
phosphorylating several structural proteins that are vital for this process.

2.3 Other cAMP and cGMP Targets

Other cAMP and cGMP targets also play key roles in cellular physiology. For
example, cyclic nucleotide-gated channels (nonselective channels that allow
many ions to flow into or out of a cell) have important functions in retinal
photoreceptors and olfactory receptor neurons (see Ch. 11 [Julius and
Nathans 2012]). Most notably, cGMP is a key second messenger in vision:
photoisomerization of the chromophore in the light receptor, rhodopsin,
produces a conformational change in the receptor that allows it to activate the
G protein transducin, which, in turn, activates a cGMP PDE. The resulting
reduction in the concentration of cGMP leads to the closure of sodium and
calcium channels and, thus, hyperpolarization of photoreceptor cells, leading
to changes in neurotransmitter release. Mutations in many components of this
pathway, including the PDE, cause blindness. Regulation of ion channels by
cAMP is particularly important in the sinoatrial node of the heart, in which
cAMP-responsive hyperpolarization-activated cyclic nucleotide-gated
(HCNs) channels help to generate pacemaker currents that control cardiac
contractility (see Ch. 13 [Kuo and Ehrlich 2014]). Other classes of HCN
channel have analogous functions in the brain and nervous system. cAMP
also controls the cAMP-responsive guanine nucleotide exchange factor
EPAC1, a protein that promotes activation of the Rap1 GTPase to regulate
cell adhesion by stimulating integrin molecules in the plasma membrane (Bos
2003).

2.4 Cyclic Nucleotide PDEs

Termination of cAMP and cGMP signals is mediated by PDEs. These
enzymes represent a vast gene family of 11 distinct subtypes and more than
100 isoforms that can break the phosphoester bond of either cyclic nucleotide
to liberate AMP or GMP (Beavo and Brunton 2002). PDE activity can be
regulated in a variety of ways. For example, calcium-dependent processes
control the activity of the PDE1 and PDE2 isoforms, PKA phosphorylation
attenuates the activity of PDE3 and PDE4 isoforms, and PKA or PKG
phosphorylation participates in the control of PDE5. Less is known about the
mechanisms of regulation for PDE6-11. More recently, there has been
considerable interest in the development and clinical application of small-
molecule PDE inhibitors. Selective PDE inhibitors that produce elevated
levels of cAMP/cGMP have been used clinically to alleviate chronic
obstructive pulmonary disease, asthma, and combat certain immune
disorders, but their most celebrated therapeutic application has been in the
treatment of male erectile dysfunction. Sildenafil citrate (Viagra) and its
relatives act by inhibiting cGMP-specific PDE5 (Beavo and Brunton 2002) in
the arterial wall smooth muscle of the penis, which elevates cGMP and
increases blood flow.

3 LIPID AND LIPID-DERIVED SECOND MESSENGERS

Two seemingly unrelated discoveries half a century ago provided the first
insights into how cells use lipids to signal. Hokin and Hokin discovered that
cholinergic stimulation of pancreatic slices promotes incorporation of 32P
from radiolabeled ATP into phospholipids, which led to an explosion of
studies showing that numerous extracellular signals promote hydrolysis of
phosphoinositides in cells and eventually the identification of DAG as a
major second messenger. During this period, Yasutomi Nishizuka purified
the enzyme PKC and showed it is the target for this lipid second messenger
(Nishizuka 1992).
We now know that many extracellular signals trigger hydrolysis of two
classes of lipids to generate second messengers: glycerolipids, whose
hydrophobic backbone is DAG (e.g., phosphatidylinositol 4,5-bisphosphate,
PIP2), and sphingolipids, whose hydrophobic backbone is ceramide (e.g.,
sphingomyelin). Both produce a variety of products that are involved in cell
signaling (Fig. 3). Binding of ligands to receptors that couple to lipid-
modifying enzymes (see below) results in activation of enzymes that
hydrolyze specific acyl chains or polar head groups from each lipid class to
generate lipid second messengers. Specifically, hydrolysis of acyl chains
generates lysophospholipids (e.g., lysophosphatidylcholine and
lysophosphatidic acid through hydrolysis catalyzed by phospholipase A2)
and lysophingolipids (e.g., sphingosine through hydrolysis catalyzed by
ceramidases). It also produces free fatty acids, such as arachidonic acid,
which are important signaling entities in their own right.
Figure 3. Lipid-derived second messengers. (A) Hydrolysis of glycerophospholipids results in the
production of lysophospholipids, free fatty acids (arachidonic acid is shown), diacylglycerol,
phosphatidic acid, and, in the case of hydrolysis of PIP2, the water soluble inositol 3,4,5-trisphosphate.
(B) Hydrolysis of sphingolipids yields ceramide, sphingosine, and sphingosine 1-phosphate.

Hydrolysis of polar head groups from glycerophospholipids by

phospholipase D and PLC results in generation of phosphatidic acid and
DAG, respectively. The polar head group released following PIP2 hydrolysis,
IP3, is a key second messenger itself. IP3 can also be further phosphorylated
to form polyphosphoinositols that serve as additional second messengers.
Hydrolysis of the polar head group from sphingomyelin, catalyzed by
sphingomyelinase, produces ceramide. Further hydrolysis of the acyl chain
by ceramidase produces sphingosine, and the subsequent phosphorylation of
this by sphingosine kinase produces sphingosine 1-phosphate.
Lipid second messengers that retain two acyl chains (e.g., phosphatidic
acid, DAG, and ceramide) remain membrane associated, but the decreased
lipophilicity of second messengers that retain only one acyl chain (lysolipids
such as lysophosphatidic acid and sphingosine) allows them to dissociate
from membranes and serve as soluble second messengers. Finally, note that
in addition to its hydrolysis, PIP2 can be phosphorylated to produce another
key second messenger, PIP3 (see below).

3.1 IP3 and DAG

Signals from both GPCRs (e.g., histamine receptors) and RTKs (e.g.,
epidermal growth factor [EGF] receptors) can result in activation of PLC,
which cleaves phospholipids to generate DAG and the lipid headgroup. In the
case of GPCR signaling, lipid hydrolysis is mediated by binding of G
proteins (notably Gq) to phospholipases (PLCβ); in the case of RTKs, lipid
hydrolysis is mediated by recruitment of phospholipases (PLCγ) to tyrosine-
phosphorylated proteins at the plasma membrane (tyrosine phosphorylation
of PLCγ by RTKs also stimulates its activity directly). If the lipid cleaved is
PIP2 (rather than phosphatidylcholine [PC]), then water-soluble IP3 is also
produced. IP3 binds to IP3 receptors on the endoplasmic reticulum (ER), and
other organelles, causing release of calcium into the cytosol (Fig. 1). It can be
further modified to yield additionally phosphorylated phosphoinositols,
including diphosphoryl inositol phosphates (Tsui and York 2010). Inositol
1,3,4,5-tetrakisphosphate (IP4) activates chloride channels. Inositol
1,2,3,4,5,6-hexaphosphate (IP6) is a compound found in plants (hence, its
name phytic acid), but it is also present in yeast and animal cells, along with
the enzymes that synthesize and metabolize it, and is gaining interest as a
potential anticancer agent because of its apoptosis-promoting properties. The
DAG sensor is a small globular domain, called the C1 domain, originally
identified in PKC. Approximately 30 other proteins contain a C1 domain
(Colon-Gonzalez and Kazanietz 2006), including protein kinase D, Ras,
gastrin-releasing polypeptides, DAG kinase, and n-chimaerin.
PKC represents a family of nine genes grouped into three families
(Newton 2009). The so-called conventional PKC isozymes require the
coordinated presence of both calcium (sensed by the C2 domain) and DAG
(sensed by the adjacent C1 domain) for activation and thus transduce signals
that trigger PIP2 hydrolysis, but not those that trigger hydrolysis of other
phospholipids, such as PC (because these do not mobilize calcium via IP3).
Novel isozymes of PKC do not have a calcium sensor, but because they bind
DAG with an affinity two orders of magnitude higher than conventional PKC
isozymes, they are efficiently activated by DAG alone. Thus, they can
transduce signals triggered by PC hydrolysis. A third class of PKC isozymes,
the atypical PKCs, do not respond to DAG or calcium. Signaling by
conventional and novel PKC isozymes is terminated by phosphorylation of
DAG by DAG kinase, which removes the second messenger. Note that PKC
isozymes are constitutively phosphorylated so, unlike many other kinases,
these phosphorylations do not acutely regulate activity.
DAG- or calcium-dependent translocation of conventional and novel PKC
isozymes to the membrane is a hallmark of their activation (see Fig. 4). In
unstimulated cells, PKC localizes to the cytosol, where it may be
concentrated on specific protein scaffolds. For example, the conventional
PKCα binds to the PDZ-domain scaffold DLG1. PIP2 hydrolysis provides
calcium, which binds to the C2 domain and thus recruits cytosolic PKC to the
plasma membrane; there, PKC binds to the membrane-embedded second
messenger DAG via its C1 domain. Binding of both the C2 and C1 domains
to membranes provides the energy to release an autoinhibitory
pseudosubstrate segment from the substrate-binding cavity, allowing PKC to
phosphorylate substrates and relay signals. Interestingly, a function common
to many of the PKC isozymes is that their phosphorylation of certain
substrates terminates signaling pathways. For example, phosphorylation of
receptors such as the EGF and insulin receptors by PKC promotes their
internalization and degradation, thus acting as a negative-feedback loop to
attenuate signaling via these pathways.
Figure 4. (A) PKC transduces signals that promote phospholipid hydrolysis. Signals that cause
phospholipase-C-mediated hydrolysis of PIP2 activate conventional PKC isozymes by a two-step
mechanism. First, calcium (whose levels increase following IP3 production) binds to the C2 domain of
PKC. This increases the affinity of the module for the plasma membrane, causing PKC to translocate to
the membrane. Here, it binds its allosteric activator, DAG. This binding produces a conformational
change that expels the autoinhibitory pseudosubstrate segment from the active site, allowing substrate
phosphorylation and downstream signaling. (B) Activation of PI3K following engagement of growth
factor receptors such as insulin receptor generates the phospholipid PIP3, which recruits the kinase Akt
to the membrane where it is phosphorylated by the upstream kinase PDK1. Subsequent phosphorylation
events lead to activation of Akt.

PKC has a unique signature of activation depending on its cellular

location (Gallegos and Newton 2008). In general, its activation kinetics
mirror those for the increase in intracellular calcium concentration, and
deactivation kinetics mirror the decay of DAG. Thus, membranes such as the
Golgi in which DAG production is sustained serve as a platform for sustained
PKC activity, whereas membranes such as the plasma membrane where DAG
is more rapidly removed by phosphorylation serve as platforms for transient
PKC activity. Although PKC can phosphorylate cytosolic targets, these
events likely occur at the membrane. Reduction in cytoplasmic calcium levels
and phosphorylation of DAG by DAG kinase effectively terminates PKC
signaling.

3.2 PIP3 and Akt Signaling

Binding of growth factors to receptor tyrosine kinases results in the activation
of PI3K isoforms (Fig. 4B), which catalyze the phosphorylation of PIP2 at the
3′ position to generate the very minor, but highly efficacious, lipid second
messenger PIP3 in the plasma membrane (Cantley 2002). PI3K also
phosphorylates phosphatidylinositol and phosphatidylinositol 4-phosphate
(PIP) at the 3′ position to generate corresponding 3′-phosphoinositides that
also serve as second messengers. PIP2 is itself a minor component of the
plasma membrane, accounting for ∼0.05% of the total phospholipid, yet the
phosphorylation of 10% of this pool effectively transduces growth factor
signals. The reason for this highly effective signaling is the use of protein
modules that bind with high affinity and specificity to the various
phosphorylated species of the inositol head group (e.g., inositol 3,4,5-
trisphosphate versus inositol 3,4-bisphosphate) that is exposed to the cytosol.
These include pleckstrin homology (PH), FYVE, and PX domains (Lemmon
2007; p. 87 [Hemmings and Restuccia 2012]).
The best defined target for PIP3 is the prosurvival kinase Akt (Manning
and Cantley 2007). Like PKC, Akt is present in the cytosol in unstimulated
cells. PIP3 produced following PI3K activation recruits Akt to the plasma
membrane via its PH domain. This triggers the phosphorylation of Akt at two
sites, leading to its activation and downstream signaling. The first
phosphorylation is catalyzed by the phosphoinositide kinase PDK1 (also
recruited to the membrane by PIP3) at a segment near the entrance to the
active site, which, in turn, leads to rapid phosphorylation at a carboxy-
terminal site. Autophosphorylation and the mTORC2 kinase complex have
been proposed to regulate this second site (see p. 91 [Laplante and Sabatini
2012]). Once phosphorylated, Akt is locked in an active and signaling-
competent conformation and can be released from the plasma membrane to
signal throughout the cell, including the nucleus. Signaling is terminated by
dephosphorylation of PIP3 and dephosphorylation of Akt. The former is
catalyzed by the tumor suppressor PTEN, a lipid phosphatase, which removes
the lipid second messenger by dephosphorylating the 3′ phosphate of PIP3.
Dephosphorylation of the two Akt sites is catalyzed by protein phosphatases
such as the tumor suppressor PHLPP.

3.3 Sphingolipid Hydrolysis and Protein Targets that Control

Apoptosis
Sphingolipids are a class of bioactive lipids that play important roles in
diverse cellular responses (Hannun and Obeid 2008). The major signaling
lipids derived from sphingomyelin are ceramide, sphingosine, and
sphingosine 1-phosphate, which are produced by the action of
sphingomyelinase, ceramidase, and sphingosine kinase, respectively (Fig. 3).
Ceramide propagates information in stress-response pathways; it is
produced following activation of receptors by stimuli such as cytokines or
death ligands and also by nonreceptor signals in response to radiation,
cytotoxic insult, or infection by pathogens. Sphingosine also has apoptotic
roles, although its direct targets in cells are unclear. In contrast to ceramide
and sphingosine, sphingosine 1-phosphate promotes prosurvival signaling.
When secreted, it binds to a class of GPCRs that promote diverse cellular
effects, including cell-survival, differentiation, inflammation, and
angiogenesis (Pitson 2011). Intracellular sphingosine 1-phosphate acts as a
second messenger, activating enzymes such as the TRAF2 E3 ligase and
some histone deacetylases.

3.4 Lysolipids, Prostaglandins, and Other Eicosanoids

Oxidation of 20-carbon fatty acids produces eicosanoids, thus signaling
molecules that bind to GPCRs to mediate inflammatory and immune
responses (Wymann and Schneiter 2008). Phospholipase-A2-mediated
hydrolysis of phospholipids containing arachidonic acid (a 20-carbon
unsaturated fatty acid) in the sn-2 position of the glycerol backbone (see Fig.
3) yields a fatty-acid product that can be oxidized by cyclooxygenases to
yield prostaglandins, or by lipooxygenases to yield leukotrienes. These
signaling molecules bind to specific receptors that couple to diverse
pathways. Many of these are involved in inflammatory responses. Notably,
leukotrienes bind to GPCRs that cause inflammatory responses common in
asthma, and, as such, leukotriene pathways serve as targets for antiasthma
drugs. Aspirin inhibits cyclooxygenases and thus the production of
prostaglandins, which are inflammatory (Venteclef et al. 2011). As
mentioned above, arachidonic acid itself also serves as a second messenger.

4 IONS AS INTRACELLULAR MESSENGERS

Ions such as sodium, potassium, calcium, magnesium, protons, chloride, iron,
and copper play essential roles in cell function. Many ions act as cofactors for
structural proteins and enzymes. Moreover, cellular processes are sensitive to
changes in their ambient ionic environment; changes in pH, for example, can
alter enzymatic activity and the behavior of cellular ion transporters
(Vaughan-Jones et al. 2009). In addition, ions control cellular activity by
spreading electrical signals, for example, action potentials in heart and
neurons.
Ions such as calcium and magnesium can also play direct roles as
dynamic intracellular messengers that regulate specific protein targets during
signal transduction (Fig. 5A). These ions are imported from the extracellular
milieu or mobilized from intracellular stores and control the activity of
protein targets by binding to specific motifs on the protein itself or upstream
regulators (e.g., calmodulin) (Berridge et al. 2000). Note that most ions
should not be considered intracellular messengers, however. For example, the
sodium ions that enter cardiac myocytes during an action potential serve only
to depolarize the cell membrane. This causes the opening of voltage-operated
calcium channels (VOCCs), allowing influx of the real messenger—calcium
ions—which control contraction by binding to proteins such as calmodulin
and troponin C (Ch. 13 [Kuo and Ehrlich 2014]). Similarly, to reverse an
action potential, potassium ions are rapidly extruded and the membrane
potential is restored, but it is the reversion of the calcium concentration to the
prestimulated state through the action of the sodium/calcium exchanger
(NCX) that represents signal termination (sodium and potassium levels are
returned to the original state by the sodium/potassium ATPase [Na+/K+-
ATPase], so that another action potential can be evoked).
Figure 5. (A) Calcium and magnesium signals. Both ions can enter cells via channels in the plasma
membrane. In addition, calcium is stored in organelles such as the ER. Calcium exerts its effects by
binding to numerous cellular protein targets, including calmodulin, whereas magnesium may function
as a calcium mimetic or have magnesium-specific effects. (B) The various ways in which calmodulin
can function to alter cellular targets. It is generally thought that cellular calmodulin is largely bound to
proteins even when the calcium concentration is low, and that there is a relatively small pool of
cytosolic calcium-free calmodulin (apocalmodulin). However, even apocalmodulin can regulate
specific cellular processes (e.g., IP3 receptors, IP3Rs). When the calcium concentration is elevated,
calcium ions bind to calmodulin. This causes calmodulin to be displaced from some targets and
associate with others. In some cases (e.g., phosphorylase kinase), calmodulin is a constitutively bound
subunit that binds calcium and activates the enzyme when the calcium concentration is elevated. Other
calcium-binding proteins, such as neuronal calcium sensors, may also display complex interactions
with their various targets. *CaM-dependent inhibition of IP3R channels.

A key reason for using ions as messengers is speed of response. Cells use
energy to maintain gradients of ions across their lipid membranes. By
activating channels or transporters, cells can use the potential energy
established by the electrochemical gradient of an ion to rapidly generate a
cellular signal (Clapham 2007). Unlike other intracellular messengers, ionic
signals can be generated with no enzymatic steps. The speed of the response
depends on the rate at which the intracellular concentration of the ion changes
and the proximity of the ions to their cellular targets. For example, action
potentials cause the fast release of neurotransmitters at nerve terminals
because the cytosolic concentration of calcium ions just beneath the plasma
membrane increases from ∼100 nM to >10 µM within milliseconds (Berridge
2006). In situations in which ions have to diffuse further before encountering
their target(s), the response will be slower. As we discuss below, the ability
to generate different spatiotemporal patterns, such as waves and oscillations,
is another important advantage of ionic intracellular messengers.

4.1 Calcium
Calcium is an extremely versatile intracellular messenger that controls a wide
range of cellular functions by regulating the activity of a vast number of
target proteins. The cellular effects of calcium are mediated either by direct
binding to a target protein, or stimulation of calcium sensors that detect
changes in calcium concentration and then activate different downstream
responses (Berridge 2004). The multitude of sensors that mediate effects of
calcium can be characterized by the nature of their calcium-binding site(s).
The most common calcium-binding motifs are EF-hands and C2 domains.
Synaptotagmin and troponin C are examples of proteins with C2 domains and
EF-hands, respectively. Calcium sensors also act by recruiting a range of
intermediary effectors, such as calcium-sensitive enzymes—for example,
calcium/calmodulin-dependent protein kinases (CaMKs), calcineurin (also
known as protein phosphatase 2B), myosin light chain kinase, and
phosphorylase kinase (see Ch. 13 [Kuo and Ehrlich 2014]).

4.2 Control of Calcium Levels

Intracellular calcium levels are controlled by an assortment of channels,
pumps, transporters, buffers, and effector moieties. At rest, cytosolic calcium
is maintained at ∼100 nM. In most cases, its levels are elevated by the
opening of channels located either on various organellar stores or in the
plasma membrane. Release of calcium from internal stores represents a major
source of signal calcium for many cells. The principal calcium stores are the
ER, sarcoplasmic reticulum (SR), Golgi, and acidic organelles of the
endolysosomal system (Bootman et al. 2002). Calcium release channels are
present on the membranes of these organelles and gate the flux of calcium
from the ER/SR lumen into the cytosol. Ubiquitous calcium-release channels
include the IP3Rs that respond to IP3 produced by hydrolysis of PIP2 (Parys
and De Smedt 2012), ryanodine receptors (Belevych et al. 2013), and two-
pore channels that respond to nicotinic acid adenine dinucleotide phosphate
(Galione 2011).
Channels that permit the influx of calcium across the plasma membrane
are typically characterized by their activation mechanism. Receptor-operated
calcium channels and second messenger-operated calcium channels are
opened by the binding of an external or internal ligand. Examples of these are
N-methyl-D-aspartate (NMDA) receptors that respond to the neurotransmitter
glutamate (see Ch. 12 [Kennedy 2013]), and Orai channels regulated by the
intracellular messenger arachidonic acid. VOCCs that are activated by a
change in membrane potential are widely expressed in excitable tissues and
can be divided into three families [CaV1 (L-type), CaV2 (N, P/Q, and R type),
and CaV3 (T type)], which have specific characteristics and functions
(Catterall 2011). The transient receptor potential (TRP) family includes a
number of calcium-permeable channels with distinct activation mechanisms
(Gees et al. 2012). Depletion of ER or SR calcium activates “store-operated”
calcium entry. This pathway for calcium influx has been known for more
than two decades, and the store-operated current (Icrac, for calcium-release-
activated current) has been well characterized. However, the molecular
players, STIM (stromal interaction molecule) and Orai, have only been
identified relatively recently through investigation of patients with severe
combined immunodeficiency caused by a lack of calcium influx into their T
lymphocytes (Hogan et al. 2010).

4.3 Calcium Buffers

Cells express a number of calcium-binding proteins that buffer calcium
changes within various cellular compartments and modulate calcium signals.
The rapid binding of calcium by the cytosolic buffers parvalbumin and
calbindin D-28k shapes both the spatial and temporal properties of
intracellular calcium signals (Schwaller 2010). ER/SR calcium-binding
proteins (e.g., calsequestrin, calreticulin, GRP78, and GRP94) facilitate the
accumulation of large amounts of calcium, which is necessary for rapid cell
signaling (Prins and Michalak 2011). Mitochondria also play a key buffering
role in that they express a calcium uniporter capable of taking up substantial
amounts of calcium whenever the cytosolic calcium concentration increases
during signaling (Rizzuto and Pozzan 2006). The uptake of calcium into the
mitochondrial matrix stimulates the citric acid cycle to produce more ATP.
This increased oxidation also enhances mitochondrial reactive oxygen
species (ROS) formation, which contributes to redox signaling pathways.
Loading of calcium into mitochondria is believed to be essential to avoid
induction of autophagy. However, exaggerated mitochondrial calcium
loading can precipitate apoptosis (Baumgartner et al. 2009).

4.4 Signal Termination

Calcium pumps and exchangers are responsible for pumping calcium out of
the cell or back into the ER/SR to terminate a calcium signal. These pumps
and exchangers operate at different times during the recovery process. NCX
proteins have low affinities for calcium, but have high capacities that enable
them to rapidly remove large quantities of calcium and are especially evident
in excitable cells (Nicoll et al. 2013). The plasma membrane calcium-ATPase
and SR/ER calcium-ATPase enzymes have lower transport rates but higher
affinities, which means that they operate at relatively low cytosolic calcium
concentrations (Vandecaetsbeek et al. 2011). Calcium/hydrogen exchangers
are important for the loading of calcium into endo/lysosomal compartments.

4.5 Spatiotemporal Organization of the Calcium Signaling

Machinery
The substantial diversity of cellular calcium signals in different cell types
reflects their use of distinct combinations and arrangements of calcium-
transport mechanisms. In neurons, for example, microscopic calcium signals
trigger the release of neurotransmitter-containing vesicles at presynaptic
terminals (Berridge 2014). The calcium signal is a plume of ions around the
mouth of an open channel that dissipates within milliseconds and can only
activate processes within a few tens of nanometers. The receiver for the
calcium signal is synaptotagmin, a calcium-binding protein that promotes
fusion of the neurotransmitter-containing vesicle with the neuronal plasma
membrane. The rapidity of this response depends on the calcium channels
and exocytotic machinery (SNARE complexes) being linked together within
a highly localized microdomain. A key component of the SNARE complex is
SNAP-25, a membrane-bound protein that interacts with both the VOCCs
and synaptotagmin. In contrast, the regular beating of the heart relies on the
sequential elevation of calcium levels within all the myocytes of the atrial and
ventricular chambers. In this case, the calcium signal is global, occurring
across the whole volume of each myocyte to evenly activate troponin C,
thereby allowing actin and myosin to engage and cause contraction (Ch. 13
[Kuo and Ehrlich 2014]).
An important driver for global calcium signaling is regenerative calcium
elevation—that is, the autocatalytic increase in cellular calcium
concentration. Regenerative calcium signals arise because several
components of the calcium signaling toolkit, such as PLC, RYRs, and IP3Rs,
are activated by calcium (Roderick et al. 2003). The opening of one calcium
channel can therefore promote a positive feed-forward reaction in which
more calcium ions enter the cytosol. This regenerative calcium-induced
calcium release (CICR) process can override cellular calcium buffers,
enabling rapid coordinated surges in calcium concentration. The triggering of
CICR is typically the critical event in causing the transition from local to
global calcium signaling. Without CICR, the global calcium signals in
cardiac myocytes that trigger contraction (described above) would not occur.
CICR leads to a rapid increase in calcium concentration. Within an individual
cell, each spike of calcium caused by CICR is evident as calcium waves, such
as the calcium waves evoked by fertilization of an oocyte (see Wakai et al.
2011). Microscopic analysis and modeling has shown that such waves
propagate in a salutatory manner, involving successive rounds of CICR and
diffusion, as the wave jumps from a cluster of calcium channels to the next
(Thul et al. 2012). Such waves can engulf the entirety of the cellular
cytoplasm, and pervade into the nucleus to activate or inhibit transcription
(Bootman et al. 2009).
If a cellular stimulus is not sufficient to trigger a global response, then
only local calcium elevations will occur. The calcium ions emanating from
channels during local calcium signaling only switch on processes within their
immediate vicinity. The sphere of action of such microdomains depends on
how long the channels are open, their ability to conduct calcium, and the
capacity of surrounding calcium buffers and homeostatic mechanisms that
remove calcium (Parekh 2008). Calcium buffers and pumps are highly
effective at restricting the diffusion of calcium so that the calcium
concentration declines exponentially with distance from a calcium source. If
local calcium events do not turn into global calcium signals, the proximity of
particular effectors and their affinity for calcium determines the response. For
example, relatively low-affinity effector molecules such as synaptotagmin
(described above) and BK potassium channels (big calcium-activated
potassium channels, also called maxi-K channels) depend on submicrometer
proximity to a channel to sense an activating calcium concentration. Sensor
proteins with relatively higher affinity, such as calmodulin and SK potassium
channels (small calcium-activated potassium channels), can be activated at
greater distances.

4.6 Magnesium
Magnesium can also be considered an intracellular messenger because its
concentration can change dynamically in response to cellular stimulation (Li
et al. 2011). Given that magnesium binds to nucleotides, oligonucleotides,
and hundreds of enzymes, it is reasonable to conclude that it is an
intracellular messenger in its own right. Of its potential binding sites, ATP is
particularly important. Cellular processes typically use ATP complexed with
magnesium as an energy source. Moreover, magnesium has been shown to
cause prolonged inhibition of potassium channels in neurons following
muscarinic acetylcholine receptor activation (an effect that is not mimicked
by calcium), thereby regulating neuronal excitability (Chuang et al. 1997).
In addition, magnesium deserves consideration because it influences the
effects of calcium. Indeed, magnesium and calcium are typically thought to
have antagonistic actions. Magnesium frequently inhibits the transport and
cellular activities of calcium and can prevent pathological consequences of
increases in calcium levels (Romani 2013). Like calcium, there is an
electrochemical gradient of magnesium across the plasma membrane that can
serve as a reservoir for signal generation. The extracellular concentrations of
magnesium and calcium are similar (1.1–1.5 mM), and magnesium can also
act as a “calcimimetic” (e.g., by binding to the calcium-sensing receptor), a
GPCR that has pleiotropic actions. Within cells, the magnesium
concentration (0.3–1.5 mM) is several orders of magnitude higher than that of
calcium. It has been suggested that mitochondria might serve as a store of
magnesium and that magnesium can potentially regulate cellular respiration
(Wolf and Trapani 2012). Moreover, magnesium in the mitochondrial matrix
inhibits permeability transition pore (PTP) activation, an increase in the
leakiness of the inner mitochondrial membrane that allows solutes <1500 Da
to pass, and can precipitate mitochondrial swelling, apoptosis, and cell death.
Calcium promotes PTP, so magnesium acts as a counteracting antagonist.
Like calcium, magnesium has a plethora of transport pathways. Of these,
TRPM6 and TRPM7 (members of the TRP family) are relatively well
understood. TRPM6 is restricted to kidney tubules and the intestinal
epithelium, and plays an important role in magnesium (re)adsorption
(defective TRPM6 function leads to hypomagnesemia), whereas TRPM7 is
ubiquitously expressed in mammals. Both TRPM6 and TRPM7 are
“chanzymes”: ion channels that incorporate a kinase domain. Interestingly,
TRPM7 is regulated by PIP2, the source of the calcium-mobilizing messenger
IP3. Moreover, it binds to PLC, the enzyme that hydrolyses PIP2.
Consequently, hormonal stimulation of cells will lead to both a calcium
signal (via IP3 production) and a change in magnesium flux (via loss of PIP2-
mediated regulation of TRPM7) (Langeslag et al. 2007). MagT1 is also
believed to be a key cellular magnesium channel. Like TRPM6 and TRPM7,
it is located on the plasma membrane of mammalian cells. However, whereas
TRPM6 and TRPM7 allow both calcium and magnesium fluxes, it is believed
that MagT1 is a specific pathway for magnesium. Individuals bearing
mutations in the MAGT1 gene show high levels of Epstein–Barr virus
infection and a predisposition to lymphoma (Chaigne-Delalande et al. 2013).

5 CONCLUDING REMARKS
Second messengers disseminate information received by cellular receptors
rapidly, faithfully, and efficaciously. They are small, nonprotein organic
molecules or ions that bind to specific target proteins, altering their activities
in a variety of ways that allow them to respond appropriately to the
information received by receptors.
A key advantage of second messengers over proteins is that, unlike
proteins, second messenger levels are controlled with rapid kinetics. Thus,
whereas it may take tens of minutes for the levels of a protein to increase
significantly, most second messenger levels increase within microseconds
(e.g., ions) to seconds (e.g., DAG), They are often produced from precursors
that are abundant in cells or released from stores that contain high
concentrations of the second messenger; so, their generation is not rate
limiting. Thus, when the appropriate signal is received, second messengers
are rapidly generated, diffuse rapidly, and alter target protein function highly
efficiently.
 Second messengers vary significantly in size and chemical character:
from ions to hydrophilic molecules such as cyclic nucleotides to hydrophobic
molecules such as diacylglycerol. Moreover, the continuing discovery of new
second messengers is expanding the repertoire of molecules known to convey
information within the cell. Indeed, only very recently, cyclic guanosine
monophosphate-adenosine monophosphate was shown to be a second
messenger that is synthesized by the enzyme cGAS in response to HIV
infection and binds to and activates a protein called STING, leading to
induction of interferon (Wu et al. 2013). The ability to respond rapidly to
information thus depends on an expanding library of small molecules.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005926
CHAPTER 4

Signaling Networks: Information Flow,

Computation, and Decision Making

Evren U. Azeloglu and Ravi Iyengar

Department of Pharmacology and Systems Therapeutics and Systems
Biology Center New York, Mount Sinai School of Medicine, New York,
New York 10029

SUMMARY

Signaling pathways come together to form networks that connect

receptors to many different cellular machines. Such networks not only
receive and transmit signals but also process information. The
complexity of these networks requires the use of computational models
to understand how information is processed and how input–output
relationships are determined. Two major computational approaches used
to study signaling networks are graph theory and dynamical modeling.
Both approaches are useful; network analysis (application of graph
theory) helps us understand how the signaling network is organized and
what its information-processing capabilities are, whereas dynamical
modeling helps us determine how the system changes in time and space
upon receiving stimuli. Computational models have helped us identify a
number of emergent properties that signaling networks possess. Such
properties include ultrasensitivity, bistability, robustness, and noise-
filtering capabilities. These properties endow cell-signaling networks
 with the ability to ignore small or transient signals and/or amplify
signals to drive cellular machines that spawn numerous physiological
functions associated with different cell states.

Outline
1 Introduction
2 Emergent properties of signaling networks
3 Information flow and processing
4 Concluding remarks
References

1 INTRODUCTION
Coordinated regulation of cellular processes allows cells to maintain
homeostatic balance and make decisions as to whether to divide, differentiate,
or die. In each case, the cell responds to chemical, mechanical, or electrical
signals, including hormones, neurotransmitters, mechanical stretch and shear,
and ion currents. The signaling pathways activate relay information to
effectors that alter subcellular processes, but they also process this
information. Information processing involves computation in which the
network of connected signaling molecules recognizes the amplitude and
duration of the incoming signal and produces an output signal of appropriate
strength and duration. This depends on the organization (topology) of the
signaling network and can change the relationship between inputs and
outputs. For example, the cell can limit responses to maintain homeostatic
balance or trigger a set of changes that takes it to another state (e.g.,
differentiation or division). The inherent computational abilities of signaling
networks thus provide the cell with decision-making capabilities.
In all signaling pathways, information flows through coupled biochemical
reactions and molecular interactions to the cellular machines that control its
output: biochemical (e.g., metabolic enzymes), mechanical (e.g., actin
cytoskeleton contractility), or electrical (e.g., ion channel activity in excitable
cells). Most of these pathways are part of wider networks (Weng et al. 1999).
Studying the flow of information (signal) through these networks (see Box 1)
is like studying traffic patterns, which depend on the population density of
the towns through which the highways pass, the state of local roads, the time
of the day, the weather, and other factors. Similarly, to understand the flow of
signals that regulate cellular machines, we must know how the pathways
involved are connected to form networks, the characteristics of the
connections, and how information is processed as it flows through these
interconnected pathways. Below, we discuss the organization of these
networks and explain how they can be modeled quantitatively.

BOX 1. INFORMATION AND SIGNALS IN CELL-SIGNALING

PATHWAYS

In the context of intracellular-signaling pathways and networks, the

terms information and signal are used interchangeably. These terms are
most often used to distinguish the activity state of a signaling
component. Biochemical information, or signal, flows from component
A to component B as the activity state of A controls the activity state of
component B. The activity state of a given molecule is defined as the
fraction of active (e.g., A* or B*) to total molecules (e.g., A or B). A
typical node A in this example could be a receptor, such as the
adrenergic receptor, that has an interaction (i.e., edge) with a target node
B, such as the G protein Gs. Communication through this edge results in
node A activating node B (i.e., β adrenergic receptor-activating Gs). It is
important to note that information content at any given time depends on
both the local concentration of interacting components and the reaction
kinetics that govern change in activity state. Green arrows in the figure
denote information flow.
 Biochemical information transfer is established through coupled
reactions, which are either noncovalent reversible binding reactions or
enzymatic reactions that involve covalent modification. However, such
information flow sometimes results in the movement of components
from one subcellular compartment to another. The most common
examples of such movements are the translocation of protein kinases or
transcription factors from the cytoplasm to the nucleus in response to
hormone or growth factor signals, where they interact with other
proteins or DNA to affect gene expression. This is considered spatial
information transfer.

1.1 Versatility of Signaling Components Enables Pathways to

Form Networks
Most signaling pathways in mammalian cells interact with one another
(Jordan et al. 2000). This so-called “cross talk” starts at the level of receptors.
Growth factor receptors with intrinsic tyrosine kinase activity, for example,
interact with multiple effector pathways, including the Ras-ERK-kinase
pathway (p. 81 [Morrison 2012]), the phosphoinositide 3-kinase (PI3K)
pathway (p. 87 [Hemmings and Restuccia 2012]), and the phospholipase-C γ
pathway (Fig. 1A) (p. 95 [Bootman 2012]). Similarly, in the case of receptors
coupled to heterotrimeric G proteins, the ability of both the Gα and Gβγ
subunits to transmit signals allows the receptors to couple to multiple
downstream effectors (Fig. 1B).
Figure 1. Interaction of multiple components with receptors leads to signal flow within multiple
signaling pathways. Good examples are the interplay of PI3K, PLCγ, and Ras ERK signaling following
activation of epidermal growth factor receptor (EGFR) (A), heterotrimeric G proteins linked to
adrenergic receptors (ARs) (B), and the small G protein Rho (C).

Small G proteins, such as Ras, Rho, Rap, and Cdc42, are also major loci
of interconnectivity (Bar-Sagi and Hall 2000). Their guanine nucleotide
exchange factors (GEFs) and GTPase-activating proteins (GAPs) can be
regulated by numerous mechanisms, including binding of ligands such as
cAMP, calcium, and diacylglycerol (DAG), or posttranslational
modifications such as phosphorylation and acylation. Multiple receptors feed
into these GTPase regulators, and the small G proteins themselves can
modulate the activity of multiple signaling pathways by targeting several
different effectors (Fig. 1C).
Protein kinases and protein phosphatases are additional loci at which
multiple signaling pathways interact. Typically, a single protein kinase or
phosphatase can be activated by multiple receptors and has multiple
substrates. Together, all these components make the signaling pathways
within a cell extensively interconnected.
Most mammalian proteins are present as multiple isoforms. These can
arise from alternative splicing of a single mRNA or use of alternative
initiation codons, or they can be products of different genes altogether.
Typically, these isoforms have different characteristics that alter both
upstream and downstream interactions (connectivity) and/or their
intracellular localization. This further increases connectivity. An early
example is adenylyl cyclases (AC1-AC8), which allow signals from multiple
types of receptors and ion channels to feed into the cAMP pathway (Pieroni
et al. 1993; p. 99 [Sassone-Corsi 2012]). These enzymes can be activated or
inhibited by signals relayed via different types of receptors (Fig. 2), thus
enabling cAMP levels to provide an integrated measure of the information
coming into the cell from various sources. cAMP in turn works through
multiple effectors (e.g., protein kinase A, the EPAC Rap-GEF, and the cyclic-
nucleotide-gated channels) to regulate numerous cellular targets to evoke
physiological responses.
Figure 2. Different adenylyl cyclase (AC) isoforms are activated by multiple different upstream
signals. This allows the second messenger cAMP to provide an integrated measure of information from
several different sources.

Why is one class of signaling component often responsible for regulating

many cellular machines? We do not yet know the answer. The cellular
machinery probably evolved to produce distinct activity patterns in response
to specific signals, whose amplitude, duration, and subcellular location evoke
signature responses from only a subset of cellular machines. There is
suggestive evidence to support this idea but no definitive proof, and this is an
area of ongoing research in systems biology.

1.2 Graph-Theory-Based Models Allow Us to Infer the

Information-Processing Ability of Signaling Networks
The complexity of signaling networks requires that we have an orderly
approach if we are to understand what these networks do, and how they do it.
For this, in addition to experiments, we need a theoretical framework.
Networks are often studied using a branch of mathematics called graph
theory that analyzes systems composed of objects (called nodes or vertices)
and binary relationships (called edges). First developed by Euler in 1736
(Biggs et al. 1986), graph theory focuses on the combinatorial study of
multiple interactions between numerous components, and it has been very
successfully used in many disciplines, including social sciences. A classic
experiment by Stanley Milgram in 1967 using chain letters showed that any
two persons in the United States are connected by a median of five
interconnected individuals and led to the concept of “six degrees of
separation” (Pool et al. 1989). Statistical analyses have since defined metrics
that provide estimates of overall connectivity and organization within
networks. In general, these focus on computing the relationships between
edges and nodes within the network (see Box 2).

BOX 2. COMPUTATIONAL MODELS OF SIGNALING

NETWORKS

The complexity that arises from having many isoforms of signaling

components, and their differential connectivity, makes it hard to
intuitively understand the organization of signaling networks and to
make accurate predictions of their outputs a priori. However,
computational analysis based on experimental observations could yield
accurate predictions while improving our understanding of the signaling
landscape. Two types of computational approaches are useful: (1)
network models, and (2) dynamical models.

1. Network Models

Network analysis, application of a field of mathematics called graph

theory, typically involves the use of statistical methods to identify
characteristics of a network. So-called network models incorporate an
additional level of complexity, the relationship between the different
entities. Although statistical and network models share many features in
terms of methods used for analysis, the outcomes are different.
Statistical models provide knowledge about relationships within a
system by analyzing the experimental background for significant
correlative changes. Network models provide knowledge about the
organization of the system, in addition to identifying the underlying
relationships. The term used to describe network organization is
topology. The organization of networks can be categorized based on
how the components are connected to one another. There are different
types of networks. In part B in the figure below, three different networks
are shown in decreasing order of clustering coefficients. Small-world
networks have densely connected topologies, in which the typical
distance between two randomly chosen nodes is proportional to the
logarithm of the number of nodes (Barabasi and Albert 1999).
The connectivity between components may be direct (i.e.,
component A chemically interacts with component B) or indirect
(wherein changes in component A modulate changes in component C
without direct chemical interactions). Direct binary relationships allow
tracking of pathways from receptors to effectors, and prediction of distal
relationships. The use of models of network topology to predict the
information-processing capability of the system relies on identification
of small organizing units, called network motifs, such as feedback and
feedforward loops. The smallest fundamental motifs have two to four
interacting components; some of the motifs that are prominently
featured in biological networks are outlined in part B in the figure
below.
 A limitation of graph-theory-based analysis is that it provides a static
view of the system. It tells us how the system is organized, but does not
tell us how such organization enables the system to change with respect
to time on receiving a signal. In signaling networks, every connection
between components in a signaling reaction represents one or more
chemical reactions, whose dynamics are regulated by both the
concentration of the reactants and the rates of the forward and reverse
reactions. Accordingly, connectivity between components does not
automatically imply that signals are propagated through them. To
understand how spatiotemporal reaction dynamics affect signal
propagation, we need a second type of analysis called dynamical
modeling.

2. Dynamical Models

Dynamical models describe how a system changes with respect to time,

based on reaction rates between components. They can be divided into
two classes: (1) deterministic models, in which the time-dependent
change in the state of the system can be predicted from the initial
concentrations of the reactants and the reaction rates; and (2) stochastic
models, in which the trajectory of the system is not fully predictable but
depends on the probability of a set of reactions occurring. Typically,
stochastic models involve systems in which the concentrations of
reactants or the volumes occupied are very small, which decrease or
increase the certainty of interactions, respectively.
Deterministic models usually use ordinary-differential-equation
(ODE)-based representations of biochemical or biophysical interactions.
ODE systems are based on the assumption that system dynamics depend
only on time. These offer a detailed and quantitative picture of
interactions. Using the known parameters of reaction stoichiometry, a
system of ODEs can be formulated to represent the flow of information
within a signaling network. These equations can then be simultaneously
solved as a function of time to reveal the behavior of individual
components as the reactions proceed (see figure above). A typical ODE
model can be used to describe signal flow upon receptor ligand
activation.
Although ODE-based models are common and very valuable as a first
approximation, they suffer from an inherent simplifying assumption that
all components are evenly distributed within the reaction space (i.e.,
time is the only independent variable). This is usually not true within
cells. Simple ODE models can be improved by introducing spatial
information transfer via multiple subcellular compartments such that the
rates at which components move between compartments are specified.
Typically, for a signal flowing from the cell surface to the nucleus, the
cell can be thought of as a three-compartment system comprising the
plasma membrane, cytoplasm, and the nucleus.
A more sophisticated, and complete, way of improving deterministic
models is to use partial differential equations, in which each component
has two associated attributes: specific reaction rates (k) and a diffusion
coefficient (D), which define system behavior as a function of time (t)
and space (x,y), respectively (see figure above). For example, a model of
signal flow from receptor tyrosine kinases to extracellular-regulated
kinase (ERK) to transcription factors involves information flow from the
cell surface membrane through the cytoplasm to the nucleus, and it is
best described by a partial-differential-equation (PDE)-based model.
Although such models provide a more realistic picture of the cell, they
have some drawbacks. For example, diffusion coefficients for most
cellular components have not been measured and they need to be
approximated from molecular weights. In addition, numerical
simulations of PDE-based models remain computationally expensive; so
such models are not widely used. Nevertheless, to fully understand how
signaling reactions occur in localized regions within the cell (especially
for cells with nonstandard geometries) one must use PDE-based models
Sometimes reactions in cells involve components that are present in
very small numbers or do not react in an easily predictable manner. The
most common example is the binding of a transcription factor to the
promoter of a gene. To model such an event accurately, we need to
consider the probability of the interaction occurring as a function of
time, volume, and number of molecules. Stochastic models incorporate
these parameters, and use Gillespie’s algorithm as the standard approach
for computations (Gillespie 2007). These models combine individual
probabilities of interactions at the molecular level under given
circumstances (e.g., probability of binding of a transcription factor to a
promoter given its concentration and volume constraints). In theory,
they can provide highly accurate representations of biochemical events;
however, because of the need for a large number of operations per unit
time, they tend to be computationally expensive even with small
spatiotemporal constraints. In addition, because of their random nature,
in most cases, they require Monte Carlo simulations that use random
sampling to determine how the system changes with respect to time or
space. Such models are also often difficult to develop owing to a paucity
of experimental data. Hence, stochastic models are not as commonly
used as deterministic models to describe large systems but could become
more popular in the future as more information becomes available.
 Signaling networks, like many other networks, are scale-free. This means
the ratio of edges to nodes follows a power law rather than a Gaussian
distribution (Barabasi and Albert 1999). In a typical network, there are a
small number of nodes with many edges, called hubs. In contrast, most nodes
have only few edges. Distribution of highly and sparsely connected nodes is
not evenly spaced. Interconnectivity between nodes is also not evenly
distributed with the overall networks. A metric called the clustering
coefficient can be used to compute the extent of interconnectivity. This is
used to identify highly or sparsely connected regions within a network, which
allows us to identify clusters or subnetworks within the larger network (see
Box 2).
The local topology within a signaling network can have numerous effects
on its input–output relationships. Signals may be amplified, dampened,
prolonged or shortened; such behavior is controlled by ubiquitously repeating
patterns of connectivity called regulatory motifs. Several classes of regulatory
motifs are observed in biological networks (Alon 2007). These include
feedforward and feedback loops and bi-fan motifs (see Box 2).
The feedforward motif has two arms from a starting node to a target node.
A good example is the signal from the MAP kinase ERK to the transcription
factor Fos (Fig. 3A). ERK stimulates transcription of the Fos gene and
consequently production of Fos protein, which it can also phosphorylate.
Because the phosphorylated form of Fos is protected from degradation, the
feedforward loop prolongs the signal from the ERK pathway. Extending
signal output is one feature of a feedforward motif. Another is redundancy,
which can make a system robust against perturbations (see below).
Feedforward loops in which all the relationships are of the same sign (e.g., all
stimulatory) are called coherent feedforward loops, and they can be used for
additive computation within the signaling network. When two arms of the
starting node have opposing effects, the motif is called an incoherent
feedforward loop. Incoherent feedforward loops can induce accelerated
transient output following a step input (Mangan and Alon 2003), which could
be useful for rapid cellular response.
Figure 3. Regulatory motifs in cell signaling networks. (A) A representative coherent feedforward
motif depicting signal flow from ERK to the transcription factor Fos, where both transcriptional
activation and posttranslational stabilization lead to prolonged signal duration. (B) Negative feedback
through β-adrenergic receptor (β-AR)-activated PKA limits the duration of response. (C) Positive-
feedback loops within ERK-PLA2-PKC pathways can lead to bistability. (D) Kinases JNK and p38
phosphorylate and activate both of the transcription factors ATF2 and Elk1, forming a bi-fan motif that
provides tight regulation, including temporal control and coincidence detection.

Feedback loops are common regulatory features of biochemical pathways

and networks. They can be both positive and negative. In negative-feedback
loops, a downstream component inhibits an upstream stimulator. This type of
regulation is very common in metabolic pathways, where a downstream
metabolite will allosterically inhibit an upstream enzyme to reduce flux. It is
also found in signaling. Phosphorylation and deactivation of the β-adrenergic
receptor by PKA to limit the duration of responses to the hormone
epinephrine is a classic example (Fig. 3B). Positive-feedback loops are also
found in signaling pathways. An example is the interaction between ERK and
phospholipase A2 (PLA2), which results in the activation of PLA2 (probably
via activation of MNK1/2, which phosphorylates PLA2 at S727), leading to
the increased production of arachidonic acid. Diacylglycerol and arachidonic
acid together can activate protein kinase C (PKC). This positive-feedback
loop results in prolonged activation of PKC as well as ERK (Fig. 3C). Such
positive-feedback loops can function as switches to move the signaling
network from an inactive to persistently active state.
Bi-fan motifs form another class of abundant motifs found in signaling
networks, especially at the interface between protein kinases and transcription
factors. Most protein kinases phosphorylate multiple transcription factors,
and many transcription factors are phosphorylated by multiple protein
kinases. The JNK and p38 MAP kinases and the transcription factors ATF2
and Elk1 are shown in Figure 3D. This crisscrossing allows a cell to integrate
multiple signals to give a cohesive pattern of gene expression. In the case of
Elk1 and ATF2, for example, the motif enables upstream kinases to
effectively synchronize the activity levels of the transcription factors.

2 EMERGENT PROPERTIES OF SIGNALING

NETWORKS
The organization of a signaling pathway and its regulatory motifs can endow
a signaling network with emergent properties that the individual components
by themselves do not possess. These are difficult to predict from examination
of any one of the components.

2.1 Ultrasensitivity
Sometimes a stimulus, such as receptor activation, can produce a switchlike
response in a downstream signaling component. In such a system, at a certain
point called the threshold, a small change in the ligand/receptor can cause a
large change in the activity of a downstream effector. Such responses are
called ultrasensitive. This was first observed with coupled enzymes switching
between activity states (Goldbeter and Koshland 1981). Subsequently, similar
ultrasensitivity has been observed in control of the cell cycle by the ERK
pathway, where small changes in stimulus cause large changes in ERK
activity (Ferrell and Machleder 1998). Ultrasensitivity can be produced by
several mechanisms such as cooperativity, multistep regulation as seen in the
ERK pathway, and by changing the levels of activators to inhibitors of a
signaling component such as a GTPase (Lipshtat et al. 2010).

2.2 Bistability
The ability of positive-feedback loops like the one involving PKC, ERK, and
PLA2 (Fig. 3C) to function as switches is an emergent property called
bistability. ODE-based models (see Box 2) of the ERK-PLA2-PKC pathway
(Bhalla and Iyengar 1999) allow one to plot the activity of ERK as a function
of protein kinase C activity and vice versa (Fig. 4). The two curves intersect
three times. In this system, if the initial stimulus is above the threshold level
(the middle intersection point) needed to increase activity of either protein
kinase C or ERK, the system will move from the lower intersection point
(basal state) to the upper intersection point (active state) for a prolonged
period. The upper and lower intersection points represent the two stable
states. The middle intersection point represents the level above which the
initial stimulus engages the feedback loop and moves the system to the
activated equilibrium state. Otherwise, when the stimulus is withdrawn the
system will revert to its basal equilibrium state. The computational modeling
analysis shows that the connectivity alone is insufficient for the switching
behavior. The concentrations of the signaling components and the reaction
rates are critical in ensuring that a feedback loop with this topology functions
as a bistable switch.
Figure 4. Activity states of components in a positive-feedback loop can be plotted as functions of each
other to study the characteristics of a bistable system that switches from an inactive basal state (state 1)
to an active state (state 2) once a threshold is reached. For example, when the concentration of active
MAPK or PKC is above the threshold levels upon growth factor receptor activation, the system moves
to state 2. The plot is obtained by computational modeling of the positive-feedback loop in Figure 3C,
and recording the steady-L-state levels of active PKC for a given fixed concentration of active MAPK
(black dashed line) upon stimulation, and vice versa (red solid line).

Bistability is involved in triggering numerous biological processes. The

positive ERK-PLA2-PKC-feedback loop described above functions as a
switch in cerebellar neurons, where it converts brief electrical signals into
long-lasting changes in cellular responsiveness called long-term depression
(Tanaka and Augustine 2008). In another example, during cell replication, a
positive-feedback loop confers bistability on cyclin B expression, enabling
the cell to enter mitosis (Sha et al. 2003). Similarly, programmed cell death is
controlled by two cooperative positive-feedback loops, which confer
bistability on mitochondrial permeability initiated by caspase-3 (Bagci et al.
2006; Ch. 19 [Green and Llambi 2014]).

2.3 Redundancy and Robustness

Because most signaling pathways are critical for survival, unsurprisingly,
cells often use multiple pathways to connect a single input with an output.
For example, activated epidermal growth factor receptors (EGFRs)
simultaneously activate PKC (via tyrosine phosphorylation of PLCγ and
generation of diacylglycerol [Ch. 3 (Newton et al. 2014)]) and the small G
protein Ras (via formation of Grb2-SOS adapter-GEF complexes), both of
which feed into the ERK pathway (p. 81 [Morrison 2012]). As shown in
Figure 1A, this redundancy provides a safety net for activation of key
processes that are necessary for cell survival or homeostasis. It further allows
signaling networks to withstand perturbations that can alter input–output
relationships, and it can ensure that cells respond only to stimuli of
appropriate duration and magnitude, increasing robustness.
Network topology plays an important role in the robustness of signaling
systems. Coherent feedforward motifs (see Box 2), for example, can be used
in transcriptional regulatory networks as “persistence monitors” that respond
to only sustained stimuli (Mangan and Alon 2003). Biochemical properties of
signaling components such as a high catalytic rate or the ability to be altered
by posttranslational modification are also major contributors to robustness of
signaling networks.

2.4 Oscillatory Behavior

Oscillatory phenomena occur in cellular events from cell division to circadian
rhythms. They generally involve negative-feedback loops; coupled positive-
and negative-feedback loops can also lead to sustained oscillations (Tyson
and Novak 2010). The cell cycle, for example, requires the cyclic expression
and degradation of cyclins (Ch. 5 [Duronio and Xiong 2013]). Control of the
cell cycle in Xenopus oocytes was one of the first oscillatory biological
systems to be modeled with numerical simulations (Novak and Tyson 1993).
Two feedback loops, one on top of the other, operate in the Xenopus oocyte
cell cycle. In this case, a negative-feedback loop is necessary and sufficient
for the oscillations of the driver, cyclin-CDK complex, but a positive-
feedback loop dampens and synchronizes the cycles and is necessary for
systems-level physiological operations (Pomerening et al. 2005).
3 INFORMATION FLOW AND PROCESSING
3.1 Information in Signaling Network Is Contextual
Although most cells share a multitude of common signaling components,
identical signals can lead to diverse biological responses from different cell
types. For instance, a pathway could have different outputs in two different
cell types because they have different numbers or types of receptors.
A good example is a small peptide hormone known by two names:
vasopressin (VP) and antidiuretic hormone (ADH). The two names arise
because the hormone has very different physiological effects in two different
cell types. In smooth muscle cells, it binds to V1 receptors that couple
through the G protein Gq and calcium to regulate contractility, leading to
contraction of blood vessels. In kidney cells, the same hormone binds to V2
receptors that couple through Gs and cAMP to regulate water uptake, leading
to an antidiuretic effect. Thus, information in the hormone molecule is very
differently interpreted by the two different cell types in different tissues in the
body.
Such contextual dependence of information can occur within the cell as
well. For example, in neurons, cAMP can bind to and activate PKA, leading
to effects on metabolic enzymes, gene expression, and channel activity.
cAMP can also directly bind to and gate a class of cation channels called
HCNs. Because the affinity of cAMP for the HCN channels is substantially
less than its affinity for PKA and because these channels are largely present
in the distal dendrites, the information in cAMP can be selectively
transmitted to metabolic enzymes and gene regulation without substantially
affecting the electrical behavior of the neurons. In effect, cAMP at low
concentrations represents information for PKA but no information for HCN
channels, because the lower affinity precludes the channels from binding
cAMP. In considering information flow within the signaling network, we
therefore always need to know the characteristics of both the transmitter of
information and the receiver.

3.2 Information Transmitted Can Be Proportional to the

Stimulus, Processed, or Dissipated
Often the subcellular localization of the components of a signaling network
and their relative affinities ensure that information flows proportionally (e.g.,
linearly) from receptor to effector. Typically, when the strength of the
extracellular signal is such that only a small proportion of receptors are
activated, the resultant downstream responses are proportional to the level of
receptor activation. In some cases, however, network topology, spatial
localization of the signaling components, and biochemical characteristics of
individual components can lead to information processing. Enzymatic
reactions, for example, often amplify signals, depending on the catalytic rate
of the enzyme and its ability to activate multiple targets (Fig. 1B).
Conversely, signal flow that involves adaptor proteins or other binding
reactions such as scaffold proteins is less likely to amplify information
because these interactions are stoichiometric. Adaptors and scaffolds play a
critical role in signal processing, however, adding spatial constraints to
information flow and defining bidirectional specificity. Network topology
such as positive-feedback loops and coherent feedforward loops can increase
the amplitude as well as the duration of output. Amplification by any of these
mechanisms can increase sensitivity of the response or reduce responses to
small or transient stimuli.
For certain signaling pathways that are ultrasensitive, dissipation of
biological information is just as important as amplification. Prolonged
signaling may lead to cell death or disease states. Drug-induced calcium
excitotoxicity, which kills neurons, and mutated signaling proteins that lead
to neoplasia, provide good examples of the deleterious effects of aberrant
signals. Hence, signal dissipation is critical for proper functioning of
signaling pathways. A range of negative regulators act on receptors,
GTPases, effectors, and protein kinase signals to control and dissipate them
before they can reach downstream effectors and produce physiological
responses. Receptors are negatively regulated by protein kinases,
phosphatases, and binding proteins called arrestins. Almost all receptors
undergo desensitization when the cell is exposed to extracellular signals for
prolonged periods (Ch. 1 [Heldin et al. 2014]). Desensitization often involves
these proteins. Similarly, GTPases are deactivated by GAPs, and the effects
of protein kinases are tightly controlled by protein phosphatases that
antagonize them. The activities of effectors such as ACs are controlled by
degradation of their products (in the case of AC, the second messenger cAMP
by phosphodiesterases). There is a large family of phosphodiesterases that is
subject to differential regulation by protein kinases, scaffolds, and by
calcium/calmodulin. Both GAPs and phosphatases can be regulated by
protein kinases and scaffolds as well. Thus, every positive signal generator in
the cell has a negative counterpart that allows signal dissipation as needed.

3.3 Signaling Pathways and Networks Can Filter Noise

The coupling of positive and negative components allows signaling networks
to filter noise in their inputs. Thus, low-level or transient signals may activate
a few upstream signaling reactions but generate no cellular response (e.g.,
gene expression). A number of regulatory motifs improve signal-to-noise
properties of networks. As described above, positive-feedback loops that
function as switches can filter out subthreshold stimuli as noise. Feedforward
motifs and interlinked feedback loops can also be configured so that they
respond to sustained stimuli but ignore transient stimuli as noise (Brandman
et al. 2005). Bi-fans that require activation of both upstream components to
produce downstream effects function as AND gates (Fig. 3D). Such a
network motif can function as a double coincidence detector that requires
multiple upstream signals to activate downstream components. Such a
configuration can help prevent activation of downstream events such as
transcription when the transcription factor is phosphorylated by only one
protein kinase (i.e., only one signal is received) (Lipshtat et al. 2008).

4 CONCLUDING REMARKS
Signaling networks in cells produce outputs that are manifested as decisions
to perform physiological functions in response to biochemical, electrical, or
mechanical stimuli. These outputs are most often controlled by protein
kinases in the cytoplasm and near the plasma membrane. These protein
kinases phosphorylate and regulate metabolic enzymes, channels,
transporters, and components of the cytoskeletal machinery. In the nucleus,
the targets are typically transcription factors and proteins that control
chromosomal organization and dynamics. The outputs, whether they are
production of glucose from glycogen by liver cells in response to
epinephrine, firing of neurons in response to neurotransmitters, or hormone-
driven changes in gene expression of ovarian cells, reflect both the
characteristics (amplitude and duration) of the external signals and
information processing within signaling networks. The ability to balance
these allows the cell to respond to varying stimuli and return to homeostatic
balance.
When signaling components are inappropriately activated or inactivated
(e.g., by mutations), however, the information-processing capability of the
networks is also altered. This may lead to sustained changes in gene
expression that push the cell into a different state, for example, an enhanced
rate of proliferation, which is often detrimental and can cause diseases such
as cancer (Ch. 21 [Sever and Brugge 2014]). Similarly, bacterial toxins can
cause disease by altering components within signaling networks (Ch. 20
[Alto and Orth 2012]). Both examples show how critical it is for us to
understand the organization and information-processing capability of
signaling networks.

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drive reliable cell decisions. Science 310: 496–498.
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005934
SECTION II

SIGNALING PATHWAYS
MAP Kinase Pathways

Deborah K. Morrison
Laboratory of Cell and Developmental Signaling, National Cancer Institute,
Frederick, Maryland 21702
Correspondence: morrisod@mail.nih.gov

Mitogen-activated protein kinase (MAPK) modules containing three

sequentially activated protein kinases are key components of a series of vital
signal transduction pathways that regulate processes such as cell
proliferation, cell differentiation, and cell death in eukaryotes from yeast to
humans (Fig. 1) (Qi and Elion 2005; Raman et al. 2007; Keshet and Seger
2010). Each cascade is initiated by specific extracellular cues and leads to
activation of a particular MAPK following the successive activation of a
MAPK kinase kinase (MAPKKK) and a MAPK kinase (MAPKK) (Fig. 1).
The MAPKKK is typically activated by interactions with a small GTPase
and/or phosphorylation by protein kinases downstream from cell surface
receptors (Cuevas et al. 2007). The MAPKKK directly phosphorylates and
activates the MAPKK, which, in turn, activates the MAPK by dual
phosphorylation of a conserved tripeptide TxY motif in the activation
segment. Once activated, the MAPK phosphorylates diverse substrates in the
cytosol and nucleus to bring about changes in protein function and gene
expression that execute the appropriate biological response. MAPKs
generally contain docking sites for MAPKKs and substrates, which allow
high-affinity protein–protein interactions to ensure both that they are
activated by a particular upstream MAPKK (Bardwell and Thorner 1996) and
that they recognize specific downstream targets (Tanoue and Nishida 2003).
 Figure 1. MAPK pathways.

The MAP kinases can be grouped into three main families. In mammals,
these are ERKs (extracellular-signal-regulated kinases), JNKs (Jun amino-
terminal kinases), and p38/SAPKs (stress-activated protein kinases). ERK
family members possess a TEY motif in the activation segment and can be
subdivided into two groups: the classic ERKs that consist mainly of a kinase
domain (ERK1 and ERK2) and the larger ERKs (such as ERK5) that contain
a much more extended sequence carboxy-terminal to their kinase domain
(Zhang and Dong 2007). The classic ERK1/2 module (Fig. 2) responds
primarily to growth factors and mitogens to induce cell growth and
differentiation (McKay and Morrison 2007; Shaul and Seger 2007).
Important upstream regulators of this module include cell surface receptors,
such as receptor tyrosine kinases (RTKs), G-protein-coupled receptors
(GPCRs), and integrins, as well as the small GTPases Ras and Rap.
MAPKKs for the classic ERK1/2 module are MEK1 and MEK2, and the
MAPKKKs include members of the Raf family, Mos, and Tpl2.
 Figure 2. The ERK MAPK pathway.

JNK family members contain a TPY motif in the activation segment and
include JNK1, JNK2, and JNK3. The JNK module (Fig. 3) is activated by
environmental stresses (ionizing radiation, heat, oxidative stress, and DNA
damage) and inflammatory cytokines, as well as growth factors, and
signaling to the JNK module often involves the Rho family GTPases Cdc42
and Rac (Johnson and Nakamura 2007). The JNK module plays an important
role in apoptosis, inflammation, cytokine production, and metabolism
(Dhanasekaran and Reddy 2008; Huang et al. 2009; Rincon and Davis 2009).
MAPKKs for the JNK module are MKK4 and MKK7, and the MAPKKKs
include MEKK1 and MEKK4, MLK2 and MLK3, ASK1, TAK1, and Tpl2.

Figure 3. The JNK MAPK pathway.

 p38 family members possess a TGY motif in the activation segment and
include p38α, p38β, p38γ, and p38δ. Like JNK modules, p38 modules (Fig.
4) are strongly activated by environmental stresses and inflammatory
cytokines. p38 activation contributes to inflammation, apoptosis, cell
differentiation, and cell cycle regulation (Cuenda and Rousseau 2007;
Cuadrado and Nebreda 2010). The primary MAPKKs for p38 modules are
MKK3 and MKK6, and the MAPKKKs include MLK2 and MLK3, MEKKs,
ASKs, TAK1, and TAO1 and TAO2. Important substrates in p38 signaling
include the downstream kinases MK2/3, PRAK, and MSK1 and MSK2, as
well as various transcription factors.
 Figure 4. The p38 MAPK pathway.

For all of the MAPK modules, specific scaffold proteins (Good et al.
2011) have been identified that dock at least two of the core kinases of the
module. These scaffolds contribute to MAPK signaling by increasing the
local concentration of the components, providing spatial temporal regulation
of cascade activation, and/or localizing the module to specific cellular sites or
substrates. Scaffold proteins involved in MAPK cascade signaling include
KSR and MP1 for the ERK module; JIP1, JIP2, JIP3, JIP4, and POSH for the
JNK module; and JIP2, JIP4, and OSM for the p38 module (Dhanasekaran et
al. 2007).
Figure 1 adapted, with permission, from Cell Signaling Technology (http://www.cellsignal.com).

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Bardwell L, Thorner J. 1996. A conserved motif at the amino termini of MEKs might mediate high-
affinity interaction with the cognate MAPKs. Trends Biochem Sci 21: 373–374.
Cuadrado A, Nebreda AR. 2010. Mechanisms and functions of p38 MAPK signalling. Biochem J 429:
403–417.
Cuenda A, Rousseau S. 2007. p38 MAP-kinases pathway regulation, function and role in human
diseases. Biochim Biophys Acta 1773: 1358–1375.
Cuevas BD, Abell AN, Johnson GL. 2007. Role of mitogen-activated protein kinase kinase kinases in
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Johnson GL, Nakamura K. 2007. The c-Jun kinase/stress-activated pathway: Regulation, function and
role in human disease. Biochim Biophys Acta 1773: 1341–1348.
Keshet Y, Seger R. 2010. The MAP kinase signaling cascades: A system of hundreds of components
regulates a diverse array of physiological functions. Methods Mol Biol 661: 3–38.
McKay MM, Morrison DK. 2007. Integrating signals from RTKs to ERK/MAPK. Oncogene 26: 3113–
3121.
Qi M, Elion EA. 2005. MAP kinase pathways. J Cell Sci 118: 3569–3572.
Raman M, Chen W, Cobb MH. 2007. Differential regulation and properties of MAPKs. Oncogene 26:
3100–3112.
Rincon M, Davis RJ. 2009. Regulation of the immune response by stress-activated protein kinases.
Immunol Rev 228: 212–224.
Shaul YD, Seger R. 2007. The MEK/ERK cascade: From signaling specificity to diverse functions.
Biochim Biophys Acta 1773: 1213–1226.
Tanoue T, Nishida E. 2003. Molecular recognitions in MAP kinase cascades. Cell Signal 15: 455–462.
Zhang Y, Dong C. 2007. Regulatory mechanisms of mitogen-activated kinase signaling. Cell Mol Life
Sci 64: 2771–2789.
Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011254
The PI3K-PKB/Akt Pathway

Brian A. Hemmings and David F. Restuccia

Friedrich Miescher Institute for Biomedical Research, Basel 4058,
Switzerland
Correspondence: david.restuccia@fmi.ch

Identification of the phosphoinositide-3-kinase–protein kinase B/Akt (PI3K-

PKB/Akt) pathway and activating receptor tyrosine kinases (RTKs) began in
earnest in the early 1980s through vigorous attempts to characterize insulin
receptor signaling (for review, see Alessi 2001; Brazil and Hemmings 2001).
These humble beginnings led to the identification of the components and
mechanism of insulin receptor signaling via insulin receptor substrate (IRS)
proteins to PI3K and consequent PKB/Akt-mediated activation by 3-
phosphoinositide-dependent protein kinase 1 (PDK1). With the discovery of
the potent contribution of PI3K and PKB/Akt activation to tumorigenesis,
intense research into the regulation of this pathway led to the discovery of the
negative regulators, the protein phosphatase 2 (PP2A), phosphatase and
tensin homolog (PTEN), and the PH-domain leucine-rich-repeat-containing
protein phosphatases (PHLPP1/2). More recently, the elusive PKB/Akt
hydrophobic motif kinases—i.e., the mammalian target of rapamycin
(mTOR), when associated with the mTOR complex 2 (mTORC2), and the
DNA-dependent protein kinase (DNA-PK)—were identified, as was the
ability of Ras to affect the PI3K-PKB/Akt pathway via PI3K, completing our
current model of the PI3K-PKB/Akt pathway.
The PI3K-PKB/Akt pathway is highly conserved, and its activation is
tightly controlled via a multistep process (as shown in Fig. 1) Activated
receptors directly stimulate class 1A PI3Ks bound via their regulatory subunit
or adapter molecules such as the insulin receptor substrate (IRS) proteins.
This triggers activation of PI3K and conversion by its catalytic domain of
phosphatidylinositol (3,4)-bisphosphate (PIP2) lipids to phosphatidylinositol
(3,4,5)-trisphosphate (PIP3). PKB/Akt binds to PIP3 at the plasma membrane,
allowing PDK1 to access and phosphorylate T308 in the “activation loop,”
leading to partial PKB/Akt activation (Alessi et al. 1997). This PKB/Akt
modification is sufficient to activate mTORC1 by directly phosphorylating
and inactivating proline-rich Akt substrate of 40 kDa (PRAS40) and tuberous
sclerosis protein 2 (TSC2) (Vander Haar et al. 2007). mTORC1 substrates
include the eukaryotic translation initiation factor 4E binding protein 1
(4EBP1), and ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (S6K1),
which, in turn, phosphorylates the ribosomal protein S6 (S6/RPS6),
promoting protein synthesis and cellular proliferation.

Figure 1. PKB/Akt activation downstream of RTKs via the P13K pathway.

Phosphorylation of Akt at S473 in the carboxy-terminal hydrophobic

motif, either by mTOR (Sarbassov et al. 2005) or by DNA-PK (Feng et al.
2004), stimulates full Akt activity. Full activation of Akt leads to additional
substrate-specific phosphorylation events in both the cytoplasm and nucleus,
including inhibitory phosphorylation of the pro-apoptotic FOXO proteins
(Guertin et al. 2006). Fully active PKB/Akt mediates numerous cellular
functions including angiogenesis, metabolism, growth, proliferation, survival,
protein synthesis, transcription, and apoptosis (as shown in Fig. 2).
Dephosphorylation of T308 by PP2A (Andjelković et al. 1996), and S473 by
PHLPP1/2 (Brognard et al. 2007), and the conversion of PIP3 to PIP2 by
PTEN (Stambolic et al. 1998) antagonize Akt signaling.
 Figure 2. Signalling events activating PKB/Akt and cellular functions regulated by PKB/Akt.

Figures adapted, with kind permission, from Cell Signaling Technology (http://www.cellsignal.com).

REFERENCES
Alessi DR. 2001. Discovery of PDK1, one of the missing links in insulin signal transduction. Colworth
Medal Lecture. Biochem Soc Trans 29: 1–14.
Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, Cohen P. 1997.
Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and
activates protein kinase Bα. Curr Biol 7: 261–269.
Altomare DA, Testa JR. 2005. Perturbations of the AKT signaling pathway in human cancer. Oncogene
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Andjelković M, Jakubowicz T, Cron P, Ming XF, Han JW, Hemmings BA. 1996. Activation and
phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB)
promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci 93: 5699–5704.
Bozulic L, Hemmings BA. 2009. PIKKing on PKB: Regulation of PKB activity by phosphorylation.
Curr Opin Cell Biol 21: 256–261.
Brazil DP, Hemmings BA. 2001. Ten years of protein kinase B signalling: A hard Akt to follow.
Trends Biochem Sci 26: 657–664.
Brognard J, Sierecki E, Gao T, Newton AC. 2007. PHLPP and a second isoform, PHLPP2,
differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol Cell
25: 917–931.
Feng J, Park J, Cron P, Hess D, Hemmings BA. 2004. Identification of a PKB/Akt hydrophobic motif
Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 279: 41189–41196.
Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M, Fitzgerald KJ,
Sabatini DM. 2006. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals
that mTORC2 is required for signaling to Akt-FOXO and PKCα, but not S6K1. Dev Cell 11: 859–
871.
Manning BD, Cantley LC. 2007. AKT/PKB signaling: Navigating downstream. Cell 129: 1261–1274.
Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. 2005. Phosphorylation and regulation of Akt/PKB
by the rictor–mTOR complex. Science 307: 1098–1101.
Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM,
Siderovski DP, Mak TW. 1998. Negative regulation of PKB/Akt-dependent cell survival by the
tumor suppressor PTEN. Cell 95: 29–39.
Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. 2007. Insulin signalling to mTOR
mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9: 316–323.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011189
mTOR Signaling

Mathieu Laplante and David M. Sabatini

Centre de Recherche de l’Institut Universitaire de Cardiologie et de
Pneumologie de Québec (CRIUCPQ), Université Laval, Québec G1V 4G5,
Canada
Correspondence: mathieu.laplante@criucpq.ulaval.ca

The scarcity of nutrients/energy interspersed with sporadic periods of

abundance means that cells must transition between anabolic and catabolic
states. An important protein that has evolved to respond to this need is the
target of rapamycin (TOR). TOR is a well-conserved serine/threonine kinase
that plays an important role in the signaling network that controls growth and
metabolism in response to environmental cues. As its name indicates, TOR is
the target of a molecule named rapamycin, an anti-fungal macrolide produced
by the bacterial species Streptomyces hygroscopicus, which was isolated
from a soil sample from the Eastern Islands in the 1970s (Vezina et al. 1975).
In addition to its anti-fungal properties, rapamycin strongly inhibits cell
growth and proliferation, making this molecule a valuable tool to study cell
growth control. In the early 1990s, yeast genetic screens led to the
identification of TOR as one mediator of the toxic effect of rapamycin in
yeast (Heitman et al. 1991; Kunz et al. 1993). Shortly after, the mammalian
TOR (mTOR), now officially known as the mechanistic TOR, was identified
as the physical target of rapamycin (Fig. 1) (Brown et al. 1994; Sabatini et al.
1994; Sabers et al. 1995).
 Figure 1. A simplified view of the mTOR pathway.

mTOR interacts with many proteins to form at least two distinct

multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex
2 (mTORC2) (Zoncu et al. 2011). In addition to their different protein
compositions, the mTOR complexes have important differences in their
sensitivities to rapamycin, in the upstream signals they integrate, in the
substrates they regulate, and in the biological processes they control
(Laplante and Sabatini 2009b). mTORC1 is sensitive to rapamycin and
integrates inputs from at least five major signals—growth factors, genotoxic
stress, energy status, oxygen, and amino acids—to regulate many processes
involved in the promotion of cell growth and proliferation (Fig. 2). With the
exception of amino acids, all of these inputs regulate mTORC1 activity by
modulating the activity of TSC1–TSC2. TSC1–TSC2 negatively regulates
mTORC1 activity by converting Rheb into its inactive GDP-bound state.
Growth factors block TSC1–TSC2 activity and activate mTORC1, whereas
genotoxic stress, energy deficit, and oxygen deprivation promote TSC1–
TSC2 action and inhibit mTORC1. Conversely, amino acids activate
mTORC1 through the action of the Rag GTPases. In the presence of amino
acids, the Rag GTPases interact with mTORC1, which promotes its
translocation from the cytoplasm to lysosomal membranes, where Rheb is
thought to reside.
 Figure 2. The mTOR pathway.

When activated, mTORC1 promotes protein synthesis mainly by

phosphorylating the kinase S6K and the translation regulator 4E-BP1 (Ma
and Blenis 2009). This complex also induces lipid biogenesis by activating
SREBP1 and PPARγ transcription factors (Laplante and Sabatini 2009a). In
addition to promoting anabolism, mTORC1 also inhibits catabolism by
blocking autophagy through the phosphorylation of the ULK1–Atg13–
FIP200 complex. Importantly, sustained mTORC1 activation blocks growth
factor signaling by activating many negative-feedback loops that inhibit PI3-
kinase (PI3K) signaling.
Compared with mTORC1, our knowledge of mTORC2 biology is still
very limited. mTORC2 is activated by growth factors through a mechanism
that is not well understood. It is insensitive to acute treatment with rapamycin
and regulates cell survival, metabolism, and cytoskeletal organization by
phosphorylating many AGC kinases, including Akt, SGK1, and PKCα
(Laplante and Sabatini 2009b; Zoncu et al. 2011).
Figures adapted from Laplante and Sabatini (2009b).

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protein targeted by G1-arresting rapamycin-receptor complex. Nature 369: 756–758.
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011593
Calcium Signaling

Martin D. Bootman
The Babraham Institute Babraham Research Campus, Cambridge CB22 3AT,
United Kingdom
Correspondence: martin.bootman@bbsrc.ac.uk

Calcium ions regulate processes as diverse as cell motility, gene transcription,

muscle contraction, and exocytosis (Berridge et al. 2000). The first
realization that they are critical for cellular function is often attributed to
Sydney Ringer, who discovered in 1883 that saline solution made up using
London tap water (which contained calcium) supported the contraction of
isolated frog hearts, whereas saline made up using distilled water (which
lacked calcium) could not. Subsequent work revealed that numerous cell
biological processes are controlled by calcium (Carafoli 2003). Particularly
important was the discovery in the 1950s that calcium triggers skeletal
muscle contraction by binding to troponin C and that calcium can be
sequestered in the sarcoplasmic reticulum. These studies led to the notion that
calcium signals inside cells oscillate: the cytoplasmic calcium concentration
increases, a particular effector is activated, and then the calcium signal is
reversed to reset the system (see Figs. 1 and 2). Cells use a “toolkit” of
channels, pumps, and cytosolic buffers to control calcium levels (Berridge et
al. 2000). Numerous proteins are modulated directly or indirectly by calcium.
These include kinases and phosphatases, transcription factors such as NF-AT,
and the ubiquitous calcium-binding protein calmodulin (CaM).
Figure 1. Calcium signaling (simplified view).
 Figure 2. Calcium signaling.

Electrical, hormonal, and mechanical stimulation of cells can produce

calcium signals by causing entry of the ion across the plasma membrane or its
release from intracellular stores. Binding of hormones to G-protein-coupled
receptors (GPCRs), for example, leads to generation of the second messenger
inositol 1,4,5-trisphosphate (IP3), which releases calcium from intracellular
stores such as the endoplasmic reticulum. By contrast, electrical or
neurotransmitter stimulation of neurons causes calcium to enter cells from
outside via channels in the plasma membrane. This can increase the average
cytosolic calcium concentration from around 100 nM to around 1 µM. Close
to an active channel the calcium concentration can reach tens of micromolar.
Such local hot-spots of calcium are used by cells to activate specific process
that are generally not sensitive to the bulk cytosolic calcium concentration
(Bootman et al. 2001).
Cellular calcium signaling proteomes are tissue-specific, producing
unique calcium signals that suit a tissue’s physiology (Berridge et al. 2003).
For example, cardiac myocytes require a rapid (hundreds of milliseconds)
whole-cell calcium transient to trigger contraction every second (Bers 2002),
whereas cells that are not electrically excitable typically display calcium
oscillations that last for tens of seconds, and can have a periodicity of several
minutes, to control gene expression and metabolism (Dupont et al. 2011).
The rapid calcium signals within myocytes are caused by calcium entering
through voltage-activated calcium channels in the plasma membrane, which
then triggers calcium release via ryanodine receptors on the sarcoplasmic
reticulum. The slower calcium signals in nonexcitable cells typically rely on
IP3, which binds to channels (InsP3Rs) on the endoplasmic reticulum, or
potentially nicotinic acid adenine dinucleotide phosphate-gated calcium
channels (two pore channels) on acidic organelles, leading to release of
calcium into the cytoplasm (Galione 2011). Calcium signals can also pass
through gap junctions to coordinate activities of neighboring cells (Sanderson
et al. 1994).
The actions of calcium can be mediated by direct binding of calcium to
effectors, such as the phosphatase calcineurin (Berridge 2006). Alternatively,
it can act via the ubiquitous calcium-binding protein CaM. The interaction of
calcium with CaM leads to a rearrangement of the protein that allows it to
bind and allosterically regulate target molecules such as the
calcium/calmodulin-dependent kinases CaMKII and CaMKIV. CaM is
mobile within cells and can associate with its targets after binding calcium.
However, in some cases, it is prebound to its target, which provides rapid
control. Ultimately, calcium signals are reversed by the action of pumps such
as the sarco/endoplasmic reticulum ATPases (SERCA) that return it from the
cytosol to intracellular stores or the external milieu.
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remodelling. Nat Rev Mol Cell Biol 4: 517–529.
Bers DM. 2002. Cardiac excitation-contraction coupling. Nature 415: 198–205.
Bootman MD, Lipp P, Berridge MJ. 2001. The organisation and functions of local Ca2+ signals. J Cell
Sci 114: 2213–2222.
Carafoli E. 2003. The calcium-signalling saga: Tap water and protein crystals. Nat Rev Mol Cell Biol 4:
326–332.
Dupont G, Combettes L, Bird GS, Putney JW. 2011. Calcium oscillations. Cold Spring Harb Perspect
Biol 3: a004226.
Galione A. 2011. NAADP receptors, Cold Spring Harb Perspect Biol 3: a004036.
Sanderson MJ, Charles AC, Boitano S, Dirksen ER. 1994. Mechanisms and function of intercellular
calcium signaling. Mol Cell Endocrinol 98: 173–187.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011171
The Cyclic AMP Pathway

Paolo Sassone-Corsi
Center for Epigenetics and Metabolism, School of Medicine, University of
California, Irvine, California 92697
Correspondence: psc@uci.edu

Cyclic adenosine 3′,5′-monophosphate (cAMP) was the first second

messenger to be identified and plays fundamental roles in cellular responses
to many hormones and neurotransmitters (Sutherland and Rall 1958). The
intracellular levels of cAMP are regulated by the balance between the
activities of two enzymes (see Fig. 1): adenylyl cyclase (AC) and cyclic
nucleotide phosphodiesterase (PDE). Different isoforms of these enzymes are
encoded by a large number of genes, which differ in their expression patterns
and mechanisms of regulation, generating cell-type and stimulus-specific
responses (McKnight 1991).
 Figure 1. PKA regulation.

Most ACs (soluble bicarbonate-regulated ACs are the exception) are

activated downstream from G-protein-coupled receptors (GPCRs) such as the
β adrenoceptor by interactions with the α subunit of the Gs protein (αs). αs is
released from heterotrimeric αβγ G-protein complexes following binding of
agonist ligands to GPCRs (e.g., epinephrine in the case of β adrenoceptors)
and binds to and activates AC. The βγ subunits can also stimulate some AC
isoforms. cAMP generated as a consequence of AC activation can activate
several effectors, the most well studied of which is cAMP-dependent protein
kinase (PKA) (Pierce et al. 2002).
Alternatively, AC activity can be inhibited by ligands that stimulate
GPCRs coupled to Gi and/or cAMP can be degraded by PDEs. Indeed both
ACs and PDEs are regulated positively and negatively by numerous other
signaling pathways (see Fig. 2), such as calcium signaling (through
calmodulin [CaM], CamKII, CamKIV, and calcineurin [also know as PP2B]),
subunits of other G proteins (e.g., αi, αo, and αq proteins, and the βγ subunits
in some cases), inositol lipids (by PKC), and receptor tyrosine kinases
(through the ERK MAP kinase and PKB) (Yoshimasa et al. 1987; Bruce et al.
2003; Goraya and Cooper 2005). Cross talk with other pathways provides
further modulation of the signal strength and cell-type specificity, and
feedforward signaling by PKA itself stimulates PDE4.
 Figure 2. The cAMP/PKA pathway.

There are three main effectors of cAMP: PKA, the guanine-nucleotide-

exchange factor (GEF) EPAC and cyclic-nucleotide-gated ion channels.
Protein kinase (PKA), the best-understood target, is a symmetrical complex
of two regulatory (R) subunits and two catalytic (C) subunits (there are
several isoforms of both subunits). It is activated by the binding of cAMP to
two sites on each of the R subunits, which causes their dissociation from the
C subunits (Taylor et al. 1992). The catalytic activity of the C subunit is
decreased by a protein kinase inhibitor (PKI), which can also act as a
chaperone and promote nuclear export of the C subunit, thereby decreasing
nuclear functions of PKA. PKA-anchoring proteins (AKAPs) provide
specificity in cAMP signal transduction by placing PKA close to specific
effectors and substrates. They can also target it to particular subcellular
locations and anchor it to ACs (for immediate local activation of PKA) or
PDEs (to create local negative-feedback loops for signal termination) (Wong
and Scott 2004).
A large number of cytosolic and nuclear proteins have been identified as
substrates for PKA (Tasken et al. 1997). PKA phosphorylates numerous
metabolic enzymes, including glycogen synthase and phosphorylase kinase,
which inhibits glycogen synthesis and promotes glycogen breakdown,
respectively, and acetyl CoA carboxylase, which inhibits lipid synthesis.
PKA also regulates other signaling pathways. For example, it phosphorylates
and thereby inactivates phospholipase C (PLC) β2. In contrast, it activates
MAP kinases; in this case, PKA promotes phosphorylation and dissociation
of an inhibitory tyrosine phosphatase (PTP). PKA also decreases the
activities of Raf and Rho and modulates ion channel permeability. In
addition, it regulates the expression and activity of various ACs and PDEs.
Regulation of transcription by PKA is mainly achieved by direct
phosphorylation of the transcription factors cAMP-response element-binding
protein (CREB), cAMP-responsive modulator (CREM), and ATF1.
Phosphorylation is a crucial event because it allows these proteins to interact
with the transcriptional coactivators CREB-binding protein (CBP) and p300
when bound to cAMP-response elements (CREs) in target genes (Mayr and
Montminy 2001). The CREM gene also encodes the powerful repressor
ICER, which negatively feeds back on cAMP-induced transcription (Sassone-
Corsi 1995). Note, however, that the picture is more complex, because
CREB, CREM, and ATF1 can all be phosphorylated by many different
kinases, and PKA can also influence the activity of other transcription
factors, including some nuclear receptors.
In addition to the negative regulation by signals that inhibit AC or
stimulate PDE activity, the action of PKA is counterbalanced by specific
protein phosphatases, including PP1 and PP2A. PKA in turn can negatively
regulate phosphatase activity by phosphorylating and activating specific PP1
inhibitors, such as I1 and DARPP32. PKA-promoted phosphorylation can
also increase the activity of PP2A as part of a negative-feedback mechanism.
Another important effector for cAMP is EPAC, a GEF that promotes
activation of certain small GTPases (e.g., Rap1). A major function of Rap1 is
to increase cell adhesion via integrin receptors (how this occurs is unclear)
(Bos 2003).
Finally, cAMP can bind to and modulate the function of a family of
cyclic-nucleotide-gated ion channels. These are relatively nonselective cation
channels that conduct calcium. Calcium stimulates CaM and CaM-dependent
kinases and, in turn, modulates cAMP production by regulating the activity of
ACs and PDEs (Zaccolo and Pozzan 2003). The channels are also permeable
to sodium and potassium, which can alter the membrane potential in
electrically active cells.
Figure 2 adapted from Fimia and Sassone-Corsi (2001).

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Bos JL. 2003. Epac: A new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4:
733–738.
Bruce JI, Straub SV, Yule DI. 2003. Crosstalk between cAMP and Ca2+ signaling in non-excitable
cells. Cell Calcium 34: 431–444.
Fimia GM, Sassone-Corsi P. 2001. Cyclic AMP signaling. J Cell Sci 114: 1971–1972.
Goraya TA, Cooper DMF. 2005. Ca2+-calmodulin-dependent phosphodiesterase (PDE1): Current
perspectives. Cell Signal 17: 789–797.
Mayr B, Montminy M. 2001. Transcriptional regulation by the phosphorylation-dependent factor
CREB. Nat Rev Mol Cell Biol 2: 599–609.
McKnight GS. 1991. Cyclic AMP second messenger systems. Curr Opin Cell Biol 3: 213–217.
Pierce KL, Premont RT, Lefkowitz RJ. 2002. Seven-transmembrane receptors. Nat Rev Mol Cell Biol
3: 639–650.
Sassone-Corsi P. 1995. Transcription factors responsive to cAMP. Annu Rev Cell Dev Biol 11: 355–
377.
Sutherland EW, Rall TW. 1958. Fractionation and characterization of a cyclic adenine ribonucleotide
formed by tissue particles. J Biol Chem 232: 1077–1091.
Tasken K, Skalhegg BS, Tasken KA, Solberg R, Knutsen HK, Levy FO, Sandberg M, Orstavik S,
Larsen T, Johansen AK, et al. 1997. Structure, function and regulation of human cAMP-dependent
protein kinases. Adv Second Messenger Phosphoprotein Res 31: 191–203.
Taylor SS, Knighton DR, Zheng J, Ten Eyck LF, Sowadski JM. 1992. Structural framework for the
protein kinase family. Annu Rev Cell Biol 8: 429–462.
Wong W, Scott JD. 2004. AKAP signaling complexes: Focal points in space and time. Nat Rev Mol
Cell Biol 5: 959–970.
Yoshimasa T, Sibley DR, Bouvier M, Lefkowitz RJ, Caron MG. 1987. Cross-talk between cellular
signaling pathways suggested by phorbol-ester induced adenylate cyclase phosphorylation. Nature
327: 67–70.
Zaccolo M, Pozzan T. 2003. cAMP and Ca2+ interplay: A matter of oscillation patterns. Trends
Neurosci 26: 53–55.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011148
Wnt Signaling

Roel Nusse
Howard Hughes Medical Institute, Stanford University Medical Center,
Stanford, California 94305-5428
Correspondence: rnusse@stanford.edu

Members of the Wnt family are secreted ligands that regulate numerous
developmental pathways (Cadigan and Peifer 2009; Van Amerongen and
Nusse 2009; and see Ch. 10 [Perrimon et al. 2013]). Wnt binds to members of
the Frizzled family, activating a canonical signaling pathway that targets
members of the LEF/TCF transcription factor family (Fig. 1). These control
gene expression programs that regulate cell fate and morphogenesis (Van
Amerongen and Nusse 2009). Wnt also activates so-called non-canonical
pathways (Fig. 2), which regulate planar cell polarity by stimulating
cytoskeletal reorganization and can also lead to calcium mobilization
(Veeman et al. 2003).
Figure 1. Canonical Wnt signaling.
 Figure 2. Non-canonical Wnt signaling pathways.

The canonical Wnt signaling pathway is regulated at many levels,

including by extensive negative control steps. In cells not exposed to Wnt
signals, the major signaling components including the receptors and the β-
catenin protein are kept in an off state (Fig. 1, left). Active Wnt signaling
rearranges these complexes (Fig. 1, right). In the inactive state, β-catenin
levels are kept low through interactions with the protein kinases GSK-3b and
CK1, the adenomatous polyposis coli tumor suppressor protein (APC), and
the scaffolding protein axin. β-Catenin is degraded, after phosphorylation by
GSK-3 and CK1 through the ubiquitin pathway, which involves interactions
with β-TrCP. β-Catenin is also regulated by adhesion complexes containing
cadherins and α-catenin. At the level of receptors, the negative regulator
DKK can bind to the LRP receptor and inhibit Wnt signaling.
During signaling (right), Wnt proteins interact with Frizzled receptors; the
transmembrane protein LRP is also required for Wnt signaling. When Wnt
proteins bind, the receptors presumably rearrange, leading to the activation of
β-catenin. The cytoplasmic tail of LRP binds to axin in a Wnt- and
phosphorylation-dependent manner. Phosphorylation of the tail of LRP is
regulated by CK1, and Dishevelled (Dvl) and Frizzled also have roles in this
process. In a current model, Wnt signaling initially leads to formation of a
complex involving Dvl, axin, and GSK3. The DIX domain in axin is similar
to the NH2 terminus in Dvl and promotes interactions between Dvl and axin.
As a consequence, GSK does not phosphorylate β-catenin anymore, releasing
it from the axin complex and allowing it to accumulate. The stabilized β-
catenin then enters the nucleus to interact with TCF/LEF transcription
factors. Note that GSK3 also participates in other pathways, such as the mTor
and Akt pathway (see p. 87 [Hemmings and Restuccia 2012] and p. 91
[Laplante and Sabatini 2012]).
In the nucleus, in the absence of the Wnt signal, TCF/LEF acts as a
repressor of Wnt target genes, in a complex with Groucho. β-Catenin can
convert TCF/LEF into a transcriptional activator of the same genes that are
repressed by TCF/LEF alone. Three other key players in this complex are
BCL9, Pygopos, and CBP. There are many target genes for the canonical
Wnt pathway. Most of these genes are cell type specific, with the possible
exception of axin 2, which acts as a negative-feedback regulator (Grigoryan
2008).
In non-canonical Wnt signaling, Wnt stimulates the planar cell polarity
pathway by activating the small GTPases Rho and Rac. These induce
cytoskeletal rearrangements that lead to the development of lateral
asymmetry in epithelial sheets and other structures. Wnt can also provoke
release of calcium from intracellular stores, probably via heterotrimeric G-
proteins. A less-well-understood mechanism involves activation of the Ror
and Ryk tyrosine kinase receptors, which control the activities of the JNK
and Src kinases, respectively (van Amerongen et al. 2008).
Figure 1 adapted, with permission, from Cell Signaling Technology (http://www.cellsignal.com).

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Cadigan KM, Peifer M. 2009. Wnt signaling from development to disease: Insights from model
systems. Cold Spring Harb Perspect Biol 1: a002881.
Grigoryan T, Wend P, Klaus A, Birchmeier W. 2008. Deciphering the function of canonical Wnt
 signals in development and disease: Conditional loss- and gain-of-function mutations of β-catenin
in mice. Genes Dev 22: 2308–2341.
* Hemmings BA, Restuccia DF. 2012. The P13K-PKB/Akt pathway. Cold Spring Harb Perspect Biol
4: a011189.
* Laplante M, Sabatini DM. 2012. mTOR signaling. Cold Spring Harb Perspect Biol 4: a011593.
* Perrimon N, Pitsouli C, Shilo B-Z. 2012. Signaling mechanisms controlling cell fate and embryonic
patterning. Cold Spring Harb Perspect Biol 4: a005975.
van Amerongen R, Nusse R. 2009. Towards an integrated view of Wnt signaling in development.
Development 136: 3205–3214.
van Amerongen R, Mikels A, Nusse R. 2008. Alternative Wnt signaling is initiated by distinct
receptors. Sci Signal 1: re9.
Veeman MT, Axelrod JD, Moon RT. 2003. A second canon. Functions and mechanisms of β-catenin-
independent Wnt signaling. Dev Cell 5: 367–377.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011163
Hedgehog Signaling

Philip W. Ingham
Institute of Molecular and Cell Biology, Singapore 138673
Correspondence: pingham@imcb.a-star.edu.sg

The Hedgehog (Hh) proteins belong to one of a small number of families of

secreted signals that play a central role in the development of most
metazoans. hh itself was originally identified as a mutation that causes a
“segment polarity” phenotype in Drosophila—as were most of the core
components of the Hh signal transduction pathway (Ingham et al. 2011).
These components have been highly conserved between flies and vertebrates
(Figs. 1 and 2); however, mammals express three related proteins, Sonic
hedgehog (Shh), Indian hedgehog, and Desert hedgehog (Dhh), and genetic
analysis in mice has uncovered a vertebrate-specific role of the primary
cilium in Hh signaling (Goetz et al. 2009).
Figure 1. Hedgehog signaling (simplified view).
 Figure 2. Hedgehog signaling.

An unusual feature of Hh proteins is their covalent coupling to

cholesterol, which occurs during autocleavage of the proprotein to yield the
signaling moiety, HhN (Beachy et al. 1997). HhN is also palmitoylated at its
amino terminus by an acyl transferase encoded by the Drosophila skinny
hedgehog gene (Skn). Secretion of lipidated HhN requires the function of a
large multipass transmembrane protein, Dispatched, that is structurally
related to the Hh receptor Patched (Ptch1) (Ingham et al. 2011). Binding of
HhN to Ptc1 is promoted by two other transmembrane proteins, CDO and
BOC (known as IHOG and BOI in Drosophila), which act redundantly to
bind HhN via one of several fibronectin III (FnIII) motifs in their
extracellular domains (Beachy et al. 2010). In its unbound state, Ptc1
localizes to the primary cilium (Rohatgi and Scott 2007), where it acts via a
poorly characterized mechanism, thought to involve the transport of one or
more lipids, to suppress the activity of the G-protein-coupled receptor
(GPCR)-like protein Smoothened (Smo) (Ayers and Thérond 2010).
The principal response of cells to HhN is the activation of target genes by
the Gli zinc finger proteins. Gli2 and Gli3 (but not Gli1) are bifunctional
transcription factors: their full-length forms function as transcriptional
activators, but they can be converted into lower-molecular-weight
transcriptional repressors. This is promoted by the phosphorylation of a series
of motifs within the carboxy-terminal domain of the protein. These motifs are
sequentially phosphorylated by PKA, GSK3, and CKI to generate recognition
signals for the F-box protein bTrCP, a component of the SCF complex. This
in turn catalyzes the ubiquitylation of the carboxyl terminus, targeting it for
degradation by the proteasome to yield the truncated amino-terminal
repressor forms, Gli2/3R (Ingham et al. 2011). These bind to Hh target genes
to repress their transcription. Gli2/3 proteins appear to shuttle up and down
the primary cilium in association with the Cos/Kif7 and SuFu proteins; in the
absence of Smo activity, this association seems to promote their processing at
the base of the primary cilium. Inactivation of Ptc by HhN results in Smo
being transported to the tip of the primary cilium (a process that requires
Kif3a and β-arrestin activity), where its activity promotes the dissociation of
the Gli2/3-Cos-SuFu complex (Tukachinsky et al. 2010), releasing the full-
length highly labile forms of the Gli proteins. These translocate to the nucleus
and activate transcription of target genes, such as Ptch1, which attenuates the
signal, Gli1, which amplifies the signal, and the genes encoding the cell cycle
regulators, Myc, cyclin D, and E.
Figures 1 and 2 adapted, with permission, from Cell Signaling Technology
(http://www.cellsignal.com).

REFERENCES
Ayers KL, Thérond PP. 2010. Evaluating smoothened as a G-protein-coupled receptor for Hedgehog
signalling. Trends Cell Biol 20: 287–298.
Beachy PA, Cooper MK, Young KE, von Kessler DP, Park WJ, Hall TM, Leahy DJ, Porter JA. 1997.
 Multiple roles of cholesterol in hedgehog protein biogenesis and signaling. Cold Spring Harb Symp
Quant Biol 62: 191–204.
Beachy PA, Hymowitz SG, Lazarus RA, Leahy DJ, Siebold C. 2010. Interactions between Hedgehog
proteins and their binding partners come into view. Genes Dev 24: 2001–2012.
Goetz SC, Ocbina PJ, Anderson KV. 2009. The primary cilium as a Hedgehog signal transduction
machine. Methods Cell Biol 94: 199–222.
Ingham PW, Nakano Y, Seger C. 2011. Mechanisms and functions of Hedgehog signalling across the
metazoa. Nat Rev Genet 12: 393–406.
Rohatgi R, Scott MP. 2007. Patching the gaps in Hedgehog signalling. Nat Cell Biol 9: 1005–1009.
Tukachinsky H, Lopez LV, Salic A. 2010. A mechanism for vertebrate Hedgehog signaling:
Recruitment to cilia and dissociation of SuFu-Gli protein complexes. J Cell Biol 191: 415–428.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011221
Notch Signaling

Raphael Kopan
Department of Developmental Biology, and Department of Medicine
(Division of Dermatology), Washington University, St. Louis, Missouri
63110
Correspondence: kopan@wustl.edu

The Notch pathway regulates cell proliferation, cell fate, differentiation, and
cell death in all metazoans. Notch itself is a cell-surface receptor that
transduces short-range signals by interacting with transmembrane ligands
such as Delta (termed Delta-like in humans) and Serrate (termed Jagged in
humans) on neighboring cells (Fig. 1). Some soluble ligands have also been
identified in Caenorhabditis elegans, but these bind to Notch together with
transmembrane adaptors (Komatsu et al. 2008). Ligand binding leads to
cleavage and release of the Notch intracellular domain (NICD), which then
travels to the nucleus to regulate transcriptional complexes containing the
DNA-binding protein CBF1/RBPjk/Su(H)/Lag1 (CSL).
 Figure 1. Notch signaling (simplified view).

Following their synthesis, Notch receptors are cleaved by protein

convertases during exocytosis at site 1 (S1), which regulates their trafficking
and signaling activity (Logeat et al. 1998; Gordon et al. 2009). During
passage through the Golgi, they can be glycosylated by glycosyltransferases
such as Fringe, which determines the subsequent response to different
subfamilies of ligands. These and other posttranslational modifications of the
receptors and ligands tune the amplitude and timing of Notch activity to
generate context-specific signals. Several proteins, including E3 ubiquitin
ligases (e.g., Deltex and Nedd4), Numb, and α-adaptin, regulate the steady-
state levels of the Notch receptor at the cell surface. In signal-sending cells,
E3 ubiquitin ligases (Neur and MIB) similarly ubiquitylate the intracellular
domain of the ligand to promote epsin-mediated endocytosis, which is
associated with ligand activation (Fig. 2).

Figure 2. Notch signaling.

 Following ligand binding, signaling is initiated when endocytosis of
ligand–receptor complexes induces unfolding of a juxtamembrane negative
control region (NRR) unique to Notch proteins. Unfolding of the NRR allows
access by the protease ADAM10 (also known as KUZ), which removes the
Notch extracellular domain by cleaving at site 2 (S2); γ-secretase then
cleaves Notch within its transmembrane domain at site 3 (S3) to release
various forms of the NICD. Those that have valine or methionine residues at
the amino terminus escape the N-end-rule degradation pathway (Tagami et al.
2008) and are stable enough to impact transcription (see below).
Interestingly, productive interactions between Notch and its ligands occur
when these are present on neighboring cells (i.e., in trans); when receptor and
ligand are present on the same cell (i.e., interactions are in cis), activation is
inhibited. cis interactions thus determine whether a cell will signal (the ligand
is more abundant than Notch) or receive (Notch is more abundant than the
ligand) (Sprinzak et al. 2010). Alternatively, in some cases ligand and
receptors can be segregated into different subdomains to allow simultaneous
transmission and reception of signals (Luty et al. 2007).
Only one nuclear protein is known to mediate the bulk of Notch signals:
CSL (Kopan and Ilagan 2009). CSL is a DNA-binding adaptor that interacts
with many proteins to build either repressor complexes, which include
histone deacetylases (HDACs) that preserve a closed chromatin
conformation, or activating complexes, which contain NICD, along with
other proteins including histone acetyltransferases (HATs) that open up
chromatin. In canonical, CSL-mediated Notch signaling, NICD translocates
to the nucleus, binds to CSL, and helps recruit the adaptor protein
Mastermind-like (MAML) (Kopan and Ilagan 2009). MAML recruits the
HAT p300 and components of the transcription machinery. Thus, every
cleaved Notch molecule generates one signaling unit, and tuning the
effectiveness of receptor–ligand interaction directly determines the amount of
NICD in the nucleus. During the transcriptional activation process, NICD is
phosphorylated on a degron within its PEST domain by kinases such as
cyclin-dependent kinase 8 (CDK8) and targeted for proteasome-mediated
degradation by E3 ubiquitin ligases such as Sel10 (also known as Fbw7).
This limits the half-life of a canonical Notch signal and resets the cell for the
next pulse of signaling.
In addition to the canonical signals, mounting evidence indicates that
CSL-independent activities of Notch also regulate vertebrate (Rangarajan et
al. 2001; Demehri et al. 2008) and invertebrate (Ramain et al. 2001)
development, but the biochemical details of this aspect of the pathway are yet
to be uncovered. In the absence of ligand, Notch may also be involved in
other cellular processes, such as regulating the stability of β-catenin (Sanders
et al. 2009), a component of the Wnt signaling pathway (p. 103 [Nusse
2012]).
Under most physiological conditions, unbound Notch receptors simply
recycle or are targeted for lysosomal degradation. With one exception
(Mukherjee et al. 2011), only pathological or experimental conditions are
known to lead to receptor activation without ligand. These include mutations
in the NRR domain (Weng et al. 2004), overexpression of Notch with
ADAM proteases, and exposure to calcium chelators (Bozkulak and
Weinmaster 2009; van Tetering et al. 2009), all of which expose Notch to
shedding by ADAM17 (also known as TACE). Notch can also become
activated when ESCRT components are mutated (Moberg et al. 2005;
Thompson et al. 2005; Vaccari and Bilder 2005), which delays entry into the
lysosome and permits ligand-independent activation. The frequent activation
of Notch by mutations in T-cell acute lymphoblastic leukemia and its
frequent inactivation in head and neck squamous cell carcinomas (Agrawal et
al. 2011; Stransky et al. 2011) illustrate the importance of the pathway for
control of cell fate and proliferation and the severe consequences of its
dysregulation.
Figures adapted with kind permission of Cell Signaling Technology (http://www.cellsignal.com).

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Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, Oh H, Aster JC, Krishna S, Metzger
D, Chambon P, et al. 2001. Notch signaling is a direct determinant of keratinocyte growth arrest
and entry into differentiation. EMBO J 20: 3427–3436.
Sanders PG, Munoz-Descalzo S, Balayo T, Wirtz-Peitz F, Hayward P, Arias AM. 2009. Ligand-
independent traffic of Notch buffers activated armadillo in Drosophila. PLoS Biol 7: e1000169.
Sprinzak D, Lakhanpal A, LeBon L, Santat LA, Fontes ME, Anderson GA, Garcia-Ojalvo J, Elowitz
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Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence
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Tagami S, Okochi M, Yanagida K, Ikuta A, Fukumori A, Matsumoto N, Ishizuka-Katsura Y,
Nakayama T, Itoh N, Jiang J, et al. 2008. Regulation of Notch signaling by dynamic changes in the
precision in S3 cleavage of Notch-1. Mol Cell Biol 28: 165–176.
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van Tetering G, van Diest P, Verlaan I, van der Wall E, Kopan R, Vooijs M. 2009. Metalloprotease
ADAM10 is required for Notch1 site 2 cleavage. J Biol Chem 284: 31018–31027.
Weng AP, Ferrando AA, Lee W, Morris JPIV, Silverman LB, Sanchez-Irizarry C, Blacklow SC, Look
AT, Aster JC. 2004. Activating mutations of NOTCH1 in human T cell acute lymphoblastic
leukemia. Science 306: 269–271.
Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011213
Signaling by the TGFβ Superfamily

Jeffrey L. Wrana
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario
M5G 1X5, Canada
Correspondence: wrana@lunenfeld.ca

The transforming growth factor β (TGFβ) superfamily was discovered in a

hunt for autocrine factors secreted from cancer cells that promote
transformation (Roberts et al. 1981). However, it soon became clear that
TGFβ and the related bone morphogenetic proteins (BMPs) regulate diverse
developmental and homeostatic processes and are mutated in numerous
human diseases. Furthermore, TGFβ-superfamily members such as activins,
Nodal, and growth differentiation factors (GDFs) were shown to control cell
fate as a function of concentration, thus defining them as a key class of
secreted morphogens (Green and Smith 1990).
TGFβ-superfamily members are highly conserved across animals and
comprise the largest family of secreted morphogens. Ligands are produced by
cleavage of a prodomain that releases active disulfide-linked homo- or
heterodimers. TGFβ (plus activin, Nodal, and GDFs) and BMPs both signal
through transmembrane serine/threonine kinase receptors. These are typically
presented as using distinct downstream pathways (see Figs. 1 and 2),
although some cross talk in certain cell types occurs. In each pathway there
are two kinds of receptors: type I and type II (Massague 1998). Ligand
binding induces formation of heterotetramers containing two type II and two
type I receptors, which allows the constitutively active type II receptor to
phosphorylate a glycine-serine (GS)-rich region in the type I receptor. This
initiates signaling through the Smad pathway (Fig. 1), with the
phosphorylated GS region providing a docking site for receptor-regulated
Smad proteins (R-Smads). In TGFβ signaling, this is promoted by Smad
anchor for receptor activation (SARA) in the endosomal compartment
(Attisano and Wrana 2002).

Figure 1. Smad signaling.

 Figure 2. Non-Smad signals.

Despite the large TGFβ superfamily (>30 members in humans), the

receptor repertoire is limited: only five type II and seven type I receptors are
encoded in mammalian genomes, with the type I receptors funneling
signaling into one of two distinct R-Smad pathways: the TGFβ-Smad
pathway (R-Smad2/3) or the BMP-Smad pathway (R-Smad1/5/8) (Massague
2012). Docking of R-Smads allows phosphorylation of the last two serines in
their carboxyl termini by the type I kinase, which induces dissociation from
the receptor, binding to the common Smad, Smad4, and nuclear accumulation
of the Smad complex. In the nucleus, most Smads regulate transcriptional
responses by direct binding of their MH1 domain to DNA (Smad2 only binds
to DNA indirectly) in cooperation with various DNA-binding partners that
include sequence-specific transcription factors. These Smad DNA-binding
partners typically bind to the R-Smad, thus maintaining specificity of the
transcriptional response. Smads also interact with transcriptional coactivators
or corepressors that modulate the transcriptional output. Interestingly, the
remaining two Smads, Smad6 and Smad7, are transcriptional targets of R-
Smads and act in a negative-feedback loop to inhibit signaling by interacting
with the receptors and recruiting Smurf and related ubiquitin ligases of the
Nedd4 family to induce receptor degradation. SnoN and the related Ski, as
well as Arkadia, a ring-finger ubiquitin ligase (also known as RNF111), are
important examples of negative and positive regulators of Smad2/3-
dependent transcription, respectively (Stroschein et al. 1999; Niederlander et
al. 2001). SnoN and Ski negatively regulate the pathway as corepressors of
Smads, whereas Arkadia promotes signaling by degrading negative regulators
of the pathway, such as Smad7 and SnoN/Ski.
The Smad pathways are the major mediators of transcriptional responses
induced by the TGFβ family, which control cell-fate determination, cell-cycle
arrest, apoptosis, and actin rearrangements. However, receptor signaling is
not restricted to R-Smad activation (Fig. 2). The type II receptor
phosphorylates Par6 polarity proteins bound to the type I receptor to dissolve
tight junctions in epithelial cells (Ozdamar et al. 2005) and specify axons (Yi
et al. 2010). Furthermore, protein phosphatase 2A (PP2A) is regulated by the
receptors (Griswold-Prenner et al. 1998), signaling via SHC-Grb2 has been
reported (Lee et al. 2007), and interactions between the kinase PAK and the
TGFβ receptor link it to cytoskeletal and focal adhesion dynamics (Wilkes et
al. 2009). BMPRII is of particular interest because it has a unique carboxy-
terminal tail that serves as a docking site for binding of the kinases LIMK
and JNK, thus linking BMP signaling to the actin cytoskeleton and the
microtubule network, respectively (Miyazono et al. 2010; Podkowa et al.
2010). These pathways are critical in neuronal dendritogenesis and may be
important in familial pulmonary hypertension, in which mutations in BMPRII
are a major cause of disease (Morrell 2011). Numerous kinase cascades,
including the ERK, JNK, and p38 MAPK pathways, are also regulated by
TGFβ signaling (Mu et al. 2012), with signaling to TRAF6 being one
important mechanism of MAPK regulation. These non-Smad pathways
combine with the gene-expression programs controlled by Smads to yield an
integrated response to TGFβ signals.
Finally, TGFβ signaling is embedded in a higher-order network of
interactions with other signaling pathways. For example, Smads interact with
the Wnt pathway (via Lef/TCF transcription factors and β-catenin), the
Hedgehog pathway (via Gli transcriptional regulators), and the Hippo
pathway (via TAZ and YAP). This provides an integrated signaling network
that allows contextual interpretation of morphogen signals in diverse
biological settings (Attisano and Wrana 2013).

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Attisano L, Wrana JL. 2002. Signal transduction by the TGF-β superfamily. Science 296: 1646–1647.
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Green JB, Smith JC. 1990. Graded changes in dose of a Xenopus activin A homologue elicit stepwise
transitions in embryonic cell fate. Nature 347: 391–394.
Griswold-Prenner I, Kamibayashi C, Maruoka EM, Mumby MC, Derynck R. 1998. Physical and
functional interactions between type I transforming growth factor β receptors and Bα, a WD-40
repeat subunit of phosphatase 2A. Mol Cell Biol 18: 6595–6604.
Lee MK, Pardoux C, Hall MC, Lee PS, Warburton D, Qing J, Smith SM, Derynck R. 2007. TGF-β
activates Erk MAP kinase signalling through direct phosphorylation of ShcA. EMBO J 26: 3957–
3967.
Massague J. 1998. TGF-β signal transduction. Annu Rev Biochem 67: 753–791.
Massague J. 2012. TGFβ signalling in context. Nat Rev Mol Cell Biol 13: 616–630.
Miyazono K, Kamiya Y, Morikawa M. 2010. Bone morphogenetic protein receptors and signal
transduction. J Biochem 147: 35–51.
Morrell NW. 2011. Role of bone morphogenetic protein receptors in the development of pulmonary
arterial hypertension. Adv Exp Med Biol 661: 251–264.
Mu Y, Gudey SK, Landström M. 2012. Non-Smad signaling pathways. Cell Tissue Res 347: 11–20.
Niederlander C, Walsh JJ, Episkopou V, Jones CM. 2001. Arkadia enhances nodal-related signalling to
induce mesendoderm. Nature 410: 830–834.
Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y, Wrana JL. 2005. Regulation of the
polarity protein Par6 by TGF-β receptors controls epithelial cell plasticity. Science 307: 1603–1609.
Podkowa M, Zhao X, Chow CW, Coffey ET, Davis RJ, Attisano L. 2010. Microtubule stabilization by
bone morphogenetic protein receptor-mediated scaffolding of c-Jun N-terminal kinase promotes
dendrite formation. Mol Cell Biol 30: 2241–2250.
Roberts AB, Anzano MA, Lamb LC, Smith JM, Sporn MB. 1981. New class of transforming growth
factors potentiated by epidermal growth factor: Isolation from non-neoplastic tissues. Proc Natl
Acad Sci 78: 5339–5343.
Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K. 1999. Negative feedback regulation of TGF-β
signaling by the SnoN oncoprotein. Science 286: 771–774.
Wilkes MC, Repellin CE, Hong M, Bracamonte M, Penheiter SG, Borg JP, Leof EB. 2009. Erbin and
 the NF2 tumor suppressor Merlin cooperatively regulate cell-type-specific activation of PAK2 by
TGF-β. Dev Cell 16: 433–444.
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development. Cell 142: 144–157.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011197
The JAK/STAT Pathway

Douglas A. Harrison
Department of Biology, University of Kentucky, Lexington, Kentucky 40506
Correspondence: DougH@uky.edu

Cellular responses to dozens of cytokines and growth factors are mediated by

the evolutionarily conserved Janus kinase/signal transducers and activators of
transcription (JAK/STAT) signaling pathway (Fig. 1). These responses
include proliferation, differentiation, migration, apoptosis, and cell survival,
depending on the signal, tissue, and cellular context. JAK/STAT signaling is
essential for numerous developmental and homeostatic processes, including
hematopoiesis, immune cell development, stem cell maintenance, organismal
growth, and mammary gland development (Ghoreschi et al. 2009).
 Figure 1. The JAK/STAT pathway (simplified view).

Janus kinases (JAKs) were identified through sequence comparisons as a

unique class of tyrosine kinases that contain both a catalytic domain and a
second kinase-like domain that serves an autoregulatory function, hence the
homage to the two-faced Roman god. They were functionally linked to
STATs and interferon signaling in powerful somatic cell genetic screens
(Darnell et al. 1994; Schindler and Plumlee 2008). The JAK/STAT cascade is
among the simplest of the conserved metazoan signaling pathways. The
binding of extracellular ligand leads to pathway activation via changes to the
receptors that permit the intracellular JAKs associated with them to
phosphorylate one another. Trans-phosphorylated JAKs then phosphorylate
downstream substrates, including both the receptor and the STATs. Activated
STATs enter the nucleus and bind as dimers or as more complex oligomers to
specific enhancer sequences in target genes, thus regulating their transcription
(Fig. 2).

Figure 2. The JAK/STAT pathway.

In mammals, there are four members of the JAK family and seven
STATs. Different JAKs and STATs are recruited based on their tissue
specificity and the receptors engaged in the signaling event (Schindler and
Plumlee 2008). In invertebrates, the Drosophila JAK/STAT pathway has
been extensively studied and comprises only one JAK and one STAT
(Arbouzova and Zeidler 2006). Although the canonical JAK/STAT pathway
is simple and direct, pathway components regulate or are regulated by
members of other signaling pathways, including those involving the ERK
MAP kinase, PI 3-kinase (PI3K), and others. Furthermore, non-canonical
JAK and STAT activities influence the global transcriptional state through
modification of chromatin structure (Li 2008; Dawson et al. 2009).
Human JAK mutations cause numerous diseases, including severe
combined immune deficiency, hyperIgE syndrome, certain leukemias,
polycythemia vera, and other myeloproliferative disorders (Jatiani et al.
2010). Because of the causative role in these diseases and their central
significance in immune response, JAKs have become attractive targets for
development of therapeutics for a variety of hematopoietic and immune
system disorders (Pesu et al. 2008; Haan et al. 2010). Owing to the pleiotropy
of the JAK/STAT pathway, agents that selectively perturb specific family
members are being sought.
Figures adapted by kind permission of Cell Signaling Technology (http://cellsignal.com).

REFERENCES
Arbouzova NI, Zeidler MP. 2006. JAK/STAT signalling in Drosophila: Insights into conserved
regulatory and cellular functions. Development 133: 2605–2616.
Darnell JE Jr, Kerr IM, Stark GR. 1994. Jak-STAT pathways and transcriptional activation in response
to IFNs and other extracellular signaling proteins. Science 264: 1415–1421.
Dawson MA, Bannister AJ, Gottgens B, Foster SD, Bartke T, Green AR, Kouzarides T. 2009. JAK2
phosphorylates histone H3Y41 and excludes HP1a from chromatin. Nature 461: 819–822.
Ghoreschi K, Laurence A, O’Shea JJ. 2009. Janus kinases in immune cell signaling. Immunol Rev 228:
273–287.
Haan C, Behrmann I, Haan S. 2010. Perspectives for the use of structural information and chemical
genetics to develop inhibitors of Janus kinases. J Cell Mol Med 14: 504–527.
Jatiani SS, Baker SJ, Silverman LR, Reddy EP. 2010. JAK/STAT pathways in cytokine signaling and
myeloproliferative disorders: Approaches for targeted therapies. Genes Cancer 1: 979–993.
Li WX. 2008. Canonical and non-canonical JAK-STAT signaling. Trends Cell Biol 18: 545–551.
Pesu M, Laurence A, Kishore N, Zwillich SH, Chan G, O’Shea JJ. 2008. Therapeutic targeting of Janus
kinases. Immunol Rev 223: 132–142.
Schindler C, Plumlee C. 2008. Inteferons pen the JAK-STAT pathway. Semin Cell Dev Biol 19: 311–
318.
Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011205
Toll-Like Receptor Signaling

Kian-Huat Lim and Louis M. Staudt

Metabolism Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892
Correspondence: lstaudt@mail.nih.gov

Toll-like receptors (TLRs) are protective immune sentries that sense

pathogen-associated molecular patterns (PAMPs) such as unmethylated
double-stranded DNA (CpG), single-stranded RNA (ssRNA), lipoproteins,
lipopolysaccharide (LPS), and flagellin. In innate immune myeloid cells,
TLRs induce the secretion of inflammatory cytokines (Ch. 15 [Newton and
Dixit 2012]), thereby engaging lymphocytes to mount an adaptive, antigen-
specific immune response (see Fig. 1) that ultimately eradicates the invading
microbes (Kawai and Akira 2010).
 Figure 1. TLR signaling (simplified view).

Identification of TLR innate immune function began with the discovery

that Drosophila mutants in the Toll gene are highly susceptible to fungal
infection (Lemaitre et al. 1996). This was soon followed by identification of a
human Toll homolog, now known as TLR4 (Medzhitov et al. 1997). To date,
10 TLR family members have been identified in humans, and at least 13 are
present in mice. All TLRs consist of an amino-terminal domain, characterized
by multiple leucine-rich repeats, and a carboxy-terminal TIR domain that
interacts with TIR-containing adaptors. Nucleic acid–sensing TLRs (TLR3,
TLR7, TLR8, and TLR9) are localized within endosomal compartments,
whereas the other TLRs reside at the plasma membrane (Blasius and Beutler
2010; McGettrick and O’Neill 2010). Trafficking of most TLRs from the
endoplasmic reticulum (ER) to either the plasma membrane or
endolysosomes is orchestrated by ER-resident proteins such as UNC93B (for
TLR3, TLR7, TLR8, and TLR9) and PRAT4A (for TLR1, TLR2, TLR4,
TLR7, and TLR9) (Blasius and Beutler 2010). Once in the endolysosomes,
TLR3, TLR7, and TLR9 are subject to stepwise proteolytic cleavage, which
is required for ligand binding and signaling (Barton and Kagan 2009). For
some TLRs, ligand binding is facilitated by coreceptors, including CD14 and
MD2.
Following ligand engagement, the cytoplasmic TIR domains of the TLRs
recruit the signaling adaptors MyD88, TIRAP, TRAM, and/or TRIF (see Fig.
2). Depending on the nature of the adaptor that is used, various kinases
(IRAK4, IRAK1, IRAK2, TBK1, and IKKε) and ubiquitin ligases (TRAF6
and pellino 1) are recruited and activated, culminating in the engagement of
the NF-κB, type I interferon, p38 MAP kinase (MAPK), and JNK MAPK
pathways (Kawai and Akira 2010; p. 81 [Morrison 2012]). TRAF6 is
modified by K63-linked autoubiquitylation, which enables the recruitment of
IκB kinase (IKK) through a ubiquitin-binding domain of the IKKγ (also
known as NEMO) subunit. In addition, a ubiquitin-binding domain of TAB2
recognizes ubiquitylated TRAF6, causing activation of the associated TAK1
kinase, which then phosphorylates the IKKβ subunit. Pellino 1 can modify
IRAK1 with K63-linked ubiquitin, allowing IRAK1 to recruit IKK directly.
TLR4 signaling via the TRIF adaptor protein leads to K63-linked
polyubiquitylation of TRAF3, thereby promoting the type I interferon
response via interferon regulatory factor (IRFs) (Hacker et al. 2011).
Alternatively, TLR4 signaling via MyD88 leads to the activation of TRAF6,
which modifies cIAP1 or cIAP2 with K63-linked polyubiquitin (Hacker et al.
2011). The cIAPs are thereby activated to modify TRAF3 with K48-linked
polyubiquitin, causing its proteasomal degradation. This allows a TRAF6–
TAK1 complex to activate the p38 MAPK pathway and promote
inflammatory cytokine production (Hacker et al. 2011). TLR signaling is
turned off by various negative regulators: IRAK-M and MyD88 short
(MyD88s), which antagonize IRAK1 activation; FADD, which antagonizes
MyD88 or IRAKs; SHP1 and SHP2, which dephosphorylate IRAK1 and
TBK1, respectively; and A20, which deubiquitylates TRAF6 and IKK
(Flannery and Bowie 2010; Kawai and Akira 2010).

Figure 2. TLR signaling.

Deregulation of the TLR signaling cascade causes several human

diseases. Patients with inherited deficiencies of MyD88, IRAK4, UNC93B1,
or TLR3 are susceptible to recurrent bacterial or viral infections (Casanova et
al. 2011). Chronic TLR7 and/or TLR9 activation in autoreactive B cells, in
contrast, underlies systemic autoimmune diseases (Green and Marshak-
Rothstein 2011). Furthermore, oncogenic activating mutations of MyD88
occur frequently in the activated B-cell-like subtype of diffuse large B-cell
lymphoma and in other B-cell malignancies (Ngo et al. 2011). Inhibitors of
various TLRs or their associated kinases are currently being developed for
autoimmune or inflammatory diseases and also hold promise for the
treatment of B-cell malignancies with oncogenic MyD88 mutations. Many
TLR7 and TLR9 agonists are currently in clinical trials as adjuvants to boost
host antitumor responses in cancer patients (Hennessy et al. 2010).
Figures adapted with kind permission of Cell Signaling Technology (http://www.cellsignal.com).

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compartmentalization. Nat Rev Immunol 9: 535–542.
Blasius AL, Beutler B. 2010. Intracellular Toll-like receptors. Immunity 32: 305–315.
Casanova JL, Abel L, Quintana-Murci L. 2011. Human TLRs and IL-1Rs in host defense: Natural
insights from evolutionary, epidemiological, and clinical genetics. Annu Rev Immunol 29: 447–491.
Flannery S, Bowie AG. 2010. The interleukin-1 receptor-associated kinases: Critical regulators of
innate immune signalling. Biochem Pharmacol 80: 1981–1991.
Green NM, Marshak-Rothstein A. 2011. Toll-like receptor driven B cell activation in the induction of
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Hacker H, Tseng PH, Karin M. 2011. Expanding TRAF function: TRAF3 as a tri-faced immune
regulator. Nat Rev Immunol 11: 457–468.
Hennessy EJ, Parker AE, O’Neill LA. 2010. Targeting Toll-like receptors: Emerging therapeutics? Nat
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Kawai T, Akira S. 2010. The role of pattern-recognition receptors in innate immunity: Update on Toll-
like receptors. Nat Immunol 11: 373–384.
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. 1996. The dorsoventral regulatory
gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell
86: 973–983.
McGettrick AF, O’Neill LA. 2010. Localisation and trafficking of Toll-like receptors: An important
mode of regulation. Curr Opin Immunol 22: 20–27.
Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. 1997. A human homologue of the Drosophila Toll
protein signals activation of adaptive immunity. Nature 388: 394–397.
* Morrison DK. 2012. MAP kinase pathways. Cold Spring Harb Perspect Biol 4: a011254.
* Newton K, Dixit VM. 2012. Signaling in innate immunity and inflammation. Cold Spring Harb
Perspect Biol 4: a006049.
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H, et al. 2011. Oncogenically active MYD88 mutations in human lymphoma. Nature 470: 115–119.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011247
Immunoreceptor Signaling

Lawrence E. Samelson
Center for Cancer Research, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892-4256
Correspondence: lsamelson@comcast.net

T cells and B cells are stimulated when antigens bind to T-cell receptors
(TCRs) and B-cell receptors (BCRs) in their respective plasma membranes
(Fig. 1). The antigens presented to T cells are in the form of short peptides
that have been processed in infected cells and are displayed on the surface
bound to major histocompatibility complex (MHC) class I or class II
molecules. BCRs, in contrast, recognize free antigens in their native form
present in the extracellular milieu or on cell surfaces.
 Figure 1. Early events in T cell and B cell receptor signaling.

Despite superficial differences, the responses in the two cell types are
remarkably similar. For both, signaling begins with engagement of a complex
receptor composed of antigen receptor subunits with immunoglobulin or
immunoglobulin-like domains. Both receptors also contain non-polymorphic
subunits, the TCRζ chain and CD3 subunits (γ, δ, and ε) for the TCR, and the
α and β chains of the BCR (Reth 1995; Wucherpfennig et al. 2010).
Coreceptors are important in both T cells (CD4 for helper T cells; CD8 for
cytotoxic T cells) and B cells (CD19). For both receptors, members of two
protein tyrosine kinase (PTK) families, Src (Lck for T cells and Lyn for B
cells) and Syk (Zap-70 for T cells and Syk for B cells) (Bradshaw 2010; Liu
et al. 2010; Wang et al. 2010), are critical signaling molecules closely
associated with the receptors and coreceptors. Activation of tyrosine kinases
is the first biochemical change that follows receptor engagement.
Following antigen engagement, activated tyrosine kinases phosphorylate
several adapter proteins and signaling enzymes, which then interact. For T
cells, this is exemplified by the phosphorylation of the adapters LAT and
SLP-76 (Fig. 2), the formation of multiprotein complexes nucleated at these
adapters, and the inclusion of such enzymes as phospholipase Cγ1 (PLCγ1)
and VAV in the protein assemblies (Balagopalan et al. 2010; Jordan and
Koretzky 2010). Activation of an additional protein tyrosine kinase, Itk in T
cells and Btk in B cells, occurs in these complexes (Andreotti et al. 2010).
Similarly in B-cells, phosphorylation of BLNK and CD19 leads to
recruitment of adapters and signaling enzymes. In both cell types these events
occur at the plasma membrane, where critical lipid substrates of enzymes
such as PLCγ1 and PI3 kinase (PI3K) are located.
 Figure 2. T cell receptor signaling.

Downstream from these initial events, a number of other protein

complexes are formed, and various signaling pathways are activated. Many
of these involve activation of protein serine kinases (Finlay and Cantrell
2010). In T cells and B cells, activation of protein kinase C (PKC) leads to
formation and activation of the CARMA1–Bcl10–MALT1 complex (Thome
et al. 2010). Stimulation of PI3K leads to activation of the kinase Akt (Huang
and Sauer 2010). Downstream from the TCR and BCR, one also observes
activation of small G proteins such as Ras, which, in turn, leads to activation
of the Raf/MEK and ERK kinases. Finally, in both cell types, these various
events lead to cytoskeletal changes and gene expression induced by
transcriptional activators such as NF-AT and NF-κB. T cells and B cells thus
activated can proliferate, differentiate, and synthesize the cytokines and
effector molecules that allow them to fulfill their roles in the adaptive
immune response.
Figures adapted with kind permission of Cell Signaling Technology (http://cellsignal.com).

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Andreotti AH, Schwartzberg PL, Joseph RE, Berg LJ. 2010. T-cell signaling regulated by the Tec
family kinase, Itk. Cold Spring Harb Perspect Biol 2: a002287.
Balagopalan L, Coussens NP, Sherman E, Samelson LE, Sommers CL. 2010. The LAT story: A tale of
cooperativity, coordination, and choreography. Cold Spring Harb Perspect Biol 2: a005512.
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Signal 22: 1175–1184.
Finlay D, Cantrell D. 2010. The coordination of T-cell function by serine/threonine kinases. Cold
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lineages by the adaptor protein SLP-76. Cold Spring Harb Perspect Biol 2: a002501.
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cell receptor signaling. Cold Spring Harb Perspect Biol 2: a002295.
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Thome M, Charton JE, Pelzer C, Hailfinger S. 2010. Antigen receptor signaling to NF-κB via
CARMA1, BCL10, and MALT1. Cold Spring Harb Perspect Biol 2: a003044.
Wang H, Kadlecek TA, Au-Yeung BB, Goodfellow HE, Hsu LY, Freedman TS, Weiss A. 2010. ZAP-
70: An essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol 2: a002279.
Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. 2010. Structural biology of the T-cell
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011510
Signaling by Nuclear Receptors

Richard Sever1 and Christopher K. Glass2

1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
2University of California San Diego, La Jolla, California 92093

Correspondence: ckg@ucsd.edu

Nuclear receptors are a family of ligand-regulated transcription factors that

are activated by steroid hormones, such as estrogen and progesterone, and
various other lipid-soluble signals, including retinoic acid, oxysterols, and
thyroid hormone (Mangelsdorf et al. 1995). Unlike most intercellular
messengers, the ligands can cross the plasma membrane and directly interact
with nuclear receptors inside the cell (Fig. 1), rather than having to act via
cell surface receptors. Once activated, nuclear receptors directly regulate
transcription of genes that control a wide variety of biological processes,
including cell proliferation, development, metabolism, and reproduction.
Although nuclear receptors primarily function as transcription factors, some
have also been found to regulate cellular functions within the cytoplasm. For
example, estrogens act through the estrogen receptor in the cytoplasm of
endothelial cells to rapidly activate signaling pathways that control vascular
tone and endothelial cell migration (Wu et al. 2011).
 Figure 1. Nuclear receptor signaling.

Studies of the salivary glands of insect larva in the 1960s first indicated
that steroid hormones regulate transcription. Subsequent work showed that
estrogen can selectively activate the genes encoding egg-white and yolk
proteins, leading to the cloning of the estrogen, glucocorticoid, and thyroid
hormone receptors in the 1980s (Hollenberg et al. 1985; Green et al. 1986;
Miesfeld et al. 1986; Sap et al. 1986; Weinberger et al. 1986). We now know
that 48 nuclear receptors are encoded in the human genome (Mangelsdorf et
al. 1995). In many cases, ligands for these have been identified, but several
“orphan receptors” remain (Burris et al. 2012). Whether all of these have
bona fide ligands is unclear, because some nuclear receptors can act in the
absence of a ligand (Table 1).
Table 1. Common nuclear receptors and their ligands
Receptor Abbreviation Ligand
Androgen receptor AR Testosterone
Estrogen receptor ER Estrogen
Estrogen-related receptor ERR ?
Glucocorticoid receptor GR Cortisol
Mineralocorticoid receptor MR Aldosterone
Progesterone receptor PR Progesterone
Retinoic acid receptor RAR Retinoic acid
Retinoid orphan receptor ROR ?
Retinoic acid-related receptor RXR Rexinoids
Liver X receptor LXR Oxysterols
Peroxisome proliferator-activated receptor γ PPARγ Fatty acid metabolites
Thyroid hormone receptor TR Thyroid hormone
Vitamin D3 receptor VDR Vitamin D3

Nuclear receptors share a common structure, comprising a highly variable

amino-terminal domain that includes several distinct transactivation regions
(the A/B domain; also referred to as AF1 for activation function 1), a central
conserved DNA-binding domain that includes two Zn fingers (the C domain),
a short region responsible for nuclear localization (the D domain), and a large
fairly well-conserved carboxy-terminal ligand-binding domain (the E domain,
or LBD) that also contributes to interactions of the subset of nuclear receptors
that form heterodimers (Mangelsdorf et al. 1995). Some also possess a highly
variable carboxy-terminal tail (the F domain) that in most cases has unknown
functions.
The receptors can exist as monomers, homodimers, or heterodimers and
recognize DNA sequences termed hormone response elements (HREs)
derived from pairs of sequences with the consensus RGGTCA (R is a purine).
They can be grouped into four subtypes based on their mode of action. Type I
receptors, such as the androgen receptor, the estrogen receptor, and the
progesterone receptor, are anchored in the cytoplasm by chaperone proteins
(e.g., HSP90) (Echeverria and Picard 2010). Ligand binding frees the
receptor from the chaperone, allowing homodimerization, exposure of the
nuclear localization sequence, and entry into the nucleus (Fig. 1). Once in the
nucleus, the ligand–receptor complex associates with transcriptional
coactivators that facilitate binding to and activation of target genes (Glass and
Rosenfeld 2000; Bulynko and O’Malley 2011). Recent genome-wide location
analysis indicates that most nuclear-receptor binding sites in the genome are
located in enhancer elements that are far away from the transcriptional start
site, as first documented for the estrogen receptor (Carroll et al. 2006).
Studies of the glucocorticoid receptor suggest that the ligand-bound receptor
rapidly exchanges with its binding sites and that increases and decreases in
receptor activity follow changes in the concentration of endogenous
glucocorticoids.
Type II receptors, such as the thyroid hormone receptor and the retinoic
acid receptor, in contrast, reside in the nucleus bound to their specific DNA
response elements even in the absence of ligand. They generally form
heterodimers with the retinoid X receptor (RXR) and in the absence of ligand
exert active repressive functions through interactions with NCoR and SMRT
corepressor complexes (Chen and Evans 1995; Horlein et al. 1995) that are
associated with histone deacetylases (HDACs) (Watson et al. 2012). Binding
of ligand to the LBD leads to dissociation of corepressors and their
replacement with coactivator complexes. Coactivator complexes typically
contain proteins with enzymatic functions, including histone
acetyltransferases, that help open up chromatin and facilitate activation of
target genes (Glass and Rosenfeld 2000). Note that several type II receptors
bind to ligands produced in the same cell (e.g., LXR responses to oxysterols),
which allows cell-autonomous feedback regulation.
Type III receptors function similarly to type I receptors except that the
organization of the HRE differs (it is a direct repeat rather than inverted) and
type IV receptors instead bind as monomers to half-site HREs (Mangelsdorf
et al. 1995).
Ligands allosterically control the interactions of nuclear receptors with
coactivators and corepressors by influencing the conformation of a short
helix, referred to as AF2 (activation function 2), at the carboxy-terminal end
of the LBD (Glass and Rosenfeld 2000). In the absence of ligand, the AF2
helix is in an open conformation that enables binding of corepressors to type
II receptors. Upon agonist binding, the AF2 helix adopts a conformation in
which it forms one side of a charge clamp that grips the ends of a short helix
of consensus sequence LxxLL present in coactivator proteins that interact
directly with the LBD (Nolte et al. 1998). Selective modulation of nuclear
receptor activities can be achieved by synthetic ligands that differentially
alter the AF2 conformation (Glass and Rosenfeld 2000). For example, the
estrogen receptor modulator tamoxifen prevents AF2 from adopting a charge-
clamp conformation, thereby blocking AF2-dependent transcriptional
activity.
The functions of nuclear receptors can also be modulated by
posttranslational modifications that include phosphorylation, ubiquitylation,
and SUMOylation (Berrabah et al. 2011; Treuter and Venteclef 2011; Lee
and Lee 2012). Phosphorylation can activate some nuclear receptors
independently of ligand binding and function as the major mechanism
regulating activities of orphan receptors (Berrabah et al. 2011). Receptor
ubiquitylation can occur in response to ligand binding and may contribute to
termination of hormonal signaling (Lee and Lee 2012). SUMOylation
typically reduces the activation function of nuclear receptors and/or promotes
repressor activity (Treuter and Venteclef 2011).
A characteristic feature of nuclear receptors with respect to their
integrative roles in development and homeostasis is their ability to regulate
different genes in different cell types. For example, estrogen receptors
regulate different sets of genes in the brain, breast, and uterus that contribute
to the distinct functions of those organs. Recent studies indicate that tissue-
specific responses are a consequence of binding of nuclear receptors to
enhancer elements that are selected in a cell-specific manner. Cell-specific
enhancer selection is conferred by the key lineage-determining factors for
each cell type, which interact in a collaborative manner to generate open
regions of chromatin that provide access points for signal-dependent
transcription factors (Fig. 2) (Heinz et al. 2010). In the case of LXRs, for
example, macrophage-specific binding sites are established by interactions
between macrophage-lineage-determining factors that include PU.1 and AP-
1, whereas in liver (Heinz et al. 2010) LXR-binding sites occur in association
with the hepatocyte-lineage-determining factors HNF4 and C/EBPα
(Boergesen et al. 2012). In each case, a complex multistep process involving
numerous coactivator proteins is involved in building a functional enhancer,
and the tissue-specific responses can be further tailored by expression of
distinct coactivator/corepressor complexes (Fig. 2) (Bulynko and O’Malley
2011).

Figure 2. Tissue-specific nuclear receptor signaling in hepatocytes versus macrophages.

Given the wide variety of processes controlled by nuclear receptors, their

dysregulation can contribute to numerous diseases, including cancer,
diabetes, and infertility. However, because they bind to small molecules, they
represent promising therapeutic targets for which selective agonists and
antagonists can be engineered (Burris et al. 2012). Tamoxifen, for example, is
an estrogen receptor antagonist currently used to treat breast cancer, and
thiazolidinediones that target peroxisome proliferator-activated receptor γ
(PPARγ) are used to treat type 2 diabetes. Because nuclear receptors regulate
many genes in many tissues, synthetic ligands usually show beneficial
therapeutic effects and unwanted side effects that limit clinical use. Major
goals in the nuclear receptor field therefore include attaining a better
understanding of the mechanisms underlying their actions in specific cell
types and ways in which to selectively modulate their activities (Burris et al.
2012).

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Glass CK, Milburn MV. 1998. Ligand binding and co-activator assembly of the peroxisome
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nuclear receptor SUMOylation. Biochim Biophys Acta 1812: 909–918.
Watson PJ, Fairall L, Schwabe JW. 2012. Nuclear hormone receptor co-repressors: Structure and
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a016709
The Hippo Pathway

Kieran F. Harvey1 and Iswar K. Hariharan2

1Peter MacCallum Cancer Centre, East Melbourne 3002, Australia
2University of California, Berkeley, Berkeley, California 94720

Correspondence: ikh@berkeley.edu

The Hippo pathway (Fig. 1), also known as the Salvador-Warts-Hippo

pathway, regulates tissue growth in a wide variety of organisms (Harvey and
Tapon 2007; Grusche et al. 2010; Oh and Irvine 2010; Pan 2010; Halder and
Johnson 2011; Zhao et al. 2011). Many components of the pathway were
identified as a result of mutations in the fruit fly Drosophila melanogaster
that resulted in tissue overgrowth (Table 1). The pathway is conserved in
vertebrates, including mammals (Fig. 2), and changing the activity of the
pathway can result in dramatic changes in the size of certain organs, most
notably the liver (Pan 2010; Halder and Johnson 2011). In addition to its role
in regulating tissue growth, the pathway has been implicated in the control of
other biological processes, such as cell-fate determination, mitosis, and
pluripotency. Deregulation of Hippo pathway activity has been reported in
many human cancers. The human homolog of D. melanogaster Merlin
(MER), also known as Neurofibromatosis Type 2 (NF2) is a bona fide tumor
suppressor, while altered activity of several Hippo pathway components has
been implicated in human tumorigenesis (Harvey and Tapon 2007).
Figure 1. The Drosophila Hippo pathway.
 Figure 2. The mammalian Hippo pathway.

Table 1. Components of the Drosophila melanogaster Salvador-Warts-Hippo

pathway and their human homologues
Drosophila Human
Upstream
Fat (FT) FAT1—FAT4
Dachsous (DS) DCHS1 and DCHS2
Discs overgrown (DCO) CK1ε and CK1δ
Lowfat (LFT) LIX1 and LIX1L
Four-jointed (FJ) FJX1
Dachs (D) ?
Approximated (APP) ZDHHC9, ZDHHC14 and ZDHHC18
Zyxin (ZYX) Zyxin, LPP and TRIP6
Merlin (MER) Merlin (also known as NF2)
Expanded (EX) Willin/FRMD6 and FRMD1
Kibra Kibra
Crumbs (CRB) CRB1—CRB3
Lethal giant larvae (LGL) LGL1 and LGl2
Discs large (DLG) DLG1—DLG4
Scribble (SCRIB) SCRIB
aPKC aPKCι and aPKCζ
STRIPAK (PP2A) PP2A (STRIPAK)
RASSF RASSF1-RASSF6
Myopic (MOP) HD-PTP
JUB Ajuba, LIMD1, WTIP
TAO1 TAO1—TAO3

Core
Hippo (HPO) MST1 and MST2
Salvador (SAV) SAV1
Mats (MTS) MOBKL1A and MOBKL1AB
Warts (WTS) LATS1 and LATS2

Downstream
Yorkie (YKI) YAP and TAZ
WBP2 WBP2
Scalloped (SD) TEAD1—TEAD4
MAD SMADs
TSH TSHZ1—TSHZ3
HTH MEIS1—MEIS3

At the core of the pathway is a module composed of two kinases—Hippo

(HPO) (Harvey et al. 2003; Jia et al. 2003; Pantalacci et al. 2003; Udan et al.
2003; Wu et al. 2003) and Warts (WTS; also known as LATS) (Justice et al.
1995; Xu et al. 1995)—and two other proteins—Salvador (SAV) (Kango-
Singh et al. 2002; Tapon et al. 2002) and Mob as Tumor Suppressor (MATS)
(Lai et al. 2005). HPO functions upstream of WTS and can directly
phosphorylate it. Mutations that inactivate any of these four proteins result in
tissue overgrowth. The first indication that some of these proteins might
function in a pathway was the observation that sav and wts mutants display
similar phenotypic abnormalities and that the two proteins can interact with
each other (Tapon et al. 2002). More recently it has been shown that activity
of this module can be regulated by RASSF, a scaffold protein that promotes
tissue growth by recruiting the serine-threonine phosphatase complex
STRIPAK to inhibit HPO autophosphorylation, and hence HPO activity
(Ribeiro et al. 2010).
The main output of the module involves the transcriptional coactivator
Yorkie (YKI) (Huang et al. 2005). Phosphorylation of YKI by WTS induces
binding of 14-3-3 proteins to YKI that limit YKI activity by preventing
nuclear accumulation. Phosphatases that counter the activity of WTS have
not been discovered but the Myopic (MOP) tyrosine phosphatase regulates
YKI activity, repressing it (Gilbert et al. 2011). YKI promotes tissue growth
by increasing expression of positive regulators of cell growth and inhibitors
of apoptosis. YKI, itself does not bind DNA but functions together with
several transcription factors, including Scalloped (SD; the homolog of TEAD
transcription factors in vertebrates), Homothorax (HTH), Teashirt (TSH), and
Mothers against DPP (MAD). Transcriptional regulatory proteins such as
WBP2 also control Hippo-pathway-dependent tissue growth (Zhang et al.
2011). WBP2 and other as-yet-unidentified proteins have been predicted to
interact with YKI via its WW domains, which are important for YKI’s
transcription activation function (Oh and Irvine 2010).
The HPO and WTS kinases appear to receive multiple inputs. The first
upstream regulators to be discovered were the Band 4.1 proteins Expanded
(EX) and MER (Hamaratoglu et al. 2006). These function together with the
WW-domain-containing protein Kibra to activate the core kinase cassette by
an unknown mechanism. EX is also thought to repress YKI by physical
interaction and sequestration. The Fat/Dachsous branch of the pathway
consists of the atypical cadherins Fat (FT) and Dachsous (DS) as well as the
downstream effector proteins Discs overgrown (DCO, a serine-threonine
kinase also known as casein kinase 1ε), Dachs (D, an atypical myosin),
Approximated (APP, a palmitoyltransferase), Lowfat (LFT), and Zyxin
(ZYX) (Grusche et al. 2010; Rauskolb et al. 2011). The Fat/Dachsous branch
impinges on pathway activity by modulating the abundance of WTS and also
modulates the Kibra-EX-MER branch by regulating EX levels. The sterile
20-like kinase, TAO1, phosphorylates and activates HPO (MST1/2 in
mammals) although it is unclear whether TAO1 activity is regulated
(Boggiano et al. 2011; Poon et al. 2011).
Increasing evidence underlines the importance of cell junctions for
regulation of Hippo pathway activity. In D. melanogaster epithelial cells,
many Hippo pathway proteins reside, at least partially, at the sub-apical
region (SAR), adherens junction (AJ) or septate junction (SJ). Examples of
such junctional proteins include the AJ protein Jub and the apical-basal
polarity proteins Discs large (DLG), Lethal giant larvae (LGL), Scribble
(SCRIB), Crumbs (CRB), and atypical protein kinase C (aPKC). In
mammalian epithelial cells, several other junctional proteins regulate Hippo
pathway activity (see Table 2), including angiomotin and α-catenin
(Schlegelmilch et al. 2011; Zhao et al. 2011). The Hippo pathway may
therefore help couple tissue growth to mechanical stresses or cell–cell
contact, which might be important for organ size regulation.

Table 2. Components of the human Salvador-Warts-Hippo pathway and their

Drosophila melanogaster homologs
Human Drosophila
Upstream
α-Catenin α-Catenin
PATJ Discs lost
PALS1 Stardust
AMOTs ?
β-TRCP Slimb

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Gilbert MM, Tipping M, Veraksa A, Moberg KH. 2011. A screen for conditional growth suppressor
genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie. Dev
Cell 20: 700–712.
Grusche FA, Richardson HE, Harvey KF. 2010. Upstream regulation of the hippo size control pathway.
Curr Biol 20: R574–582.
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Hamaratoglu F, Willecke M, Kango-Singh M, Nolo R, Hyun E, Tao C, Jafar-Nejad H, Halder G. 2006.
The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate
cell proliferation and apoptosis. Nat Cell Biol 8: 27–36.
Harvey K, Tapon N. 2007. The Salvador-Warts-Hippo pathway—an emerging tumour-suppressor
network. Nat Rev Cancer 7: 182–191.
Harvey KF, Pfleger CM, Hariharan IK. 2003. The Drosophila Mst ortholog, hippo, restricts growth and
cell proliferation and promotes apoptosis. Cell 114: 457–467.
Huang J, Wu S, Barrera J, Matthews K, Pan D. 2005. The Hippo signaling pathway coordinately
regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP.
Cell 122: 421–434.
Jia J, Zhang W, Wang B, Trinko R, Jiang J. 2003. The Drosophila Ste20 family kinase dMST functions
as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev 17:
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Justice RW, Zilian O, Woods DF, Noll M, Bryant PJ. 1995. The Drosophila tumor suppressor gene
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cell shape and proliferation. Genes Dev 9: 534–546.
Kango-Singh M, Nolo R, Tao C, Verstreken P, Hiesinger PR, Bellen HJ, Halder G. 2002. Shar-pei
mediates cell proliferation arrest during imaginal disc growth in Drosophila. Development 129:
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Poon CL, Lin JI, Zhang X, Harvey KF. 2011. The sterile 20-like kinase Tao-1 controls tissue growth by
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011288
SECTION III

SIGNALING PROCESSES
CHAPTER 5

Signaling Pathways that Control Cell

Proliferation

Robert J. Duronio1,2,3 and Yue Xiong2,3,4

1Department of Biology and Genetics, University of North Carolina, Chapel Hill, North Carolina
27599
2Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North
Carolina 27599
3Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina
27599
4Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North
Carolina 27599
Correspondence: duronio@med.unc.edu; yxiong@email.unc.edu

SUMMARY

Cells decide to proliferate or remain quiescent using signaling pathways

that link information about the cellular environment to the G1 phase of
the cell cycle. Progression through G1 phase is controlled by pRB
proteins, which function to repress the activity of E2F transcription
factors in cells exiting mitosis and in quiescent cells. Phosphorylation of
pRB proteins by the G1 cyclin-dependent kinases (CDKs) releases E2F
factors, promoting the transition to S phase. CDK activity is primarily
regulated by the binding of CDK catalytic subunits to cyclin partners
and CDK inhibitors. Consequently, both mitogenic and antiproliferative
signals exert their effects on cell proliferation through the transcriptional
 regulation and ubiquitin-dependent degradation of cyclins and CDK
inhibitors.

Outline
1 Introduction
2 Transcriptional regulation of G1 cyclins by mitogenic signals
3 Transcriptional regulation of CDK inhibitors
4 Control of G1 cyclins by the ubiquitin–proteasome system
5 Control of G1 CDK inhibitors by the ubiquitin–proteasome system
6 Concluding remarks
References

1 INTRODUCTION
Control of cell proliferation generally occurs during the first gap phase (G1)
of the eukaryotic cell division cycle (see Box 1). Multiple signals, ranging
from growth factors to DNA damage to developmental cues, influence the
decision to enter S phase, when DNA is replicated (Fig. 1). Hence, G1 phase
cell cycle control is intrinsically linked with a diverse set of pathways
controlling differentiation, stem and progenitor cell quiescence, senescence,
and responses to a variety of stresses. The decision to enter S phase from G1
represents a point of no return that, in the absence of stress such as DNA
damage, commits cells to complete the cell cycle and divide, and is therefore
tightly controlled. This decision is made at what is called the “restriction
point” in mammalian cells and “START” in yeast, after which cells become
largely refractory to extracellular signals and will complete S phase and
proceed through a second gap phase (G2 phase) and then mitosis. In
multicellular organisms, most differentiated cells exit the active cell cycle
during G1 phase and enter G0 phase, in which they remain metabolically
active for days or even years, performing specialized functions. Postmitotic
nerve and skeletal muscle cells provide good examples. Some G0 cells, such
as quiescent T cells, can be stimulated by mitogenic signals to reenter the cell
cycle.

Figure 1. G1 cell cycle control by the pRB pathway. Many cellular signaling events are intrinsically
linked to G1 phase of the cell cycle, which is controlled by the RB pathway. Signaling to the RB
pathway, and thus G1 control by different cellular processes, is achieved mainly through the regulation
of cyclins and CDK inhibitors (CKIs). In mammalian cells, mitogenic signals first induce the synthesis
of D-type cyclins, leading to activation of cyclin-D-dependent CDK4 and CDK6, and then induce E-
type cyclins to activate CDK2. Cyclin-D–CDK4/6 and cyclin-E–CDK2 cooperatively phosphorylate
RB-family proteins, derepressing E2F to allow transcription of E2F-target genes, thereby promoting the
G1/S transition. The INK4 proteins specifically inhibit CDK4 and CDK6, whereas the p21 (CIP/KIP)
family of CKIs inhibits multiple CDKs. Although the schematic illustration is based on mammalian
cells, the regulation of both G1 cyclins and CDK inhibitors is evolutionarily conserved.

BOX 1. THE EUKARYOTIC CELL CYCLE

The classical cell cycle comprises four phases—G1, S, G2, and M—and
 is controlled by cyclin-dependent kinases (CDKs) and their cyclin
partners. The commitment to divide occurs in G1 phase, which is
controlled by cyclin-D–CDK4/6 and cyclin-E–CDK2 at the so-called
G1/S transition. DNA is then replicated in S phase. This is followed by a
second gap phase, G2, at the end of which cyclin-B–CDK1 controls
entry into M phase (mitosis), when the cell divides. Cells can exit the
cell cycle in G1 phase and enter G0 phase (quiescence). In some cases,
they can reenter the cell cycle and begin dividing again (see main text).

The restriction point is primarily controlled in mammalian cells by the RB

pathway, named after the first tumor suppressor identified, the retinoblastoma
protein (pRB) (Weinberg 1995). pRB is a member of a highly conserved
family of proteins, encoded by a single gene in the single-celled green alga
Chlamydomonas (MAT3), Caenorhabditis elegans (LIN-35), and Arabidopsis
(RBR1); two genes in Drosophila (RBF1 and RBF2); and three genes in
mammalian cells (RB1; p107, also known as RBL1; and p130, also known as
RBL2) (Weinberg 1995; van den Heuvel and Dyson 2008). Budding yeast
cells contain a protein (Whi5) that, although it does not share sequence
similarity with pRB, functions at START in a similar manner (Costanzo et al.
2004; de Bruin et al. 2004). pRB proteins are present as hypophosphorylated,
active forms in cells exiting mitosis and in quiescent cells, where they use a
conserved pocket to bind to LxCxE motifs in numerous chromatin-associated
proteins and transcription factors, particularly members of the E2F family.
pRB proteins negatively regulate the expression of E2F-target genes, many of
which are required for entry into and progression through S phase, by
recruiting various repressive chromatin regulatory complexes and histone-
modifying enzymes or by blocking the transactivation function of E2F
proteins. Phosphorylation of the pRB family proteins by CDKs during G1
phase causes pRB to dissociate from E2Fs, allowing the transcription of
target genes that stimulate progression into S phase (Fig. 1) (Dyson 1998).
The principal kinases that phosphorylate pRB family proteins during G1
phase in mammalian cells are three cyclin-dependent kinases (CDKs)5:
cyclin-D-dependent CDK4 and CDK6 (Ewen et al. 1993; Kato et al. 1993)
and cyclin-E-dependent CDK2 (Akiyama et al. 1992; Hinds et al. 1992). As
many as eight distinct mammalian G1 CDK–cyclin complexes can be formed
from combinatorial association of three D-type cyclins (cyclins D1, D2, and
D3) with CDK4 and CDK6 and two E-type cyclins (cyclins E1 and E2) with
CDK2, and these phosphorylate as many as 16 sites in pRB proteins
(Akiyama et al. 1992; Kitagawa et al. 1996). Regulation of pRB-E2F by G1
CDKs has been evolutionarily conserved in plants, worms, flies, and
mammals (Inze 2005; van den Heuvel and Dyson 2008). The complexity of
the pRB pathway reflects the need to meet the demand to integrate diverse
signals from different signaling pathways into a central G1 control
mechanism. Disruption of this mechanism results in a wide range of
developmental defects and human diseases, particularly cancer. Indeed,
disruption of G1 control probably represents a common event in the
development of most types of human cancer (Sherr 1996).
The critical role of pRB and G1 CDKs in controlling the G1/S transition is
further illustrated by the studies of three DNA tumor viruses: adenovirus,
human papilloma virus (HPV), and simian virus 40 (SV40). Although
evolutionarily distant from each other, these viruses encode unrelated
proteins (E1A in adenovirus, E7 in HPV, and large T in SV40) that bind to
and inactivate pRB via an LxCxE motif to promote cell proliferation and viral
replication. Primate herpesvirus saimiri and human Kaposi’s sarcoma virus
encode cyclin D homologs (v-cyclins) that preferentially bind to and activate
CDK6, creating complexes that are resistant to CDK inhibitors (CKIs; see
below).
The steady-state levels of CDK2, CDK4, and CDK6 proteins remain
relatively constant during the normal cell cycle and in quiescent, aging, and
even terminally differentiated cells. Signaling pathways that affect G1 phase
progression thus do not affect CDK levels and instead act mainly through
regulation of CDK activity by controlling the abundance of their cyclin
partners and a group of CKIs. Although both cyclins and CKIs can be
regulated at the level of mRNA stability, translational control, and subcellular
localization, the two major control mechanisms are transcriptional regulation
and ubiquitin-dependent proteolysis. We discuss these mechanisms below,
focusing on the regulation of expression and ubiquitylation of G1 cyclins and
CKIs by different signal transduction pathways.

2 TRANSCRIPTIONAL REGULATION OF G1 CYCLINS

BY MITOGENIC SIGNALS
2.1 D-Type Cyclins
D-type cyclins were simultaneously isolated initially from mammalian cells
in a genetic screen for genes capable of complementing G1 cyclin deficiency
in yeast, as the product of a gene whose expression is induced by colony-
stimulating factor (CSF1), and as the product of the potential oncogene BCL1
that is clonally rearranged and overexpressed in a subset of parathyroid
tumors (Matsushime et al. 1991; Motokura et al. 1991; Xiong et al. 1991).
These findings provided early evidence linking the activation of a G1 cyclin
with mitogenic growth factors and implicating abnormal expression of G1
cyclins in tumorigenesis. However, subsequent genetic analyses revealed
only a relatively minor role of cyclin-D-dependent CDK activity in cell
proliferation and development (Meyer et al. 2000; Kozar et al. 2004;
Malumbres et al. 2004), although mouse embryonic fibroblasts (MEFs) from
mice lacking CDK4 and CDK6 do have a reduced rate of exiting from
quiescence in response to mitogenic stimulation. Hence, the D-type cyclins,
although not an obligate component of the cell cycle machinery, couple
extracellular mitogenic signals to the G1/S transition (Sherr and Roberts
2004).
The canonical Ras–Raf–MEK–ERK mitogen-activated protein kinase
(MAPK) pathway is the best characterized pathway for the activation of
cyclin D transcription (p. 81 [Morrison 2012]). It stimulates the expression of
AP1 transcription factors such as the proto-oncogene products Jun and Fos,
which bind directly to an AP1 site in the cyclin D1 promoter (Albanese et al.
1995). D-type cyclins can also be induced by other signaling pathways,
including mitogen-stimulated Rac and NF-κB signaling, cytokine signaling,
signaling by receptors for extracellular matrix (ECM) proteins (e.g.,
integrins), and the Wnt and Notch pathways (p. 109 [Kopan 2012] and p. 103
[Nusse 2012]). Multiple transcription factors directly regulate cyclin D genes,
including Jun, Fos, STAT3, β-catenin, and NF-κB (Fig. 2A). Cyclin D genes
are expressed at very low levels in most differentiated tissues, in part because
of transcriptional repression by proteins such as Jumonji and SIP (Klein and
Assoian 2008). Repression of G1 cyclin expression is an important part of
cell cycle exit and terminal differentiation, and inappropriate reactivation of
D- or E-type cyclins can drive differentiated cells back into S phase (Buttitta
et al. 2007; Korzelius et al. 2011).
Figure 2. Transcriptional regulation of G1 cyclins. (A) The expression of cyclin genes is tightly
regulated at the level of transcription by different signals, including many mitogens. The figure uses
human cyclin D1 as an example. (B) Cyclin E expression is also highly regulated and responds to two
types of developmental signals, those that are cell-type specific and those that all cells use to control
proliferation in response to their environment. MAPK, Mitogen-activated protein kinases; ECM,
extracellular matrix; STAT, signal transducers and activators of transcription; KLF, Krüppel-like
factor; CSL, CBF-1/suppressor of hairless/LAG-1; TCF, ternary complex factor; NF-κB, nuclear
factor-κB; SIP1, SMAD interacting protein 1; HH, Hedgehog; Ci, Drosophila cubitus interruptus; YKI,
Yorkie.

In contrast to cyclin D repression, inappropriate cyclin-D-dependent

CDK4/6 activity represents the most frequent alteration of human cyclins in
cancer and bears clear pathological significance. Human cyclin D1 is
amplified in an estimated 13% of neoplasms of different types, including
breast cancer, esophageal cancer, and lymphoma (Bates and Peters 1995).
Mice transgenically expressing cyclin D1 develop mammary gland tumors
and conversely are protected against mammary tumors if cyclin D1 is deleted
(Wang et al. 1994; Yu et al. 2001). Likewise, CDK4 and CDK6 are also
frequently amplified in diverse human cancers. Mouse cells lacking either
combination of the three cyclin D proteins or CDK4/6 are more resistant to
oncogenic transformation (Sherr and Roberts 2004; Malumbres and Barbacid
2009). These observations indicate that whereas a low level of G1 CDK
activity is sufficient to support cell proliferation in response to normal
physiological levels of mitogens, significantly higher levels of G1 CDK
activity are required to sustain hyperproliferative stimulation, such as those
elicited by activated oncogenes.

2.2 Cyclin E Expression

Cyclin E is encoded by a single gene in C. elegans (CYE-1) and Drosophila
(CycE) and by two genes in mammalian cells (E1 and E2). The worm and fly
cyclin E genes are essential for cell cycle progression and development
(Knoblich et al. 1994; Fay and Han 2000). In contrast, mice lacking both
cyclin E1 and E2 or CDK2 are viable and display relatively minor defects
late in development, owing to compensation by other CDKs (Berthet et al.
2003; Geng et al. 2003; Ortega et al. 2003; Parisi et al. 2003). In well-fed
proliferating cells, cyclin E expression is cyclical, peaking at the G1/S
transition and being low or absent at other times in the cell cycle (Lew et al.
1991; Dulic et al. 1992; Koff et al. 1992). Conversely, MEFs lacking both
cyclins E1 and E2 proliferate more slowly than normal cells and have a
significantly reduced response to mitogenic stimulation, and cyclin E gene
expression is repressed in serum-deprived cells, all of which suggest that
cyclin E responds to growth factors (Herrera et al. 1996; Geng et al. 2003).
This regulation is important, because forced overexpression of cyclin E can
shorten G1 phase and drive cells into S phase, in part by causing
phosphorylation of pRB family proteins (Hinds et al. 1992; Ohtsubo and
Roberts 1993; Resnitzky et al. 1994). In vivo, transgenic expression of cyclin
E under the control of the β-lactoglobulin promoter in mice results in
mammary tumorigenesis (Smith et al. 2006), and overexpression of cyclin E
is frequently observed in various human cancers and correlates with
increased tumor aggression (Hwang and Clurman 2005). Hence, tight control
of the levels of cyclin E is critically important for normal cell physiology and
for preventing a neoplastic cell cycle. This notion is supported by
biochemical and genetic analyses of the regulation of cyclin E by
phosphorylation and by its regulatory protein FBW7 (see below).
Cyclin E transcription is directly controlled by E2F (Duronio and
O’Farrell 1995; Ohtani et al. 1995; Geng et al. 1996). Thus, one important
way that signaling regulates cyclin E is through the pRB/E2F pathway, which
also integrates the output from the growth factor signals that control D-type-
cyclin-dependent CDK activity. Indeed, if the mouse cyclin E gene is
engineered to respond to the signals that control cyclin D1 gene expression,
then cyclin D1 is no longer needed (Geng et al. 1999). Because cyclin-E–
CDK2 can phosphorylate and inactivate pRB, resulting in E2F activity, a
positive-feedback amplification is an important part of G1/S control (Fig.
1B). This helps produce the switch-like behavior needed for unidirectional
decisions like the G1/S transition (Xiong and Ferrell 2003; Ferrell et al.
2009).
Control of cyclin E transcription via E2F is a cornerstone of G1/S cell
cycle control, but the cyclin E gene also responds directly to signaling
pathways. This often occurs when developmental programs coordinate cell
cycle progression with cell differentiation. In the Drosophila eye, for
example, Hedgehog signaling induces cyclin E at the G1/S transition of the
last cell cycle before differentiation of specialized cell types such as
photoreceptors (p. 107 [Ingham 2012]). The Drosophila CycE gene contains
multiple enhancer elements that respond to and integrate various signals (Fig.
2B), including those from the pRB/E2F, Hedgehog and Wnt signaling
pathways, in different cell types at different stages of development (Jones et
al. 2000; Deb et al. 2008; p. 107 [Ingham 2012]).
In Drosophila, CycE is also a target of the growth-inhibitory Hippo
pathway (p. 133 [Harvey and Hariharan 2012]), whose main target is the
inactivation of the transcriptional coactivator Yorkie (YKI) (Huang et al.
2005). Tissue overgrowth upon disruption of the Hippo pathway is
accompanied by increased expression of cyclin E, probably through direct
regulation of CycE transcription by transcription factors associated with YKI.
In vertebrates, the Hippo-pathway-mediated regulation of cell proliferation
appears to be largely mediated by cyclin D1 (Cao et al. 2008).
 Transcription of the cyclin E gene thus responds to two types of
developmental signals: those that are cell type specific and essential for cell
cycle progression (e.g., Hedgehog and Wnt signals), and those that are not
cell type specific or strictly essential for cell cycle progression but instead
modulate the rate of growth and cell proliferation in response to the cellular
environment (e.g., E2F-mediated responses and Hippo) (Fig. 2B).

2.3 Posttranscriptional Regulation of CDKs

Posttranscriptional mechanisms also regulate CDK activity in response to
various signals. The mitotic CDK, CDK1 (also known as CDC2), is inhibited
during interphase by phosphorylation at two adjacent residues within its
catalytic pocket, T14 and Y15, and is activated by CDC25-mediated
dephosphorylation to bring about a sudden burst of CDK1 activity that
triggers mitosis (Ch. 6 [Rhind and Russel 2012]). Both CDK2 and CDK4 are
also phosphorylated at analogous residues to mediate the responses to
different signals: phosphorylation of T14 and Y15 of CDK2 is important for
regulating the timing of DNA replication and centrosome duplication (Zhao
et al. 2012), and phosphorylation of Y17 of CDK4 is required for G1 arrest
upon UV irradiation, which could cause DNA damage that should be repaired
before entry into S phase (Terada et al. 1995).

3 TRANSCRIPTIONAL REGULATION OF CDK

INHIBITORS
CKIs play an important role in arresting the cell cycle in G1 phase in response
to a variety of stimuli, ranging from growth factor deprivation to DNA
damage, cellular stress, differentiation, and senescence. Failure to arrest the
cell cycle resulting from loss of function of a CKI can cause developmental
defects or hyperplasia and tumorigenesis. The first CKI characterized was
mammalian p21 (also known as CDKN1A, CIP1, or WAF1), which binds to
and inhibits the activity of multiple CDK–cyclin complexes (Xiong et al.
1992, 1993a; Harper et al. 1993). The p21 family (also known as the CIP/KIP
family) includes three related proteins: p21, p27 (also known as CDKN1B or
KIP1), and p57 (also known as CDKN1C or KIP2). A distinct CKI, p16 (also
known as INK4A), was isolated around the same time and is a specific
inhibitor of CDK4 (Serrano et al. 1993). p16 is the founding member of a
separate family of INK4 CKIs that includes three additional proteins: p15
(also known as INK4B), p18 (also known as INK4C), and p19 (also known
as INK4D) (Sherr and Roberts 1995).
These two families of CKIs inhibit CDK via different mechanisms. The
INK4 proteins bind selectively to the catalytic subunits of two CDKs, CDK4
and CDK6, preventing cyclin binding; and the p21 CKIs bind to the cyclin–
CDK complex by contacting both subunits via different motifs to block
kinase activity and substrate binding. CKIs of both families are localized
predominantly in the nucleus in most tissues, but p21 family CKIs have also
been frequently observed in the cytoplasm, where they have been linked to
CDK-independent functions and tumor development. In particular, reduced
nuclear p27 and accumulation of cytoplasmic p27 have been observed in
multiple types of human cancers and are associated with poor prognosis of
breast cancer (Wander et al. 2011).
The two separate families of multiple CDK inhibitors evolved to meet the
increasing need to integrate numerous different antiproliferative signals that
can arrest cells in G1 phase. Mice lacking CKI genes have various
phenotypes, ranging from a compromised DNA damage response (p21
mutants) to widespread hyperplastic cell proliferation and organomegaly
(p18- and p27-null mice), spontaneous tumor development (p16-null mice),
and perinatal lethality and widespread developmental defects (in p57-null
mice) (Ortega et al. 2002). Furthermore, genetic studies of p21-type CKIs in
worms and flies have revealed various functions from control of cell cycle
progression to cell cycle exit in specific cell types at various times in
development (de Nooij et al. 1996; Lane et al. 1996; Hong et al. 1998; Firth
and Baker 2005).
One major difference between the two CKI families is their stability. The
p21 family inhibitors are intrinsically unstable (t1/2 < 30 min) as a result of
ubiquitin-dependent, and in most cases phosphorylation-promoted,
proteasomal degradation, and cause a rapid and transient cell cycle arrest, for
example, following DNA damage. In contrast, the INK4 proteins are stable
(t1/2 > 4–6 h) and are subject to minimal posttranslational regulation. INK4
proteins therefore maintain a long-term or permanent cell cycle arrest in stem,
progenitor, senescent, and postmitotic cells. Accordingly, whereas p21 family
CKIs are regulated both transcriptionally and posttranscriptionally, the INK4
members are regulated primarily at the level of transcription.

3.1 p21 Transcription Regulation by p53-Dependent and -

Independent Mechanisms
Cells use signaling pathways to respond to a variety of exogenous and
intrinsic stresses that have the potential to damage the genome. The tumor
suppressor p53 functions as a transcription factor to activate the expression of
many genes involved in stress responses, and defects in p53-mediated stress
responses are associated with most types of human cancer. p53-mediated
transcriptional activation of p21 following DNA damage was the first
identified example of G1-phase regulation of a CKI gene (El-Deiry et al.
1993; Xiong et al. 1993b). Given that none of the other six CKI genes is a
direct target of p53, the p53–p21–CDK regulatory module constitutes a major
mechanism for DNA-damage-induced cell cycle arrest. Indeed, knocking out
the p21 gene compromises the DNA damage response despite having little
effect on overall mouse development (Brugarolas et al. 1995; Deng et al.
1995).
Transcriptional regulation of the p21 gene has also been linked to p53-
independent cell cycle exit during development. In the Drosophila embryonic
epidermis, activation of the dacapo (dap) gene, which encodes a p21-type
CKI, triggers cell cycle exit (de Nooij et al. 1996; Lane et al. 1996). In
Caenorhabditis elegans, the insulin-like growth factor signaling pathway
similarly induces p21 expression in response to starvation, which results in
cell cycle arrest in stem cells (Baugh and Sternberg 2006), and Ras/MAPK
signaling activates p21 to control cell cycle exit in vulval precursor cells
(Clayton et al. 2008). This diversity of responses probably relies on the
existence of multiple, modular enhancers for the p21 gene that respond to
different signaling pathways (Liu et al. 2002; Meyer et al. 2002).
3.2 INK4 Repression in Stem and Progenitor Cells
INK4 genes have distinct expression patterns during development in adult
tissues and in response to different conditions (Roussel 1999). p16 is a target
of Polycomb group (PcG) transcriptional repressors: deletion of the
Polycomb gene Bmi1 retards cell proliferation, and this is associated with up-
regulation of p16 and can be partially rescued by deletion of p16 (van
Lohuizen et al. 1991; Jacobs et al. 1999). Furthermore, both PcG repression
complexes (PRC1 and PRC2) collaborate with pRB proteins to bind to the
p16 locus and trimethylate histone H3 lysine 27 (H3K27) to repress the
expression of p16 (Bracken et al. 2007; Kotake et al. 2007). These findings
explain how the up-regulation of p16 in aging stem cells results from
decreased expression of Polycomb genes and reveal a negative-feedback loop
between p16 and pRB.6 In many different types of human tumors, p16
expression is silenced by promoter DNA methylation (Merlo et al. 1995).
Unlike p16 mRNA, which is undetectable in young tissues and is induced
during aging, p18 mRNA is present early in embryogenesis and maintains a
high level throughout life in many adult tissues (Zindy et al. 1997). Deletion
of p18 in mice results in spontaneous development of various tumors
(Franklin et al. 1998; Pei et al. 2009) and increases self-renewing division of
hematopoietic stem cells and expansion of mammary luminal progenitor cells
(Yuan et al. 2004; Pei et al. 2009). p18 thus seems to suppress tumorigenesis
by maintaining a quiescent state in stem and progenitor cells of different
organs. GATA3, a transcription factor specifying mammary luminal cell fate,
binds to the p18 locus and represses p18 transcription (Pei et al. 2009). It
provides an example of a lineage-specifying factor that regulates cell
differentiation in part by repressing the expression of an INK4 gene to allow
quiescent progenitor cells to exit G0/G1 arrest, reenter the cell cycle, and
proliferate.

4 CONTROL OF G1 CYCLINS BY THE UBIQUITIN–

PROTEASOME SYSTEM
Like their mitotic counterparts, G1 cyclins undergo rapid turnover and are
degraded by the ubiquitin–proteasome pathway. This process is tightly
regulated through the phosphorylation of cyclins and, in some cases, by
proteins that target cyclins to E3 ubiquitin ligases, which provide
mechanisms for extracellular factors to signal to the G1-phase cell cycle
control machinery.
The level of cyclin E, and associated CDK2 activity, oscillates during the
cell cycle (Dulic et al. 1992; Koff et al. 1992). Cyclin E begins to accumulate
during the middle of G1 phase (as a result of E2F-mediated transcriptional
activation), peaks at the G1/S transition, and then is destroyed during S phase
following ubiquitylation. FBW7 (also known as Cdc4 or Ago) is an F-box
protein that is the substrate-recognition component of the E3 ubiquitin ligase
SCF (also known as CRL1) and recognizes two phosphodegrons in cyclin E:
a carboxy-terminal degron centered on T380 and an amino-terminal degron
centered on T62 (Fig. 3) (Welcker and Clurman 2008). Both cyclin E degrons
are phosphorylated by GSK3 and CDK2 itself, creating two independent
FBW7-binding sites. Cyclin-E–CDK2 is thought to phosphorylate cyclin E
first at T384, creating a “priming phosphate” that is needed for GSK3 to
phosphorylate T380 upstream, thus generating the doubly phosphorylated
phosphodegron that is specifically recognized by the FBW7 targeting subunit
of SCF-FBW7.
Figure 3. Targeting ubiquitin-dependent degradation of cyclin E. F-box protein FBW7 specifically
recognizes two separate phosphodegrons in cyclin E and targets cyclin E for ubiquitin-dependent
proteasome degradation by the SCF-FBW7 E3 ligase complex. The phosphorylation of both amino-
and carboxy-terminal degrons in cyclin E is catalyzed by GSK3 and CDK2 and creates two separate
binding sites for FBW7. Both mitogenic and antiproliferative signals exert their effect on the cell cycle
through cyclin E ubiquitylation by inhibiting the activity of GSK3 or stimulating the expression of
FBW7, respectively.

Because GSK3 plays critical roles in diverse signals, including those

activated by insulin, mitogenic growth factors, Wnts, Hedgehog, and
cytokines, GSK3 activity can link the regulation of cyclin E and thus G1
progression to different signaling pathways. For example, GSK3 is regulated
by the phosphoinositide 3 kinase (PI3K)–AKT pathway, which allows a
major mitogen signaling pathway (p. 87 [Hemmings and Restuccia 2012]) to
couple cell growth to G1 regulation. Transgenic expression of mutant cyclin
E (T380A) in mammary glands causes more widespread hyperplasia than that
of wild-type cyclin E and promotes p53 loss of heterozygosity and
tumorigenesis (Smith et al. 2006). Knock-in mutations that ablate both T62
and T380 result in disruption of cyclin E periodicity, increased cyclin E
activity, and abnormal proliferation in multiple cell types (Minella et al.
2008).
Studies of Fbw7-mutant mice and loss-of-function mutations of FBW7 in
human cancer support a role for SCF-FBW7 in negative regulation of cell
proliferation by targeting cyclin E, as well as Myc, Notch, and Jun (Welcker
and Clurman 2008). Mitogen signaling can also influence the activity of
FBW7 itself. In mammalian cells, activated Ras increases cyclin E levels by
inhibiting binding of cyclin E to FBW7 (Welcker and Clurman 2008), and
Notch and Hedgehog signaling suppresses cyclin E accumulation by inducing
FBW7 expression in Drosophila eye imaginal discs (Nicholson et al. 2011).
Therefore, both oncogenic and developmental signals can control the level of
cyclin E protein by regulating components of the E3 ubiquitin ligase that
targets cyclin E for destruction (Fig. 3).
Cyclin D is phosphorylated at T286, a site analogous to T380 in cyclin E,
and T286 phosphorylation promotes cyclin D destruction (Diehl et al. 1998).
Multiple F-box proteins, such as Fbxo41, Fbxw8, SKP2, and Fbxo31, have
been implicated in targeting cyclin D for destruction, but the E3 ligase
responsible remains to be definitively identified (Kanie et al. 2012).
Promoting the destruction of both D- and E-type G1 cyclins by GSK3-
mediated phosphorylation, however, could allow cells to effectively couple
the PI3K–AKT pathway to G1 cell cycle control. T286-phosphorylated cyclin
D1 can also be recognized and stabilized in the nucleus by Pin1, a prolyl
isomerase that regulates the function of proteins by causing conformational
change of their S/T-phosphorylated forms (Liou et al. 2002).
Progression through G1 phase is also controlled by other E3 ligases. In
particular, the anaphase-promoting complex (APC), which promotes the
ubiquitin-dependent proteasomal degradation of multiple mitotic regulatory
proteins, remains active in G1 phase to suppress accumulation of mitotic
cyclins until cyclin-E–CDK2 is activated at the G1/S transition.

5 CONTROL OF G1 CDK INHIBITORS BY THE

UBIQUITIN–PROTEASOME SYSTEM
Some CKIs are also regulated by the ubiquitin–proteasome pathway. Again,
this regulation involves phosphorylation of these CKIs, which provides a
mechanism linking extracellular signaling to the G1 cell cycle control
machinery.

5.1 Phosphorylation-Dependent Ubiquitylation and

Degradation of a Yeast CKI
In Saccharomyces cerevisiae, a single CDK, Cdc28, forms multiple B-type
cyclin–CDK complexes to drive both S phase and mitosis. Cdc28 is inhibited
by Sic1, a CKI that is unrelated in sequence to either the p21 or INK4 family
of CKIs. Sic1 is targeted for ubiquitylation (Fig. 4) following
phosphorylation by the G1 cyclin–CDK complex Cln–Cdc28 (Schwob et al.
1994). Inactivation of Sic1 rescues the inviability of yeast cells lacking the
G1 cyclins Cln1, Cln2, and Cln3 (Schneider et al. 1996), and mutation of
CDK phosphorylation sites in Sic1 causes stabilization of Sic1 and blocks
DNA replication. These observations indicate that the primary function of
these three G1 cyclins, once mitogenically activated, is to promote Sic1
ubiquitylation to bring about the G1/S transition. Phosphorylated, but not
unmodified, Sic1 binds to the F-box protein Cdc4, which, through a linker
protein, Skp1, brings Sic1 to the Cul1 (also known as Cdc53)–Roc1 (also
known as Rbx or Hrt1) E3 ligase complex for ubiquitylation by the E2
enzyme Cdc34 (Feldman et al. 1997; Skowyra et al. 1997). Nine sites in Sic1
are phosphorylated, and each contributes to Cdc4 binding, with any six being
required (Nash et al. 2001). This multisite phosphorylation requirement
makes Sic1 ubiquitylation ultrasensitive to the level of G1 CDK activity,
enabling cells to measure the strength of mitogens and set the level of CDK
activity that determines the timing of DNA replication. It transforms a
gradual accumulation process, such as protein synthesis during G1 phase, into
an irreversible switch for the onset of DNA replication. Sic1 is also
phosphorylated by its target, the B-type cyclin–CDK complex Clb5–CDK1,
which may ensure irreversibility of the G1/S transition once DNA replication
has been initiated.

Figure 4. Targeting ubiquitin-dependent degradation of CDK inhibitors. The p21 family of CKIs is
regulated by the ubiquitin–proteasome pathway. In many cases, this involves phosphorylation of these
CKIs. Phosphorylated CKIs are recognized by F-box proteins such as Cdc4 in budding yeast or SKP2
in human cells, which, through the SKP1 linker protein, recruits the CKI substrate to the SCF E3 ligase
for ubiquitylation.

In response to mating pheromones, budding yeast cells arrest their cycle

in G1 phase and fuse cytoplasms and nuclei to generate a diploid cell. This G1
cell cycle arrest is regulated by the Fus3 MAPK pathway, which leads to
phosphorylation and activation of Far1, a second budding yeast CDK
inhibitor that is unrelated to Sic1 and other CKIs in sequence. Far1
selectively inhibits G1 cyclin–Cdc28, leading to the inhibition of Cln–Cdc28-
induced Sic1 degradation and G1 arrest.
The distantly related fission yeast, Schizosaccharomyces pombe, contains
a single CKI, Rum1, that is unrelated to Sic1, p21, or INK4 CKIs in
sequence. Rum1 inhibits the cyclin B–CDK complex Cdc13–Cdc2 and is an
essential G1 regulator whose deletion causes premature S-phase initiation
immediately after mitosis (Correa-Bordes and Nurse 1995). Rum1 is
degraded following ubiquitylation by the SCF-Pop1 ligase, which uses Pop1,
an ortholog of budding yeast Cdc4, to target Rum1 (Kominami and Toda
1997). Hence, the mechanism for targeting G1 CDK inhibitors for
ubiquitylation has been conserved between two yeast species that are as
evolutionarily divergent from each other as either is from animals.

5.2 Regulation of Mammalian CIP/KIP by E3 Ligases

The mammalian CKI p27 is also regulated by ubiquitin-dependent proteolysis
(Pagano et al. 1995). p27 and its close relative p57 are phosphorylated by
cyclin-E–CDK2 at analogous sites (T187 in p27 and T310 in p57), which
promotes their binding to the F-box protein SKP2 and subsequent
ubiquitylation by the SCF-SKP2 E3 ligase. The recognition of T187-
phosphorylated p27 by SKP2 requires CKS1, a small evolutionarily
conserved protein whose function is essential for yeast cell viability and
normal mouse development (Fig. 3). A second p27 E3 ligase, KIP1-
ubiquitylation-promoting complex (KPC), preferentially recognizes free p27
and is competed off by the binding of cyclin-E–CDK2 (Kamura et al. 2004).
Mitogen-stimulated cyclin E expression and thus the formation of the cyclin-
E–CDK2 complex may switch cells from KPC-mediated degradation of p27
during early G0/G1 transitions to SCF-mediated degradation at the G1/S
transition. Likewise, p57, which plays important roles in development, is also
ubiquitylated by the SCF-SKP2 E3 ligase and a second E3 ligase, SCF-
FBL12, containing FBL12. FBL12 is induced by TGFβ1 and binds only to
p57, providing a mechanism for TGFβ1-induced degradation of p57, but not
p27 or p21 (Kim et al. 2008a).
p21 expression oscillates twice during each cell cycle: it is high in G1
phase, decreases during S phase, reaccumulates during G2 phase, and then
decreases at early mitosis. The protein has a very short half-life (<30 min)
and is rapidly turned over by ubiquitin-dependent proteolysis. Several E3
ligases can target p21 ubiquitylation at different phases of the cell cycle in
both phosphorylation-dependent and phosphorylation-independent manners.
During G1 phase, sustained activation of the ERK2 MAPK by mitogenic
stimuli such as epidermal growth factor (EGF) results in T57 and S130
phosphorylation on p21, leading to its ubiquitin-dependent degradation (Fig.
3) (Hwang et al. 2009). During S phase, WD40 protein CDT2 and the F-box
protein SKP2 target p21 for ubiquitylation by the CRL4-CDT2 and SCF-
SKP2 E3 ligases to prevent DNA rereplication (Bornstein et al. 2003; Abbas
et al. 2008; Kim et al. 2008b; Nishitani et al. 2008). The SCF-SKP2-mediated
p21 ubiquitylation requires S130 phosphorylation by cyclin-E–CDK2
(Bornstein et al. 2003). During early mitosis, Cdc20 binds to p21 and targets
it for ubiquitylation by APC. CRL4 also targets p21 for ubiquitylation after
low-dose UV irradiation, to delay the cell cycle, allowing time for optimal
DNA repair (Bendjennat et al. 2003; Havens and Walter 2011; Starostina and
Kipreos 2012). Hence, the mechanism for targeting G1 CKIs for
ubiquitylation has been conserved from yeast to animals and links the
regulation of CKI stability to signals from different pathways via the
phosphorylation of CKI proteins and their targeting molecules.

6 CONCLUDING REMARKS
Precise cell cycle regulation is an essential aspect of normal development and
adult homeostasis. To achieve this, cells in G1 phase integrate inputs from
major cellular signaling pathways to decide whether or not to enter S phase,
which is an irreversible cell cycle step. This integration of signals is
transformed into an appropriate level of CDK activity in large part via
changes in the level of cyclins and CKIs achieved through the regulation of
both transcription and protein stability. One challenge for the future is to
understand how multiple signaling pathways cooperate to precisely regulate
cyclin and CKI activity in various cell types, particularly stem cells, in intact
tissues. Another is to use this information to develop novel therapeutics for
the treatment of cancer, which arises in part because of disruptions to
signaling pathways that affect cell cycle regulation.

ACKNOWLEDGMENTS
We thank Alan Diehl, Andrew Koff, Kun-Liang Guan, Michele Pagano, DJ
Pan, and Xin-Hai Pei for discussions, and Tadashi Nakagawa and Ruiting Lin
for helping with figure preparation.

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5CDKs are a family of kinases that regulate the cell cycle and that require binding to noncatalytic
partner proteins termed cyclins for activity.
6Linked to p16, both structurally in the genome and through regulation by Polycomb group proteins, is
the product of the ARF tumor suppressor gene, which is transcribed from an alternative promoter and
translated in an alternative reading frame from p16. ARF does not share any amino acid sequence
similarity with INK4 proteins and instead acts as a p53 activator by binding to and inhibiting the
activity of MDM2, the principle E3 ubiquitin ligase for and negative regulator of p53. As a result, any
signal, such as oncogenic stimulation, that induces the expression of ARF will stabilize p53 and
activate p21, leading to G1 cell cycle arrest.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a008904
CHAPTER 6

Signaling Pathways that Regulate Cell

Division

Nicholas Rhind1 and Paul Russell2

1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical
School, Worcester, Massachusetts 01605
2Department of Molecular Biology, Department of Cell Biology, The Scripps Research Institute, La
Jolla, California 92037
Correspondence: nick.rhind@umassmed.edu

SUMMARY

Cell division requires careful orchestration of three major events: entry

into mitosis, chromosomal segregation, and cytokinesis. Signaling
within and between the molecules that control these events allows for
their coordination via checkpoints, a specific class of signaling pathways
that ensure the dependency of cell-cycle events on the successful
completion of preceding events. Multiple positive- and negative-
feedback loops ensure that a cell is fully committed to division and that
the events occur in the proper order. Unlike other signaling pathways,
which integrate external inputs to decide whether to execute a given
process, signaling at cell division is largely dedicated to completing a
decision made in G1 phase—to initiate and complete a round of mitotic
cell division. Instead of deciding if the events of cell division will take
place, these signaling pathways entrain these events to the activation of
 the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the
opportunity for checkpoint proteins to arrest cell division if things go
wrong.

Outline
1 Introduction
2 CDK1 and entry into mitosis
3 Regulation of the G2/M transition
4 Checkpoint regulation of the G2/M transition
5 The DNA damage and replication checkpoints
6 The DNA damage checkpoint triggered by DSBs
7 The DNA replication checkpoint triggered by stalled replication forks
8 Chk1 and Chk2 target Cdc25
9 The MAPK-dependent stress checkpoint
10 Resolution of G2 checkpoint arrests
11 Regulation of mitosis
12 Dissecting the network
13 Anaphase entry
14 Checkpoint regulation of mitosis
15 Regulation of cytokinesis
16 Concluding remarks
References

1 INTRODUCTION
The cell cycle (see Fig. 1) consists of DNA synthesis (S) and mitosis (M)
phases separated by gap phases in the order G1–S–G2–M (Murray and Hunt
1993; Nurse 2000; Morgan 2006). Cell division involves two connected
processes triggered at the end of G2 phase: mitosis itself (segregation of the
chromosomes, which duplicate in S phase) and cytokinesis (division of the
cell, per se). Mitosis can be subdivided into six distinct phases (see Box 1):
(1) prophase, in which the spindle begins to assemble in the cytoplasm and
chromosomes begin to condense in the nucleus; (2) prometaphase, in which
the nuclear envelope breaks down and chromosomes attach to the spindle; (3)
metaphase, in which chromosomes align at the spindle midzone; (4) anaphase
A, in which chromosomes move to the centrosomes, which form the spindle
poles; (5) anaphase B, in which the spindle elongates; and (6) telophase, in
which the nuclear envelope reforms around the new daughter nuclei. Mitosis
in yeasts differs in that the nuclear envelope does not break down; instead the
spindle-pole body, the yeast equivalent of the centrosome, spans the nuclear
envelope, allowing the spindle to access both the nucleus and the cytoplasm.
Signals during telophase trigger cytokinesis, which separates the daughter
nuclei into two daughter cells.

Figure 1. The major events of the cell cycle. The major events of the cell cycle are regulated by
successive waves of kinase and ubiquitin ligase activity. G1-cyclin–CDK activity is required to initiate
the cell cycle and activate B-type-cyclin–CDK activity. Low levels of B-type-cyclin–CDK activity are
sufficient to trigger S phase, but tyrosine phosphorylation by Wee1 prevents full activation, preventing
premature mitosis. Full CDK activation triggers mitosis and activates APC, which triggers anaphase
and feeds back to inactivate CDK activity. Inactivation of CDK allows exit from mitosis and the
reestablishment of interphase chromosome and nuclear structure in G1 phase. See Box 1 for description
of the stages of mitosis.
BOX 1. THE MAIN STAGES OF MITOSIS

Prophase Triggered by activation of CDK1, CDK1–cyclin-B is

imported into the nucleus, chromosomes condense from their diffuse
interphase state to compact rods, and the centrosomes begin to separate,
forming a spindle between them.
Prometaphase The nuclear envelope breaks down, the mature
spindle is formed with centrosomes on either side of the cell, and so-
called kinetochore microtubules from the spindle poles interact with the
kinetochore protein complexes that form at chromosome centromeres,
moving chromosomes to the spindle midzone. In yeasts, which do not
break down their nuclear envelopes for mitosis, the spindle forms inside
the nucleus, and the spindle poles span the nuclear envelope to connect
nuclear and cytoplasmic microtubules.
Metaphase When each of the kinetochores on a pair of sister
chromosomes is attached to microtubules from opposite spindle poles,
the opposing force pulls the pair to the metaphase plate at the middle of
the spindle in a “bi-oriented” configuration. Until all the chromosomes
are bi-oriented, unattached kinetochores produce a checkpoint signal
that prevents the metaphase/anaphase transition.
Anaphase Once the mitotic checkpoint has been satisfied, the APC
ubiquitin ligase is activated. Ubiquitin-dependent proteolysis of the
inhibitor securin leads to activation of separase—a protease that cleaves
the cohesin proteins that hold sister chromatids together. This cleavage
causes a loss of chromosome cohesion, allowing the chromosomes to
separate. They do so in two movements: anaphase A, in which
chromosomes are pulled toward the spindle poles by contraction of the
kinetochore microtubules; and anaphase B, in which the spindles are
 pushed away from each other by the elongation of inter-spindle-pole
microtubules.
Telophase After the chromosomes have been segregated to opposite
sides of the cell, CDK1 activity is inhibited by APC-mediated
destruction of cyclin B and activation of the CDK1-antagonizing
phosphatase Cdc14. The reduction in CDK1 activity allows for
reformation of the nuclear envelope, decondensation of chromosomes,
and entry into G1.

The signaling pathways that operate in G2 phase to control the onset of

mitosis, and those that operate during mitosis to control chromosome
segregation and the initiation of cytokinesis, are somewhat different from
other cellular signaling pathways. Instead of being involved in decisions
about potential cell fates or responses to varying environmental conditions,
they are involved in executing a decision that is made in G1 phase—to enter
and complete another cell cycle. These signaling pathways have two key
roles: to coordinate potentially independent events, such as mitosis and
cytokinesis; and to provide quality-control checkpoints that arrest the process
when things go wrong. The cell division signaling pathways are the
archetypical checkpoints, defined as signaling pathways that ensure a
dependency for the execution of later cell-cycle events on the successful
completion of preceding events (Hartwell and Weinert 1989).
Two major transitions are required for cell division: the G2/M transition
and the metaphase/anaphase transition. These are regulated by the protein
kinase cyclin-dependent kinase 1 (CDK1) and the anaphase-promoting
complex (APC, an E3 ubiquitin ligase), respectively (Murray and Hunt 1993;
Nurse 2000; Morgan 2006). During G2 phase CDK1 is maintained at a low
level of activity by mechanisms described below. Activation of CDK1 is
necessary and sufficient to trigger entry into mitosis. Thus, CDK1 activation
is the focal point of many signaling pathways that control the commitment to
cell division. These pathways have both positive effects, such as activating
CDK1 when cells reach a critical size, and negative effects, such as delaying
CDK1 activation in the presence of DNA damage. In general, the negative
regulatory pathways, known as checkpoints, are much better understood than
the positive regulatory pathways. Activation of CDK1 and entry into mitosis
lead to activation of the APC. The APC in turn causes separation of sister
chromatids, which allows them to segregate to opposite spindle poles at
anaphase and the complete inactivation of CDK1, which allows exit from
mitosis and the resetting of the cell cycle to G1 phase. Being necessary and
sufficient for the initiation of anaphase, the APC is the major target of the
checkpoints that arrest cells in metaphase (Fig. 1).
Below we examine these G2 and mitotic signaling pathways and the
checkpoints that regulate them, explaining how they ensure that events take
place in the correct order even in the face of cell-cycle disruptions.

2 CDK1 AND ENTRY INTO MITOSIS

The onset of mitosis is brought about by the activation of CDK1, a 34-kDa
proline-directed serine/threonine protein kinase (Nurse 1990). CDK1, also
known as Cdc2 (cell division cycle 2), phosphorylates hundreds of different
substrates at an S/TPx(x)R/K consensus, including downstream effector
kinases, to initiate the events required for progression through mitosis.
Unsurprisingly, it is tightly regulated at many levels. CDK1 is maintained at
a constant concentration throughout the cell cycle; however, its periodic
activation and inactivation is achieved by interactions with specific
regulatory subunits and by dephosphorylation of critical residues.
As their name indicates, CDKs such as CDK1 require binding of a
regulatory cyclin subunit (Murray and Hunt 1993; Morgan 1995; Bloom and
Cross 2007), which activates the kinase by causing conformational changes
near the active site. These structural changes allow the kinase to bind ATP
and substrates in an orientation that promotes the transfer of the terminal γ
phosphate of ATP to the target serine or threonine residue in the substrate.
Different CDK–cyclin heterodimers regulate the different cell-cycle
transitions (Morgan 2006). The G1 cyclins, in association with CDK4, CDK6,
and CDK2, regulate entry into the cell cycle (Duronio and Xiong 2012),
whereas S-phase events and the G2/M transition are primarily regulated by
CDK1 (CDK2 also functions in S phase) bound to a members of the B-type
family of cyclins (the CDK1–cyclin-B complex is also known as M-phase
promoting factor [MPF]).
The periodic accumulation and disappearance of cyclins has a central role
in regulating CDK1 activity during the cell cycle. B-type cyclin genes are
generally expressed during S and G2 phases, leading to the accumulation of
CDK1–cyclin complexes during these phases of the cell cycle. At the
metaphase/anaphase transition, APC ubiquitylates B-type cyclins, which
targets them for rapid destruction by the proteasome. The destruction of
cyclins causes a precipitous drop in CDK activity, resetting the cell cycle as
cells enter G1 phase.
B-type cyclins comprise a large and quickly evolving protein family
(Table 1). Most species express multiple B-type cyclins, which are believed
to confer different substrate specificities on CDK1–cyclin complexes.
However, these differences do not appear to be crucial. For example, cyclin
A (a mammalian B-type cyclin) is expressed during S phase and is
responsible for triggering DNA replication in conjunction with CDK2, but in
its absence cyclin B can provide this function (Kalaszczynska et al. 2009).
More dramatically, in the fission yeast Schizosaccharomyces pombe, the cell
cycle can be regulated by a single cyclin–CDK1 complex, demonstrating that
differential B-type-cyclin–CDK1 specificities are not required for S-phase
and M-phase events (Coudreuse and Nurse 2010). Likewise, a single B-type
cyclin can drive S phase and M phase in budding yeast (Haase and Reed
1999).

Table 1. Key proteins in cell division control

Protein Budding yeast Fission yeast Human
Cyclin-dependent kinase 1 Cdc28 Cdc2 CDK1a
S-phase-expressed B-type cyclin Clb5,6 Cig2 Cyclin A
M-phase-expressed B-type cyclin Clb1,2 Cdc13 Cyclin B
CDK-inhibitory kinase Swe1 Wee1, Mik1 WEE1, MYT1
CDK-activating phosphatase Mih1 Cdc25 CDC25B, CDC25C
Checkpoint kinase Tel1 Tel1 ATM
Checkpoint kinase Mec1 Rad3 ATR
Checkpoint effector kinase Chk1 Chk1 CHK1
Checkpoint effector kinase Rad53, Dun1 Cds1 CHK2
MRN nuclease Mre11 Rad32 MRE11
MRN scaffold Rad50 Rad50 RAD50
MRN regulator Xrs2 Nbs1 NBS1
ATR targeting subunit Ldc1 Rad26 ATRIP
9-1-1 checkpoint clamp Ddc1 Rad9 RAD9
9-1-1 checkpoint clamp Rad17 Rad1 RAD1
9-1-1 checkpoint clamp Rad24 Hus1 HUS1
Checkpoint mediator –b – MDC1
Checkpoint mediator Rad9 Crb2
Checkpoint mediator Dpb11 Rad4/Cut5 TopBP1
Checkpoint mediator Mrc1 Mrc1 Claspin
Fork protection complex Tof1 Swi1 Timeless
Fork protection complex Csm3 Swi3 Tipin
Mitotic kinase Cdc5 Plo1 Plk1-4
Mitotic kinase Ipl1 Ark1 Aurora A/B
APC regulator Cdc20 Slp1 CDC20
APC regulator Cdh1 Srw1 CCH1
Mitotic checkpoint regulator Mad1 Mad1 MAD1
Mitotic checkpoint regulator Mad2 Mad2 MAD2
Mitotic checkpoint regulator Mad3 Mad3 BUBR1
Mitotic checkpoint kinase Bub1 Bub1 BUB1
Mitotic checkpoint regulator Bub3 Bub3 BUB3
MEN/SIN scaffold Nud1 Cdc11 ?c
MEN/SIN GTPase Tem1 Spg1 ?
MEN/SIN GAP Bub2 Cdc16 ?
MEN/SIN GAP cofactor Byr4 Byr4 ?
MEN/SIN GEF Lte1 Etd1 ?
MEN/SIN kinase Cdc15 Cdc7 MST1/2
MEN/SIN kinase – Sid1 MST1/2
MEN/SIN kinase regulator – Cdc14 ?
MEN/SIN kinase Dbf2 Sid2 LATS1/2
MEN/SIN kinase regulator Mob1 Mob1 MOB1A,B
Phosphatase Cdc14 Clp1 CDC14

aNames used in the text are in bold.

b–, No ortholog is believed to exist.
c?, An ortholog has not been identified.

These results have led to a quantitative model of cell-cycle regulation, in

which relatively high CDK1 activity is required to bring about the onset of M
phase, whereas a much lower level of CDK1 activity is sufficient to catalyze
the events of S phase (Stern and Nurse 1996; Coudreuse and Nurse 2010).
Nonetheless, different cyclins are expressed at different times and confer
differential substrate specificities on CDK1, allowing layers of fine-tuning to
the basic quantitative model (Uhlmann et al. 2011).
Cyclin binding is necessary but not sufficient for robust CDK1 activity.
Full activation of CDK1 also requires phosphorylation of a threonine residue
near the active site, which causes further realignment of active-site residues
into an active conformation. This phosphorylation is catalyzed by a CDK-
activating kinase (CAK), which requires the CDK to be cyclin bound
(Morgan 1995). Curiously, the identity of CAK varies, depending on the
species, and in some organisms there are multiple CAKs. CAK activity does
not change during the cell cycle, nor is the dephosphorylation of the CAK-
phosphorylated CDK–cyclin complexes regulated in a cell-cycle-dependent
manner. Thus, phosphorylation of CDK1 by CAK is dependent on cyclin
binding and is essential for cell division, but it does not control when CDK1
is activated.
Full activation of CDK1, and thus entry into M phase, is restrained by
Wee1 and related protein kinases (Fig. 2), which inhibit the activity of
CDK1–cyclin complexes that accumulate during S and G2 phases (Russell
and Nurse 1987; Lundgren et al. 1991; Mueller et al. 1995). Wee1 family
kinases resemble serine/threonine protein kinases, but actually phosphorylate
CDK1 on a tyrosine residue (Y15), as well as the adjacent threonine residue
(T14) once it is bound to a cyclin (Gould and Nurse 1989; Featherstone and
Russell 1991; Parker and Piwnica-Worms 1992; Mueller et al. 1995). These
phosphorylations probably inhibit CDK1 kinase activity by interfering with
substrate binding (Welburn et al. 2007). Once CDK1 is bound to a cyclin and
phosphorylated by CAK and Wee1, it is primed and ready to be activated
through the dephosphorylation of T14 and Y15. Cdc25, a dual-specificity
protein phosphatase, catalyzes this dephosphorylation reaction to bring about
the G2/M transition (Gautier et al. 1991; Kumagai and Dunphy 1991;
Strausfeld et al. 1991; Beausoleil et al. 2006).
Figure 2. Signaling at the G2/M transition. The rate-limiting step for the transition from G2 to mitosis
is the dephosphorylation of CDK1 on Y15, and in some organisms T14. This phosphorylation is
catalyzed by the Wee1 family of dual-specificity kinases and the phosphate is removed by Cdc25
phosphatases. Most of the many signaling pathways that affect the G2/M transition regulate Wee1 or
Cdc25. The DNA damage and replication checkpoints inactivate Cdc25; the morphogenesis and
nutritional checkpoints activate Wee1. CDK1 regulates its own activation as part of a feedback loop by
directly phosphorylating Wee1 and Cdc25 or doing so indirectly through Plk1.

Studies of fission yeast established that Wee1 and Cdc25 together

determine when cells initiate mitosis (Nurse 1975; Russell and Nurse 1986,
1987). Mutants lacking Wee1 divide prematurely at about half the size of
wild-type cells. Strains that have extra copies of wee1 divide at progressively
larger sizes that directly correlate with increasing wee1 gene dosage. In
contrast, elimination of Cdc25 activity generates cells that grow progressively
larger and cannot initiate mitosis. Strains that overexpress Cdc25 look similar
to wee1 mutants.
The opposing activities of Wee1 and Cdc25 underlie the rapid
“switchlike” activation of CDK1 that occurs at the G2/M transition (Fig. 2).
This behavior is controlled by feedback regulation from CDK1 (Pomerening
et al. 2003). CDK1 can directly phosphorylate Wee1 and Cdc25. It can also
activate Polo-like kinase 1 (Plk1), which in turn leads to the degradation of
Wee1 and stimulates Cdc25 phosphatase activity (Kumagai and Dunphy
1996). These feedback loops create a bi-stable regulatory mechanism in
which passage through a tipping point ensures the rapid and stable transition
from a low-activity CDK1 state to a high-CDK1-activity state.
In addition to the feedback loops that activate CDK1, a feedback loop in
animal cells inactivates the protein phosphatase 2A (PP2A), which
antagonizes CDK phosphorylation (Wurzenberger and Gerlich 2011). CDK1
activates the Greatwall kinase, which in turn activates Arpp19 and α-
endosulfine, two small inhibitors of the B55 isoform of PP2A (Fig. 3)
(Castilho et al. 2009; Gharbi-Ayachi et al. 2010; Mochida et al. 2010).
Inhibition of PP2A-B55 increases the effective activity of CDK1 by reducing
the rate at which CDK1 substrates are dephosphorylated. These interacting
feedback loops are believed to be critical for ensuring the commitment to
mitosis that occurs at the G2/M transition (Domingo-Sananes et al. 2011).
Figure 3. Signaling at the M/A transition. To establish metaphase, CDK1 directly, and indirectly
through a network of kinases including Plk1 and Aurora A, phosphorylates substrates that trigger
nuclear envelope breakdown, chromosome condensation, centrosome separation, and spindle assembly.
In addition, in animal cells CDK1 activates the Greatwall kinase, which indirectly inactivates the CDK-
antagonizing PP2A-B55 phosphatase via phosphorylation of the small phosphatase inhibitors Arpp19
and endosulfine. The rate-limiting step for the transition from metaphase to anaphase is the activation
of the APC ubiquitin ligase. The APC is activated by binding of the Cdc20 regulatory subunit (and later
Cdh1) and by CDK1 phosphorylation. Active APC targets securin for destruction, activating separase
to release chromatid cohesion and trigger anaphase. In the presence of unattached kinetochores,
anaphase is delayed by the mitotic checkpoint complex (MCC), a soluble inhibitor of the APC
produced at unattached kinetochores. The APC also feeds back to inactivate CDK1 by targeting cyclin
B for degradation and activates phosphatases (CDC14 in yeasts and PP1 and PP2A-B55 in animals)
that oppose CDK activity, resetting the cell cycle to a CDK-free G1 state.
 The regulation of protein subcellular localization, in addition to the
regulation of protein activity, is important in the control of the G2/M
transition. In particular, CDK1–cyclin-B complexes, which have critical
nuclear targets, are maintained in the cytoplasm during G2 phase, where they
can be inhibited by Wee1, but sequestered away from activation by nuclear
Cdc25. One of the early events of the G2/M transition is the nuclear
localization of CDK1–cyclin-B (Porter and Donoghue 2003). However, it is
unclear whether nuclear localization of CDK1–cyclin-B causes its activation
or is a consequence of activation that enforces the CDK1–Cdc25 positive-
feedback loop. Subcellular localization of regulatory proteins also plays a
crucial role in the control of cytokinesis, as described below.

3 REGULATION OF THE G2/M TRANSITION

The G2/M transition that commits cells to division is, in general, a default
consequence of initiating the cell cycle at the G1/S transition; examples of
cells voluntarily arresting in G2 phase are rare. However, what ultimately
triggers the activation of CDK1 is unclear. Plk1 may do so (Pomerening et al.
2003; Barr et al. 2004), but such a model just begs the question of what
activates Plk1. Moreover, the involvement of Plk1 in the feedback loops that
regulate CDK1 makes it difficult to disentangle cause and effect.
One important input is cell size (Kellogg 2003; Tzur et al. 2009). In
steady-state growth conditions, cell division is coordinated with cellular
growth in such a way that a newly born cell doubles its mass before
undergoing division. This coordination can be achieved by linking the
activation of CDK1 at the G2/M transition to attainment of a particular cell
size (Jorgensen and Tyers 2004). A number of potential mechanisms have
been explored over the years (Turner et al. 2012), including size-dependent
accumulation of activators (Tyson 1983), spatial gradients (Martin and
Berthelot-Grosjean 2009; Moseley et al. 2009), and translational capacity
(Polymenis and Schmidt 1997). Nonetheless, the mechanism by which cell
size is measured is still unknown.
Another regulator, and one that is intimately entwined with cell size, is
cell morphology. In budding yeast, defects in cell morphology or cytoskeletal
architecture delay cell division (Howell and Lew 2012). This cell-cycle delay
is enforced by the accumulation of Swe1, the budding yeast Wee1 ortholog.
Swe1 is normally degraded at the end of S phase, which allows the
dephosphorylation of CDK1 by Mih1, the budding yeast Cdc25 homolog.
Swe1 degradation requires its localization to the bud neck; thus, disruption of
bud-neck structure leads to Swe1 accumulation and cell-cycle arrest. In
addition, disruption of the actin cytoskeleton also leads to Swe1 stabilization,
further tying cell-cycle progression to cell morphology.
An unrelated signaling pathway required for proper morphology in fission
yeast also affects the timing of division (Martin and Berthelot-Grosjean 2009;
Moseley et al. 2009). Wee1 localizes at cortical nodes near the middle of
cells. Pom1, a serine/threonine protein kinase, forms a concentration gradient,
spreading out from the ends of cells, and indirectly activates Wee1 via
inhibitory phosphorylation of Cdr2, a kinase that colocalizes with, and
negatively regulates, Wee1 (Hachet et al. 2011). In newly born cells, which
are short, the concentration of Pom1 near the center of the cells is reasonably
high, which results in higher Wee1 kinase activity. As cells grow, the
concentration of Pom1 decreases near the middle of the cells, decreasing
Wee1 activity and tipping the Wee1–Cdc25 balance in favor of Cdc25 and
activation of CDK1. This model explains how Pom1 can influence cell size at
division; however, cells lacking Pom1 divide at a well-defined length, albeit
about 15% shorter than that of wild type, which implies that Pom1 is not
required to maintain accurate size control. Because Pom1-deficient cells
frequently display asymmetrical division, instead of controlling cell size per
se, Pom1 appears to function in a morphogenesis checkpoint that arrests cell-
cycle progression if the incipient division site is too close to one of the cell
tips.
The size at which cells initiate mitosis is also linked to nutrient conditions
(Fantes and Nurse 1977). Although nutritional signaling via the PI3K and
TOR pathways has profound effects on G1 cell-cycle progression (Ch. 5
[Duronio and Xiong 2012] and Ch. 7 [Ward and Thompson 2012]), it can
also affect the G2/M transition. For instance, in fission yeast, poor nitrogen
availability activates the Spc1/Sty1 mitogen-associated protein kinase
(MAPK), which in turn activates Plk1, thereby inhibiting Wee1 and
activating Cdc25 to delay CDK1 activation (Shiozaki and Russell 1995;
Petersen and Nurse 2007). Note that this nutritional signaling may be
considered part of the G2 checkpoints discussed below.

4 CHECKPOINT REGULATION OF THE G2/M

TRANSITION
Although arrest in G2 phase in normal, unperturbed cells is uncommon, it
occurs in response to activation of a variety of quality-control checkpoints.
Because Y15 dephosphorylation of CDK1 is the rate-limiting step for entry
into mitosis, these checkpoints target the regulators of this step: Wee1 and
Cdc25. The best-understood checkpoints are those activated by DNA damage
and problems with DNA replication.

5 THE DNA DAMAGE AND REPLICATION

CHECKPOINTS
The purpose of the cell division cycle is to distribute complete and accurate
copies of the genome to daughter cells. Genome instability arises if cells
initiate mitosis when chromosomes are only partially replicated or are
damaged by a double-strand DNA break (DSB). The consequences of
genome instability can be cell death or neoplastic transformation. DNA
damage and replication checkpoints ensure that the onset of nuclear division
is delayed when chromosomes are broken or incompletely replicated. The
cellular responses to DNA damage and replication-fork stalling are controlled
by ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and
DNA-PK (DNA-dependent protein kinase) (Shiloh 2003; Cimprich and
Cortez 2008). These protein kinases belong to the PIKK (phosphoinositide-3-
like-kinase kinase) family, whose members include mTOR (p. 91 [Laplante
and Sabatini 2012]). PIKKs strongly prefer to phosphorylate serine and
threonine residues followed by glutamine (S/TQ). Of the checkpoint kinases,
ATM and ATR are conserved from yeasts to humans, whereas DNA-PK is
found only in metazoans (but not all).
ATM, ATR, and DNA-PK are nuclear proteins that rapidly localize to
sites of DNA damage through interactions with targeting factors. Acting with
accessory proteins, these kinases set up signaling platforms on the chromatin
that flanks DNA lesions. DNA-PK associates with the Ku70–Ku80
heterodimeric protein complex that binds to DNA ends and promotes repair
by nonhomologous end joining. DNA-PK does not have a clearly established
role in regulating cell-cycle progression. ATM and ATR, in contrast, control
multiple responses to DNA damage, including regulation of DNA synthesis
and repair proteins, regulation of transcription of the genes involved in DNA
synthesis and repair, and regulation of cell-cycle progression. ATM and ATR
regulate some of these activities directly, whereas others are controlled by
their effector kinases: Chk1 and Chk2. ATM functions specifically in the
signaling downstream from DSBs, whereas ATR is activated in response to
formation of single-stranded DNA (ssDNA), which can occur through
resection of DSBs, by endonucleolytic removal of other types of DNA
lesions, or by the stalling or collapse of replication forks.

6 THE DNA DAMAGE CHECKPOINT TRIGGERED BY

DSBs
DSBs are especially dangerous DNA lesions because they disrupt the
physical continuity of chromosomes. Initiating mitosis with broken
chromosomes results in gross genome instability, which is a feature of cancer
in humans and is usually lethal in single-celled microorganisms (Hartwell
and Weinert 1989; Harper and Elledge 2007; Jackson and Bartek 2009).
DSBs therefore generate a potent checkpoint response. Indeed, in yeasts a
single unrepaired DSB is sufficient to induce a robust checkpoint response
that prevents the onset of mitosis even as cells continue to grow beyond the
size at which they normally divide (Harrison and Haber 2006).
ATM initiates the checkpoint response to DSBs (Shiloh 2003). It
associates with DSBs by interacting with the MRN protein complex, which
consists of Mre11 (a nuclease), Rad50 (a dimeric scaffolding protein with
ATPase activity), and Nbs1 (an adaptor protein) subunits (Fig. 2). Mre11 and
Rad50, which are conserved in eubacteria and archaea, directly engage DNA
ends. Nbs1, which is only found in eukaryotes, directly binds ATM (Stracker
and Petrini 2011).
Once localized at DNA ends, ATM phosphorylates proteins involved in
cell-cycle checkpoints, DNA repair, and chromatin structure (Fig. 2).
Phosphoproteomic studies suggest that it has hundreds of substrates, but only
a few have been studied in detail (Matsuoka et al. 2007). One of these is
Chk2, which is activated by ATM. Another ATM target is an SQ motif in the
exposed carboxy-terminal tail of the histone variant H2AX. Phospho-H2AX
(also known as γH2AX) serves as a marker for DNA damage in large multi-
kilobase domains of chromatin flanking DSBs (Bonner et al. 2008). In
mammals, γH2AX establishes a recruitment platform for mediator of DNA-
damage checkpoint protein 1 (Mdc1), which is critical for amplifying and
maintaining the checkpoint signal (Stewart et al. 2003). The carboxy-terminal
region of Mdc1 contains a pair of breast cancer susceptibility protein 1
(BRCA1) carboxy-terminal (BRCT) domains that form a phosphopeptide-
binding pocket for the phosphorylated γH2AX tail (Stucki et al. 2005). Once
Mdc1 binds to γH2AX, it recruits more MRN complex through an interaction
involving phosphorylated motifs in Mdc1 and the amino-terminal forkhead-
associated (FHA) domain of Nbs1 (Melander et al. 2008; Spycher et al.
2008). These constitutive phosphorylations of Mdc1 appear to be catalyzed
by the kinase CK2. The MRN complex that binds to Mdc1 recruits additional
ATM, thus establishing a checkpoint signal amplification loop that activates
Chk2.
In addition to recruiting ATM to DSBs, the MRN complex functions with
the DNA-end-processing factor CtIP (known as Sae2 in budding yeast and
Ctp1 in fission yeast) to initiate the 5′-3′ resection of DSBs (Mimitou and
Symington 2011). This resection generates ssDNA tails that are extended
through further resection by Exo1 exonuclease and the Dna2–Sgs1 (also
known as BLM) DNA endonuclease–helicase. These ssDNA tails are rapidly
bound by heterotrimeric replication protein A (RPA). RPA is essential for
homology-directed repair of DNA damage, but it is also a recruitment factor
for ATR (Cimprich and Cortez 2008). ATR associates with RPA through its
targeting subunit ATRIP (Fig. 2). ATM promotes CtIP recruitment to DSBs
in mammals, thereby enhancing resection; hence, ATM is required for
recruitment and activation of ATR at DSBs (You et al. 2009). Recruitment of
the CtIP orthologs in budding yeast (Sae2) or fission yeast (Ctp1) does not
require the ATM ortholog Tel1, which is a key reason why the ATR
orthologs have a more dominant checkpoint role in these organisms.
DNA damage is recognized independently by a heterotrimeric ring-
shaped protein complex known as the 9-1-1 (Rad9–Hus1–Rad1) checkpoint
clamp. Rad17 and four subunits of replication factor C (RFC) form a protein
complex that loads the 9-1-1 checkpoint clamp onto resected DNA ends. The
9-1-1 complex associates with TopBP1, which also interacts with ATR and
enhances its kinase activity (Navadgi-Patil and Burgers 2009). Both ATR and
9-1-1 are required for checkpoint signaling. Furthermore, in budding yeast,
ectopically targeting both complexes to the same locus is sufficient for
checkpoint activation, which suggests that recruitment of the two complexes
to sites of damage is necessary and sufficient for checkpoint signaling
(Bonilla et al. 2008).
ATM and ATR share multiple substrates, including histone H2AX. In
fission yeast the checkpoint mediator protein Crb2 has carboxy-terminal
BRCT domains that bind to γH2AX. In addition, Crb2 also has Tudor
domains that bind to histone H4 dimethylated on K20. Crb2 can also localize
to DSBs by binding to the TopBP1 ortholog, which interacts with the 9-1-1
checkpoint clamp. Either Crb2-recruitment pathway is sufficient to mount a
partial checkpoint response, but loss of both pathways abrogates the
checkpoint because Crb2 localization at DSBs is required for activation of
Chk1 by the ATR ortholog (Du et al. 2006).

7 THE DNA REPLICATION CHECKPOINT TRIGGERED

BY STALLED REPLICATION FORKS
During DNA replication, ssDNA is exposed at the replication fork when
DNA lesions, DNA-bound protein complexes, or limiting supplies of
deoxynucleotide triphosphates (dNTPs) cause replicative DNA polymerases
to slow down or stall. This ssDNA is bound by RPA, which elicits a
checkpoint response by ATR/ATRIP and their orthologs (Bartek et al. 2004;
Branzei and Foiani 2010). This checkpoint also requires the 9-1-1 checkpoint
clamp and TopBP1, similarly to the DNA damage checkpoint activated by
DSBs, but the checkpoint mediators are different. In budding and fission
yeast Mrc1 (mediator of the replication checkpoint 1) travels with the
replication fork and is required for activation of Chk2. In addition to other
functions, the activated Chk2 arrests cell-cycle progression and maintains the
S-phase pattern of gene expression (de Bruin et al. 2008; Dutta et al. 2008),
which is critical for completing DNA replication. A similar checkpoint
pathway operates in mammals, which use an Mrc1-related protein known as
claspin to mediate ATR-dependent activation of Chk1 in response to stalled
replication forks (Kumagai and Dunphy 2000). Another protein complex, the
fork protection complex, comprising Tof1–Csm3 in budding yeast, Swi1–
Swi3 in fission yeast, and Timeless–Tipin in mammals, also associates with
stalled forks. The fork protection complex stabilizes stalled forks in a manner
that promotes the replication checkpoint response (McFarlane et al. 2010).

8 Chk1 AND Chk2 TARGET Cdc25

Chk1 and Chk2, the two (unrelated) key effectors of the DNA damage and
replication checkpoints, share Cdc25 as a primary target (Fig. 2). This
mechanism was first discovered in fission yeast, where Chk1 is essential for
reducing the rate of CDK1 Y15 dephosphorylation in response to DNA
damage (Rhind and Russell 2000). Chk2 similarly regulates Y15
dephosphorylation in response to replication fork arrest (in mammals this is
done by Chk1). Both kinases directly phosphorylate Cdc25, the key effects
being inhibition of its protein phosphatase activity and exclusion from the
nucleus (Karlsson-Rosenthal and Millar 2006). The latter involves binding of
14-3-3 proteins to Cdc25.

9 THE MAPK-DEPENDENT STRESS CHECKPOINT

In addition to DNA damage, a variety of other cellular stresses, such as
osmotic shock, oxidative stress, and microtubule depolymerization, can delay
cells in G2 phase. Many of these stresses activate the p38 MAPK pathway (p.
81 [Morrison 2012]; Ch. 18 [Hotamisligil and Davis 2014]). Although the
details are not well understood, p38 MAPK is believed to inactivate Cdc25
(Shiozaki and Russell 1995; Karlsson-Rosenthal and Millar 2006).

10 RESOLUTION OF G2 CHECKPOINT
ARRESTS
G2 checkpoint arrests are generally reversible. Once the problem is resolved,
the checkpoint is inactivated and the cell can proceed with mitosis. This
inactivation appears to be a passive process in which resolution of the
checkpoint-initiating event (e.g., the repair of the DNA damage) removes the
signal that activates the checkpoint kinases, allowing constitutive
phosphatases to remove the phosphates from their substrates and reset the
system. In situations in which the problem cannot be resolved, cells can adapt
to the checkpoint signal and enter mitosis in the presence of ongoing
checkpoint signaling. Such adaptation generally appears to be a failure of
checkpoint function, as opposed to a regulated attenuation of checkpoint
signaling (Harrison and Haber 2006). As cells continue to grow during a
checkpoint arrest, it is likely that the mitosis-promoting activities that link the
onset of mitosis to attainment of a sufficient cell size eventually overcome
mitosis-inhibiting activity of the checkpoint. Alternatively, in metazoans,
transcriptional circuits involving p53 and p21 can be activated to drive cells
into senescence or apoptosis in response to prolonged G2 arrest (Kastan and
Bartek 2004). A failure to activate these circuits can lead to premature
resumption of cell division in the presence of DNA damage (Bunz et al.
1998).

11 REGULATION OF MITOSIS
If the G2 checkpoints are not triggered, cells fully activate CDK1 and proceed
through the G2/M transition into mitosis. This decision to commit to cell
division is implemented by signaling pathways that regulate the various
processes of cell division, in particular mitosis and cytokinesis. They also
regulate organelles and other cytoplasmic components in ways that are less
well understood.
The transition from G2 phase to mitosis involves reorganization of the
nucleus, the condensation of the chromosomes, and the formation of the
mitotic spindle (see Box 1 and Fig. 3). These events are triggered by CDK1
and culminate with the mitotic chromosomes aligned on the metaphase plate
(Morgan 2006). How it coordinates these processes is a long-standing
question in the field.

12 DISSECTING THE NETWORK

The challenge of dissecting this regulatory network is twofold. First, CDK1
has dozens, if not hundreds, of mitotic substrates, many of which are not
essential for mitosis. This apparent redundancy is presumably because the
transition to metaphase is driven by many phosphorylation events, each one
of which slightly biases a single protein toward its mitotic state, but no one of
which is necessary or sufficient to drive mitosis, per se. This complexity
makes validating the importance of any single phosphorylation difficult.
Second, CDK1 activates a network of other kinases that are involved in
various steps in the G2/M transition, and some of these feed back to ensure
full activation of CDK1 (Fig. 3). The major mitotic kinases are the Polo and
Aurora families (Ma and Poon 2011). In addition to functioning in feedback
loops that stimulate CDK1, Plk1 is essential for executing mitotic
progression, especially centrosome separation and formation of a bipolar
spindle (Archambault and Glover 2009). Plk1 phosphorylates numerous
substrates at the centrosome and kinetochore complexes that link them to
spindle microtubules, many of which are also CDK1 substrates. Furthermore,
CDK1 primes Plk1 substrates. The Polo-box domain of Plk1 binds to
phospho-S/TP sites (the preferred CDK1 phosphorylation site), and therefore
CDK1 phosphorylation of a protein can increase its affinity for Plk1, priming
it for Plk1 phosphorylation.
The Aurora A and Aurora B kinases are also essential for mitosis. Aurora
A acts earlier in CDK1 activation, centromere separation, and spindle
formation. Aurora B acts later to detect and correct improperly attached
kinetochores (see below). In addition to Plk1 and Aurora kinases, the NIMA
and Greatwall families of kinases are involved in executing the G2/M
transition and in the feedback loops that ensure and maintain the full
activation of CDK1.

13 ANAPHASE ENTRY
Once chromosomes are properly aligned on the metaphase plate, anaphase is
triggered via the activation of the APC (Fig. 3) (Morgan 2006). The APC is a
large multisubunit E3 ubiquitin ligase that regulates the stability of a range of
mitotic proteins by targeting them for ubiquitin-dependent proteolysis. It is
regulated by the binding of one of two substrate-selectivity subunits: Cdc20
at the metaphase/anaphase transition and Cdh1 during telophase and into G1
phase (Pesin and Orr-Weaver 2008). APC–Cdc20 promotes the degradation
of several key substrates that trigger the irreversible transition from
metaphase to anaphase and the subsequent exit from mitosis. One key
substrate is securin, a stoichiometric inhibitor of separase, the protease that
cleaves the cohesin complexes that hold sister chromatids together during
metaphase. Another key substrate is cyclin B, degradation of which
inactivates CDK1. In addition, APC activation leads to the indirect activation
of the Cdc14 phosphatase, which dephosphorylates many CDK1 targets,
further enforcing the inactivation of CDK1 (Clifford et al. 2008). PP1 and
PP2A also play a role (Wurzenberger and Gerlich 2011).
In yeasts, there appears to be an intrinsic delay between the activation of
CDK1 and the activation of APC, which usually gives the cells enough time
to set up the metaphase plate. APC is activated by CDK1 phosphorylation
(Kraft et al. 2003). The lag may give chromosomes adequate time to align. If
not, a checkpoint signal (described below) prevents APC activation until the
problem is resolved. In most metazoans, metaphase is rarely achieved in
time, presumably because the metazoan spindle is larger and more
complicated, and the checkpoint is activated in most cell cycles to delay
anaphase until the chromosomes are properly aligned. In effect, the regulation
of anaphase has gone from being a quality-control checkpoint in yeast to
being a central signaling pathway in metazoans that triggers anaphase upon
the successful completion of metaphase.

14 CHECKPOINT REGULATION OF MITOSIS

The signaling pathway that delays initiation of anaphase until the successful
alignment of metaphase chromosomes is called the spindle assembly
checkpoint or mitotic checkpoint (Fig. 3). The checkpoint acts to prevent
action of APC–Cdc20, thus preventing the degradation of securin and cyclin
B (Musacchio and Salmon 2007). While securin remains stable, sister-
chromatid cohesion is maintained, preventing separation of chromosomes;
while cyclin B remains stable, CDK1 stays active, maintaining the mitotic
phosphorylation state of the nucleus and preventing mitotic exit.
The spindle assembly checkpoint monitors chromosome alignment during
the stages leading up to metaphase. In so-called bi-oriented attachment, the
two sister chromatids are attached to spindle microtubules emanating from
opposite poles; the checkpoint is triggered by sister-chromatid pairs lacking
such attachments. The identity of the trigger that senses this lack of bi-
orientation and activates the checkpoint has been controversial. Early
experiments showed that a single unattached chromosome can activate the
checkpoint, and that pulling on such a chromosome with a glass needle is
sufficient to stop the checkpoint signal (Li and Nicklas 1995; Rieder et al.
1995). The interpretation of this result was that tension across the kinetochore
is sufficient to stop activation of the checkpoint and thus that it monitors
kinetochore tension. However, subsequent data were difficult to reconcile
with the tension model and instead supported an occupancy model in which
the checkpoint monitors proper binding of microtubules to the kinetochore,
independently of the tension they generate (Khodjakov and Pines 2010).
These two models can be reconciled by the existence of an Aurora-B-
dependent mechanism that recognizes chromosomes that are not bi-oriented
(including those with kinetochores lacking tension) and destabilizes their
kinetochore–microtubule attachments, allowing proper attachments to reform
(Tanaka et al. 2002). In such a model, during reattachment cycles, lack of
occupancy at the kinetochore activates the checkpoint. The checkpoint thus
monitors kinetochore occupancy, but stable occupancy requires kinetochore
tension (Khodjakov and Pines 2010).
A single unattached kinetochore can activate the spindle assembly
checkpoint and inhibit all of the APC–Cdc20 in the cell, which suggests that
unattached kinetochores produce a diffusible inhibitor of APC–Cdc20. That
diffusible inhibitor is believed to be the mitotic checkpoint complex (MCC),
which contains three checkpoint proteins—Mad2, Mad3/BubR1, and Bub3—
as well as Cdc20 itself (Musacchio and Salmon 2007). The components of
MCC interact dynamically with unattached kinetochores in a manner
dependent on the more stable interaction of other checkpoint proteins,
including Mad1 and Bub1. A crucial step in MCC formation is believed to be
the conversion of the soluble open form of Mad2 to a closed form, which can
bind stably to Cdc20. This conformational conversion of Mad2 is catalyzed
by binding to Mad1 at unattached kinetochores and facilitates the assembly of
MCC, which is then released to bind to and inhibit the APC. In metazoans
MCC formation is inhibited in the absence of unattached kinetochores by
p31-commet, which is believed to prevent open-Mad2 from binding to
closed-Mad2 (Musacchio and Salmon 2007). The MCC-dependent
checkpoint signal prevents anaphase until all kinetochores are properly
attached. Once all kinetochores are occupied, the production of MCC ceases,
the MCC-dependent checkpoint inhibition of the APC is relieved, and
anaphase ensues.
Another signaling pathway that can block the metaphase/anaphase
transition is the DNA damage checkpoint. In most organisms studied, the
damage checkpoint arrests cells in G2 phase by preventing the activation of
CDK1, as described above. However, in budding yeast the DNA damage
checkpoint directly targets the metaphase/anaphase transition by preventing
separase activity via Chk1 phosphorylation and stabilization of securin
(Sanchez et al. 1999). Metazoan cells also appear to regulate metaphase
progression in response to DNA damage (Rieder 2011). However, because
these cells also display robust G2 damage arrest, the significance of the
response is less clear.
Following the completion of anaphase, the CDK1 phosphorylation events
that established the mitotic state are reversed and the cell returns to an
interphase state in a process known as mitotic exit (Clifford et al. 2008;
Rieder 2011; Wurzenberger and Gerlich 2011). CDK1 activity is reduced by
APC-mediated proteolysis of cyclin B. In addition, the rate of CDK1
substrate dephosphorylation is increased by the activation of phosphatases
that antagonize CDK1 phosphorylation. In yeast, Cdc14 is activated to
reverse Cdk1-catalyzed phosphorylations, as described below. In animal
cells, PP1 and PP2A appear to be the major phosphatases antagonizing CDK
phosphorylation (Wurzenberger and Gerlich 2011). One mechanism for
activation of PP2A-B55 at mitotic exit is the inactivation of Greatwall, but
the discovery of other regulatory loops involving phosphatases seems likely.
The process of mitotic exit is intimately connected to the final stage of cell
division, cytokinesis.

15 REGULATION OF CYTOKINESIS
The proper coordination of cytokinesis with mitosis is essential to ensure
faithful chromosome segregation and avoid aneuploidy or polyploidy. This
coordination requires the septation initiation network (SIN) in fission yeast
and the mitotic exit network (MEN) in budding yeast (McCollum and Gould
2001; Goyal et al. 2011; Meitinger et al. 2012). Cytokinesis is entrained to
mitosis; thus, the signaling pathways that regulate cytokinesis are not
involved so much in deciding when cytokinesis should occur as in
coordinating cytokinesis with mitosis and providing opportunities for
checkpoint regulation.
The MEN and SIN pathways are GTPase-triggered kinase cascades that
culminate in the activation of the Sid2 kinase in fission yeast and the Dbf2
kinase in budding yeast (Goyal et al. 2011; Meitinger et al. 2012).
Components of the MEN/SIN pathways are organized on the spindle-pole
body, the yeast equivalent of the centrosome, making it a nexus for signaling
pathways that control cell division. Sid2 is necessary and sufficient for
initiating cytokinesis in fission yeast, although its exact targets are not
known. Therefore, it is essential to restrain SIN signaling until after the
successful completion of anaphase. Initiation of SIN signaling by the Spg1
GTPase is inhibited by the GTPase-activating protein Cdc16. Full activation
of SIN signaling is antagonized by CDK1 activity (Guertin et al. 2000),
which prevents cytokinesis until after activation of the APC and ensures that
activation of the spindle assembly checkpoint will also delay cell division. In
budding yeast, Dbf2 regulates cytokinesis by promoting localization of the
chitin synthase Chs2 and the cytokinesis regulator Hof1 to the bud neck; this
activity is antagonized by CDK1 activity (Meitinger et al. 2012).
In return, the MEN/SIN pathways antagonize the activity of CDK1
(Goyal et al. 2011; Meitinger et al. 2012). This reciprocal regulation allows
cytokinesis errors, which prolong SIN signaling, to restrain CDK1 activity in
the subsequent cell cycle, thus arresting cells in G2 phase and preventing the
next mitosis until the previous cytokinesis is successfully completed. An
important component of the MEN/SIN pathways is the Cdc14 (Clp1 in
fission yeast) phosphatase (Clifford et al. 2008). Cdc14 directly
dephosphorylates CDK1 targets, facilitating mitotic exit and resetting the cell
to an interphase state at the beginning of G1 phase.
Many proteins in the MEN/SIN pathways are conserved in metazoans. In
particular, the LATS kinases, relatives of Sid2/Dbf2 that also function in the
Hippo pathway (p. 133 [Harvey and Hariharan 2012]), appear to have roles in
the regulation of cytokinesis (Yang et al. 2004). Although the details have yet
to be established, similar signaling pathways probably coordinate mitosis and
cytokinesis in metazoans.
In addition to coordinating the timing of cytokinesis, signaling during cell
division is required to determine the location of cytokinesis. Although the
details differ between organisms, the location and orientation of the
cytokinesis furrow is generally determined by the mitotic spindle, except in
fission yeast, in which the cleavage plane is determined directly by the
location of the nucleus (Almonacid and Paoletti 2010). How the signal is
transmitted from the spindle or the nucleus to the cortex to establish the site
of cytokinesis has yet to be established. Budding yeast is unusual in this
context because the cleavage plane (the bud neck) is established before
mitosis. Therefore, instead of using the spindle to orient cell division,
budding yeast uses cell division to orient the spindle. Specifically, the MEN
GTPase Tem1 is localized to the spindle-pole body, whereas Tem1’s
inhibitors, Bub2, Bfa1, and Kin4, are localized to the mother cell, and its
activator Lte1, a guanine nucleotide exchange factor (GEF) relative, is
localized to the daughter cell (Bardin et al. 2000). Thus, the MEN is only
activated once the spindle is oriented such that one end of the spindle is
through the bud neck and in the daughter cell. However, this strategy of
triggering cytokinesis as a spatial consequence of spindle elongation may be
general, as a similar mechanism functions in fission yeast (Garcia-Cortes and
McCollum 2009).

16 CONCLUDING REMARKS
The major cell-cycle transitions that constitute cell division—the G2/M
transition, the metaphase/anaphase transition, and cytokinesis—provide
important decision points that are regulated by a number of signaling
pathways. These pathways ensure that the critical events of cell division
occur in the proper order and provide the quality controls that prevent cells
from dividing with damaged DNA or misaligned chromosomes. As such,
they are instrumental in maintaining genomic integrity and, in metazoans,
preventing cancer.
These negative regulatory signaling pathways were the original
inspiration for the checkpoint paradigm of active negative regulation of cell-
cycle events (Hartwell and Weinert 1989). The initial model posited that
checkpoints delayed cell-cycle transitions to allow time for checkpoint-
independent processes to fix whatever problem had triggered the checkpoint.
Since then, these same pathways have been shown to regulate many other
aspects of cell metabolism, such as DNA repair, and the term “checkpoint” is
now used much more broadly than originally intended. Nonetheless, these
signaling pathways serve as prime examples of how cells reorganize their
metabolism and cell cycle to damage and other perturbations.
Although the well-studied signaling pathways that regulate cell division
inhibit transitions in response to signals of damage or other problems, there is
evidence for at least one positive signaling pathway, the one that regulates
cell size. One of the enduring mysteries of cell biology is how cells measure
size and how that information is used to regulate cell-cycle transitions such as
cell division. Notwithstanding the current lack of mechanistic insight, the
way size is measured and the pathways that transduce that signal are poised
to be areas of future progress in the field.
The signal transduction pathways that regulate cell division continue to be
the focus of significant experimental effort. That effort looks set to continue
as work continues on the discovery of new regulators of cell division, the
increasingly detailed mechanistic understanding of the major checkpoint
pathways, and the translation of our understanding of these pathways into
diagnostic and therapeutic advances in fields such as fertility, cancer, and
aging.

ACKNOWLEDGMENTS
We thank Dan McCollum for valuable insight. N.R. is supported by NIH
R01-GM069957 and an ACS Research Scholar Grant. P.R. is supported by
NIH R01-GM59447, CA77325, and CA117638.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005942
CHAPTER 7

Signaling in Control of Cell Growth

and Metabolism

Patrick S. Ward1,2 and Craig B. Thompson1

1Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New
York 10065
2Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Correspondence: thompsonc@mskcc.org

SUMMARY

Mammalian cells require growth-factor-receptor-initiated signaling to

proliferate. Signal transduction not only initiates entry into the cell
cycle, but also reprograms cellular metabolism. This instructional
metabolic reprogramming is critical if the cell is to fulfill the anabolic
and energetic requirements that accompany cell growth and division.
Growth factor signaling mediated by the PI3K/Akt pathway plays a
major role in regulating the cellular uptake of glucose, as well as the
incorporation of this glucose carbon into lipids for membrane synthesis.
Tyrosine-kinase-based regulation of key glycolytic enzymes such as
pyruvate kinase also plays a critical role directing glucose carbon into
anabolic pathways. In addition, the Myc transcription factor and mTOR
kinase regulate the uptake and utilization of amino acids for protein and
nucleic acid synthesis, as well as for the supply of intermediates to the
mitochondrial Krebs cycle. However, the relationship between cellular
 signaling and metabolism is not unidirectional. Cells, by sensing levels
of intracellular metabolites and the status of key metabolic pathways,
can exert feedback control on signal transduction networks through
multiple types of metabolite-derived protein modifications. These
mechanisms allow cells to coordinate growth and division with their
metabolic activity.

Outline
1 Introduction
2 PI3K/AKT signaling controls glucose metabolism and the
incorporation of carbon into macromolecules
3 HIF1 signaling provides additional regulation of glucose metabolism
in response to both oxygen availability and nutrient status
4 Tyrosine kinase signaling and pyruvate kinase: Regulation of a
metabolic switch in proliferating cells
5 Amino acid metabolism is also regulated by cellular signaling
cascades
6 Metabolically sensitive protein modifications link growth factor
signaling to cellular responses
7 Perturbations of cellular metabolism in disease
8 Concluding remarks
References

1 INTRODUCTION
Unicellular organisms have evolved to grow and divide rapidly when
nutrients are abundant, and they take up nutrients in a cell-autonomous
manner. The macromolecular precursors and free energy derived from
metabolism of these nutrients are used to synthesize the new biomass
required for cell growth and division. When the nutrient supply dwindles,
anabolic metabolism in these organisms decreases. The cells then shift to
catabolic pathways that maximize the efficiency of energy production to
survive periods of nutrient limitation (Vander Heiden et al. 2009).
In multicellular organisms, cells are generally surrounded by sufficient
nutrients to engage in continuous cell growth and proliferation. However,
organismal integrity requires that proliferation not be a cell-autonomous
process dictated by available nutrients. Mammalian cells require receptor-
mediated signal transduction initiated by extracellular growth factors to leave
the quiescent state and enter the cell cycle. The onset of cell growth and
division introduces a metabolic requirement for sufficient carbon, nitrogen,
and free energy to support synthesis of the new proteins, lipids, and nucleic
acids needed by a proliferating cell. Recent studies have shown that this
additional uptake of nutrients is regulated by signal transduction pathways
(Fig. 1). This growth-factor-directed uptake of nutrients is critical to
supporting a rate of macromolecular synthesis sufficient for growth
(DeBerardinis et al. 2008; Vander Heiden et al. 2009).
Figure 1. Growth-factor-initiated signaling reprograms metabolism in proliferating cells. (A) In
multicellular organisms, cells that are not instructed to proliferate by extracellular growth factors are
generally quiescent. In these cells, glucose carbon is predominantly metabolized to carbon dioxide in
the mitochondrial Krebs cycle when oxygen is available. This mitochondrial oxidation maximizes free-
energy generation in the form of ATP. (B) When cells are instructed to proliferate by growth factor
signaling, they increase their nutrient uptake, particularly that of glucose and glutamine. Much of this
increased nutrient uptake is used to fulfill the lipid, protein, and nucleotide synthesis (biomass) required
for cell growth, and the excess carbon is secreted as lactate. Proliferating cells also may adopt strategies
to increase their ATP consumption to maintain glycolytic flux. Metabolic pathways are indicated by
green arrows.

Mammalian cells instructed to proliferate via signal transduction are

generally successful at avoiding metabolic collapse. Assuming that
extracellular nutrients are abundant, these signaling-instructed cells will
increase both uptake of nutrients and nutrient flux through anabolic
pathways. However, if the regulation of cell growth by signaling pathways
goes unchecked, problems can rapidly develop. The availability of a key
extracellular nutrient could be limited in a particular context, or an important
enzyme in a critical anabolic pathway may, for some reason, be deficient.
Thus, to ensure that cell growth is properly coordinated with both the
availability of key nutrients and with the cellular capacity to use them
effectively, cells need a way to slow their own growth if their metabolic state
cannot support biomass production. Such a brake on anabolic metabolism
must be able to function even in the presence of growth-factor-initiated
signaling. Metabolically sensitive posttranslational modifications, including
acetylation and glycosylation, of signaling proteins provide an important
mechanism by which cellular metabolism can exert feedback control on the
output of signal transduction cascades. The relationship between cell
signaling and metabolism is thus bidirectional.

2 PI3K/AKT SIGNALING CONTROLS GLUCOSE

METABOLISM AND THE INCORPORATION OF
CARBON INTO MACROMOLECULES
A highly conserved signal transduction pathway initiated by extracellular
growth factors is the phosphoinositide 3-kinase (PI3K)/Akt pathway, whose
components are conserved throughout metazoan species (p. 87 [Hemmings
and Restuccia 2012]). In mammals, the pathway plays a particularly critical
role downstream from insulin signaling to facilitate glucose uptake in insulin-
dependent tissues such as fat and muscle. In these tissues, the PI3K/Akt
pathway promotes the trafficking of the glucose transporter GLUT4 to the
cell surface (Kohn et al. 1996; Ch. 14 [Hardie 2012]). However, this pathway
plays multiple other roles in glucose metabolism, and its activity is not
limited to those tissues classically described as insulin dependent.
In the normal, non-cancerous, setting, PI3K is activated in cells when cell
membrane receptor tyrosine kinases (RTKs), as well as G-protein-coupled
receptors (GPCRs) and cytokine receptors, are stimulated by extracellular
growth factors. Following activation, PI3K phosphorylates membrane
phosphatidylinositol lipids, which, in turn, leads to the recruitment and
activation of additional kinases, most notably Akt (Fig. 2). One of the major
effects of Akt on glucose metabolism is at the level of glucose uptake through
increased expression of GLUT1 on the cell surface (Rathmell et al. 2003).
GLUT1 is the major glucose transporter in most cell types. The translation of
GLUT1 mRNA is also increased by Akt signaling through mTORC1 and
4EBP (Taha et al. 1999). In addition, Akt signaling stimulates the activity of
several glycolytic enzymes. Hexokinase, which performs the first enzymatic
reaction of glycolysis—glucose → glucose 6-phosphate (G6P)—is more
active when associated with mitochondria, and this association is promoted
by Akt (Gottlob et al. 2001). Akt also directly phosphorylates
phosphofructokinase 2, and the resulting increase in fructose 2,6-
bisphosphate levels enhances the activity of the glycolytic enzyme
phosphofructokinase 1 (PFK1) (Deprez et al. 1997).
Figure 2. Glucose carbon flux is regulated by PI3K/Akt signaling. PI3K generates phosphatidylinositol
3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate (PIP2). PIP3 recruits Akt to the
cell membrane, permitting its activation by upstream kinases. Akt can then activate a variety of proteins
that increase glycolytic metabolism and direct glucose carbon flux toward biosynthesis. Akt enhances
glucose transporter 1 (GLUT1) translation and localization to the cell surface to increase glucose
uptake and increases the activity of the glycolytic enzymes hexokinase and phosphofructokinase 1
(PFK1). Akt also promotes lipid synthesis from glucose carbon by multiple mechanisms. It can activate
ATP-citrate lyase (ACL) to increase the availability of cytosolic acetyl-CoA. Akt activation can also
indirectly enhance the proteolytic release of the sterol regulatory element-binding protein transcription
factors (SREBPs), thereby promoting the expression of genes involved in the pathways for fatty acid
and cholesterol/isoprenoid synthesis. Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-
phosphate; F1,6P2, fructose 1,6-bisphosphate.

Akt signaling may also increase glycolytic flux indirectly by

transcriptionally up-regulating the endoplasmic reticulum enzyme ENTPD5
(Fang et al. 2010). ENTPD5 promotes proper protein glycosylation and
folding and does so through a cycle of reactions coupled to ATP hydrolysis.
Thus, Akt-mediated up-regulation of ENTPD5 can increase the cellular
ADP:ATP ratio. Increasing the ADP:ATP ratio can relieve allosteric
inhibition of PFK1 by ATP. It can also lead to increased activity of
downstream glycolytic enzymes that require ADP as a cofactor. Enhanced
ATP consumption caused by PI3K/Akt signaling may therefore enhance the
proliferating cell’s ability to rapidly metabolize glucose carbon.
Through these multiple effects on glycolytic gene expression and enzyme
activity, activated Akt is able to increase the overall glycolytic rate in the cell.
Even under aerobic conditions, cells with activated Akt display a greatly
increased rate of glycolysis and secrete a high proportion of glucose carbon
from the cell in the form of lactate (Elstrom et al. 2004). Originally described
by Otto Warburg as a phenomenon specific to tumor cells, “aerobic
glycolysis” is now known to be a general characteristic of rapidly
proliferating cells, both cancerous and non-cancerous (Bauer et al. 2004).
The PI3K/Akt pathway also regulates the fate of glucose carbon beyond
glycolysis for biosynthetic purposes. When glycolysis metabolizes glucose to
pyruvate, the pyruvate can enter mitochondria and be converted to acetyl-
CoA. This acetyl-CoA can then condense with oxaloacetate to form citrate.
The best-understood fate of mitochondrial citrate is oxidation within the
mitochondrial matrix through the Krebs citric acid cycle for ATP production.
Indeed, this fate predominates in tissues such as skeletal muscle and heart,
where the demand for free energy in the form of ATP is high. However,
proliferating cells with high levels of glucose uptake do not oxidize all of
their mitochondrial citrate, but instead transport some to the cytosol. This
cytosolic citrate can be converted to cytosolic acetyl-CoA by ATP-citrate
lyase (ACL), another enzyme stimulated by Akt (Berwick et al. 2002; Bauer
et al. 2005).
Cytosolic acetyl CoA can initiate and/or elongate fatty acid chains as part
of de novo lipid synthesis. By activating ACL, Akt signaling thus increases
the availability of acetyl-CoA precursors for lipid synthesis. It also induces
lipogenic genes involved in isoprenoid, cholesterol, and fatty acid
biosynthesis. These actions depend on mTOR-mediated activation of protein
synthesis. Enhanced synthesis of ER-exported proteins leads to depletion of
ER-Golgi lipids. This, in turn, promotes ER-to-Golgi transport and
proteolytic release of the sterol regulatory element-binding protein (SREBP)
transcription factors (Porstmann et al. 2005; Bobrovnikova-Marjon et al.
2008; Ch. 14 [Hardie 2012]). The additional lipids synthesized as a
consequence of PI3K/Akt activation play a critical role as components of the
plasma and organelle membranes that must be synthesized if cells are to grow
and proliferate. Some of them obviously also play important signaling roles
within the cell.

3 HIF1 SIGNALING PROVIDES ADDITIONAL

REGULATION OF GLUCOSE METABOLISM IN
RESPONSE TO BOTH OXYGEN AVAILABILITY AND
NUTRIENT STATUS
The HIF1 (hypoxia inducible factor 1) signaling pathway also plays a central
role in the regulation of cellular glucose metabolism. HIF1 is a transcription
factor that was initially identified through its role in the adaptive cellular
response to hypoxia (low oxygen tension). When oxygen is limited, the Krebs
cycle in the mitochondria will cause mitochondrial redox stress if ATP
continues to be produced solely by oxidative phosphorylation. Under these
conditions, HIF1 promotes the expression of genes whose products are
involved in anaerobic glycolysis (Fig. 3). This leads to increased generation
of ATP in the cytosol from the conversion of glucose to pyruvate (Semenza
et al. 1994).
Figure 3. HIF1 signaling responds to both hypoxia and growth factors to regulate glucose metabolism.
The stability of the transcription factor HIF1 is regulated through hydroxylation of proline residues on
the HIF1α-subunit by prolyl hydroxylase enzymes (PHDs). Molecular oxygen (O2) is a substrate for
PHD activity, which targets HIF1α for recognition by the Von Hippel–Lindau (VHL) ubiquitin E3
ligase and subsequent degradation by the proteasome. Inhibition of PHD activity and HIF1 degradation
occurs in the presence of elevated levels of reactive oxygen species (ROS) or the Krebs cycle
intermediates succinate and fumarate. HIF1 can also be positively regulated transcriptionally and
translationally downstream from growth factor signaling. When not degraded, HIF1 can promote the
expression of genes encoding glucose transporters and most enzymes of glycolysis. It also inhibits
glucose carbon flow into the mitochondria by promoting the expression of pyruvate dehydrogenase
kinase (PDK), which acts to inhibit pyruvate dehydrogenase (PDH). Furthermore, HIF1 up-regulates
lactate dehydrogenase A (LDH-A) expression to increase the conversion of pyruvate to lactate. This
conversion regenerates the ratio of NAD+/NADH necessary for continued flux through glycolysis.
 To maintain a high rate of glycolysis, cells must also have a high rate of
pyruvate → lactate conversion, which regenerates NAD+ from NADH. This
is important for maintaining a sufficiently high cytoplasmic NAD+:NADH
ratio to facilitate flux through the glycolytic enzyme glyceraldehyde-3-
phosphate dehydrogenase. HIF1 promotes lactate production and NAD+
regeneration by positively regulating the expression of the enzyme lactate
dehydrogenase A. HIF1 also reduces the rate of pyruvate entry into the
mitochondria by promoting the expression of pyruvate dehydrogenase kinase,
an enzyme that phosphorylates and inactivates the mitochondrial pyruvate
dehydrogenase complex (PDH) responsible for the formation of
mitochondrial acetyl CoA from pyruvate (Kim et al. 2006; Papandreou et al.
2006). HIF1-mediated regulation of PDH flux serves to limit the flow of
glucose-derived carbon into the mitochondrial, oxidative, Krebs cycle when
excessive levels of pyruvate are generated. If pyruvate is oxidized in the
Krebs cycle and increases electron transport beyond the cell’s ability to
assimilate the resulting electron flux using molecular oxygen and/or the
electrochemical potential of hydrogen in ATP generation, a considerable
increase in the levels of potentially damaging reactive oxygen species (ROS)
will result. HIF1-mediated regulation of PDH also limits the flow of glucose
carbon into biosynthetic pathways that emanate from mitochondrial Krebs
cycle intermediates (Lum et al. 2007).
HIF1 is a heterodimer composed of a labile α-subunit and a stable β-
subunit (Majmundar et al. 2010). At normal oxygen levels, the α-subunit of
HIF1 (HIF1α) is posttranslationally modified by hydroxylation of specific
proline residues by a prolyl hydroxylase (PHD). This proline hydroxylation
reaction requires both α-ketoglutarate and O2 as reactants, and generates
succinate and CO2 as products. Proline hydroxylation of HIF1α promotes its
association with a ubiquitin E3 ligase complex that is composed of the von
Hippel–Lindau (VHL) tumor suppressor as the substrate specificity subunit
together with a cullin-Rbx1 ligase (CRL). Ubiquitylation of HIF1α by the
CRL–VHL complex then targets it for degradation by the proteasome. At low
oxygen levels, the prolyl hydroxylation of HIF1α does not occur, because the
PHD reaction requires molecular oxygen (O2) as a substrate. This permits
stabilization of HIF1α during hypoxia and enhanced expression of HIF1
target genes.
In proliferating cells, several mechanisms increase HIF1 signaling even at
normal oxygen tensions. Activated Akt, through mTORC1, enhances
translation of HIF1α mRNA (Zhong et al. 2000; Hudson et al. 2002; p. 91
[Laplante and Sabatini 2012]). Increased cellular nutrient uptake can also
lead to inhibition of the proline hydroxylation reactions that would otherwise
target HIF1α for degradation. Excess nutrient metabolism can lead to
increased cellular levels of reactive oxygen species (ROS) if reactions in the
Krebs cycle occur at a rate exceeding the capacity of the electron transport
chain to capture the electrons generated (Wellen and Thompson 2010). ROS
are potent inhibitors of the HIF-targeting PHDs (Shatrov et al. 2003). Levels
of succinate and fumarate may also increase with signaling-induced
elevations in nutrient metabolism; both of these substrates can also inhibit
HIF-targeting PHDs (Isaacs et al. 2005; Selak et al. 2005). PHD inhibition by
succinate is an example of product inhibition. The result of PHD inhibition
by ROS, succinate, and/or fumarate is a feedback mechanism that decreases
the flow of glucose carbon into the mitochondria and thus inhibits further
generation of ROS through mitochondrial metabolism.

4 TYROSINE KINASE SIGNALING AND PYRUVATE

KINASE: REGULATION OF A METABOLIC SWITCH
IN PROLIFERATING CELLS
A unique aspect of glycolysis in proliferating cells is the isoform specificity
of pyruvate kinase, the enzyme that converts phosphoenolpyruvate (PEP) to
pyruvate with the concomitant generation of ATP. Several alternatively
spliced isoforms of pyruvate kinase exist, but most cells in the adult
predominantly express the M1 isoform (PKM1). However, in early
development, embryonic tissues predominantly express an alternatively
spliced M2 isoform (PKM2). Furthermore, in all cancer cells examined to
date, PKM2 is the predominant isoform found (Christofk et al. 2008a). The
splicing factors PTB, hnRNPA1, and hnRNPA2 that regulate the alternative
splicing of PKM mRNA to favor PKM2 over PKM1 expression are
themselves up-regulated by the Myc transcription factor, a proto-oncogene
product (Clower et al. 2010; David et al. 2010). Myc-regulated transcription
of these splicing factors thereby ensures a high PKM2/PKM1 ratio in cells
with activated growth factor signaling. Myc also regulates the expression of
several glycolytic enzymes (Shim et al. 1997; Osthus et al. 2000).
Unlike the constitutively active PKM1 and the other pyruvate kinase
isoforms, PKM2 is uniquely sensitive to regulation by tyrosine kinase
signaling pathways downstream from growth factor receptors (Fig. 4). PKM2
can bind to the phosphorylated tyrosine residues in tyrosine-phosphorylated
proteins and is also a target for tyrosine phosphorylation on itself (Christofk
et al. 2008b; Hitosugi et al. 2009). These events lead to inhibition of PKM2
enzymatic activity, at least partly by promoting the release of PKM2’s
allosteric activator fructose 1,6-bisphosphate. Binding of the latter to PKM2
causes it to form an active tetramer, but tyrosine-phosphorylated peptides
displace fructose 1,6-bisphosphate and cause the PKM2 tetramer to
dissociate, resulting in enzyme inactivation. The sensitivity of PKM2 to
inhibition by tyrosine kinase signaling allows it to act as a gatekeeper for the
metabolic fate of glucose carbon. When growth factors are present, PKM2 is
inhibited, which can promote the channeling of upstream glycolytic
intermediates into anabolic pathways such as nucleotide biosynthesis. When
growth factors are absent, PKM2 is active and can generate pyruvate for
subsequent catabolism in the Krebs cycle (Vander Heiden et al. 2009). All of
this is consistent with the growth advantage that PKM2-expressing cells
show in vivo compared with cells exclusively expressing PKM1 (Christofk et
al. 2008a).
Figure 4. Pyruvate kinase as a glycolytic switch, and mTOR and Myc regulate amino acid uptake and
metabolism. Pyruvate kinase catalyzes the late step of glycolysis that converts phosphoenolpyruvate
(PEP) to pyruvate, with the concomitant generation of ATP. The M2 isoform of pyruvate kinase
(PKM2) is specific to proliferating cells. Its activity is allosterically activated by the upstream
glycolytic intermediate fructose 1,6-bisphosphate (F1,6P2). Tyrosine kinase signaling downstream
from growth factor receptors causes the release of F1,6P2 from PKM2, which results in decreased
pyruvate kinase enzyme activity. This enzymatic inhibition can promote the redistribution of upstream
glycolytic intermediates into anabolic pathways like nucleotide biosynthesis. The transcription factor
Myc positively regulates the expression of PKM2 versus the constitutively active PKM1 isoform by up-
regulating several RNA splicing factors. Glutamine uptake and metabolism are also regulated by the
Myc transcription factor, which can be activated downstream from growth factor signaling pathways,
such as those involving Ras. Myc positively regulates the expression of glutamine transporters as well
as the enzyme glutaminase. Glutamate, the product of glutaminase activity, can be further metabolized
to α-ketoglutarate to supply intermediates for the mitochondrial Krebs cycle. Another major regulator
of amino acid metabolism is mTOR complex 1 (mTORC1), which can be activated downstream from
PI3K/Akt signaling as well as through the sensing of essential amino acids. mTORC1 positively
regulates protein synthesis in response to these inputs by activating S6 kinase (S6K), inhibiting
eukaryotic initiation factor 4E binding protein (4E-BP), and promoting the expression of ribosomal
proteins. A distinct mTOR complex, mTORC2, lies upstream of Akt and is positively regulated by
PI3K signaling.

The full range of effects that PKM2 may have on cell growth and
metabolism, however, remains incompletely characterized. Recently, an
alternative glycolytic pathway has been proposed to be present in PKM2-
expressing cells (Vander Heiden et al. 2010). When inactive PKM2 cannot
effectively convert PEP to pyruvate, PEP can donate its high-energy
phosphate to H11 of the upstream glycolytic enzyme phosphoglycerate
mutase. This pathway may allow the generation of pyruvate from PEP in a
step that is independent of ATP generation. Although further work is needed
to characterize and purify an enzyme that can catalyze this proposed activity,
the pathway may serve as an additional mechanism, besides Akt-mediated
up-regulation of ENTPD5, by which proliferating cells can decrease their
ATP:ADP ratio. The potential importance of PKM2 as a binding partner for a
variety of signaling kinases and transcription factors has also received
attention. For example, PKM2 translocates to the nucleus and potentiates the
transcriptional activity of the Oct4 transcription factor involved in
maintaining pluripotency (Lee et al. 2008). Many additional PKM2-
interacting proteins have been reported, most recently HIF1 and β-catenin,
but the significance of each of these interactions for facilitating cell growth
and proliferation requires further study.
The regulation of PKM2 activity by metabolites is also continuing to be
investigated. For example, ROS have recently been proposed to cause
oxidation, dissociation, and inactivation of the PKM2 tetramer (Anastasiou et
al. 2011). Conversely, the non-essential amino acid serine allosterically
activates PKM2 (Eigenbrodt et al. 1983). Ongoing work is addressing
whether inactivation of PKM2 and the resultant accumulation of glycolytic
intermediates can lead to enhanced flux through the serine synthetic pathway
and thereby promote a feedback loop through serine synthesis that can
modulate the flux of glycolytic intermediates to support cellular biosynthetic
requirements.

5 AMINO ACID METABOLISM IS ALSO REGULATED

BY CELLULAR SIGNALING CASCADES
Proliferating cells require a nitrogen source for protein and nucleotide
biosynthesis. Mammalian cells acquire nitrogen through the uptake and
metabolism of amino acids, using mechanisms that are also highly regulated
by growth factor signaling. Myc, which is downstream from several signaling
pathways, including those involving Ras and Hedgehog (see p. 81 [Morrison
2012] and p. 107 [Ingham 2012]), plays an especially important role in
regulating metabolism of the amino acid glutamine (Yuneva et al. 2007; Wise
et al. 2008; Gao et al. 2009). It is perhaps surprising that glutamine
metabolism is highly regulated by growth factor signaling, because it is
traditionally considered a non-essential amino acid that can be synthesized
from other amino acids through enzymes such as glutamine synthetase.
However, observations made by Eagle and others revealed more than 50
years ago that glutamine is uniquely critical for the proliferation of
mammalian cells in culture (Wise and Thompson 2010). Myc stimulates
glutamine uptake by promoting the expression of glutamine transporter
proteins. It can also promote the intracellular metabolism of glutamine by
increasing the expression of the enzyme glutaminase, which converts
glutamine to glutamate and ammonia (Fig. 4).
Glutamine’s carbon backbone is important for replenishing the supply of
mitochondrial Krebs cycle intermediates (anaplerosis). This is accomplished
by the conversion of glutamine to glutamate and then to the Krebs cycle
intermediate α-ketoglutarate (DeBerardinis et al. 2007). Importantly, treating
cells that are glutamine-addicted because they express oncogenic Myc with a
cell-permeable form of α-ketoglutarate can maintain cell viability in the
absence of glutamine. However, for the cells to proliferate, the complete
glutamine molecule that includes nitrogenous groups is required (Wise et al.
2008). Myc-induced glutamine catabolism can provide both the amide and
amino groups needed for non-essential amino acid synthesis. It can also
generate precursors for the biosynthesis of nucleotides and nicotinamide.
Myc provides additional stimulation of nucleotide biosynthesis by promoting
the expression of several key enzymes including TS, IMPDH2, and PRPS2
(Tong et al. 2009).
Growth factor signaling also regulates amino acid metabolism through the
mTOR kinase. mTORC1, one of two protein complexes containing mTOR
(see p. 91 [Laplante and Sabatini 2012]), is stimulated by PI3K/Akt, which
phosphorylates and consequently disrupts the tuberous sclerosis complex
(TSC). When disrupted, the TSC complex can no longer hydrolyze the GTP-
binding protein Rheb to its GDP-bound form, thus leaving the GTP-bound
Rheb free to interact with and activate mTORC1 (Sengupta et al. 2010).
mTORC1 inhibition can also be relieved by Akt phosphorylation of PRAS40
at T246 (Sancak et al. 2007).
mTORC1 promotes protein synthesis by phosphorylating and inhibiting
the activity of 4E-BPs, which normally sequester eIF4E and thus block cap-
dependent mRNA translation (Hara et al. 1997). mTORC1 can also
phosphorylate and activate S6Ks to enhance translational elongation by
relieving the suppression of eEF2 (Wang et al. 2001). In addition, mTORC1
regulates the translation of the 5′TOP mRNAs, an abundant class of mRNAs
that includes many encoding components of the translational apparatus (Tang
et al. 2001). mTORC1 also increases ribosome synthesis by activating RNA
polymerase I to transcribe rRNAs (Mayer et al. 2004).
Through its responsiveness to PI3K/Akt signaling, mTORC1 activity
enhances protein synthesis when a cell is directed to grow. However,
mTORC1 is also highly sensitive to inputs that reflect the cell’s metabolic
state. For example, upon depletion of cellular ATP levels, AMP-activated
protein kinase (AMPK) can inhibit mTORC1 through multiple mechanisms,
including activation of the TSC complex and inhibition of the mTOR binding
partner Raptor (Corradetti et al. 2004; Gwinn et al. 2008). This provides a
means for the cell to down-regulate the energetically costly process of protein
synthesis when ATP is limiting. In contrast, mTORC1 can be activated by the
availability of essential amino acids including leucine. Essential amino acids
induce the Rag GTPase complex to assume an active conformation on
lysosomal membranes. Active Rag can then recruit mTORC1 to the
lysosomal surface, where it may be more amenable to activation by Rheb
(Sancak et al. 2008; Sancak et al. 2010). Interestingly, recent work shows that
cellular leucine uptake is coupled with glutamine efflux (Nicklin et al. 2009),
linking Myc-regulated glutamine uptake with regulation of mTORC1 by
essential amino acid availability.
The much less well understood mTORC2 complex also has important
roles in growth factor signaling and metabolic regulation. Like mTORC1, it
is activated by growth factors such as insulin. But, in contrast to mTORC1,
mTORC2 lies upstream of Akt. Recent data indicate that growth-factor-
stimulated activation of mTORC2 involves a direct association with
ribosomes, which may ensure that mTORC2 is active only in cells that are
growing and undergoing protein synthesis (Oh et al. 2010; Zinzalla et al.
2011). Once active, mTORC2 can phosphorylate Akt at S473, which is
considered important both for enhancing the strength of Akt activation
downstream from PI3K and for widening the range of effective Akt
substrates.

6 METABOLICALLY SENSITIVE PROTEIN

MODIFICATIONS LINK GROWTH FACTOR
SIGNALING TO CELLULAR RESPONSES
Although many metabolic pathways that facilitate cell growth and
proliferation are up-regulated in response to growth-factor-initiated signaling,
the cell is not merely a passive recipient of instructions from growth factors.
Rather, intracellular metabolites can exert feedback control on signaling
initiated by growth factors through posttranslational modifications of critical
signaling proteins (Metallo and Vander Heiden 2010).
One area where this regulation occurs is at the level of the growth factor
receptor. Recent studies of cells whose ability to die by apoptosis upon
nutrient withdrawal has been eliminated have shown that cells normally
directed to take up nutrients upon stimulation by the growth factor interleukin
3 (IL3) can no longer take up glutamine when glucose is withdrawn from the
medium (Wellen et al. 2010). This deficiency is due to the cell’s inability to
continue displaying the IL3 receptor at the cell surface, necessary for IL3-
dependent regulation of glutamine uptake and metabolism. Metabolism of
glucose to UDP-N-acetylglucosamine (UDP-GlcNac) via the hexosamine
biosynthetic pathway, which branches off glycolysis, is necessary for the
proper N-linked glycosylation of the IL3 receptor α-subunit. This
glycosylation is critical for the proper folding of the IL3 receptor and its
localization to the cell surface. The production of UDP-GlcNac through the
hexosamine pathway also requires glutamine as a nitrogen donor. Thus, the
dependency of IL3 receptor signaling on receptor glycosylation ensures that
this pathway remains active only when there are adequate sources of both
glucose and glutamine, as well as intact enzymatic pathways for their
metabolism (Fig. 5). Appearance of the TGFβ, epidermal growth factor
(EGF), insulin-like growth factor, and Her2 receptors at the cell surface also
responds to glucose availability and/or N-glycosylation (Wu and Derynck
2009; Fang et al. 2010).
Figure 5. Hexosamine and acetyl-CoA metabolite levels integrate signaling with cell growth and
proliferation. In addition to being metabolized through glycolysis, fructose 6-phosphate can act as a
nitrogen acceptor from glutamine and be converted to glucosamine 6-phosphate. Further metabolism
through the hexosamine biosynthetic pathway results in UDP-N-acetylglucosamine (UDP-GlcNac).
UDP-GlcNac can be used for N-linked-glycosylation reactions, including that involving growth factor
receptor subunits as they are being processed in the ER. This glycosylation promotes the expression
and localization of receptor subunits at the cell membrane, where they can respond to growth factor.
Another metabolically sensitive protein modification occurs when acetyl-CoA is used as a substrate for
the acetylation of histones in the nucleus, a modification that alters gene expression. The supply of
acetyl-CoA for acetylation reactions depends on the activity of ATP-citrate lyase (ACL).

Histone acetylation is another metabolically sensitive protein modification

that influences signaling outputs. Acetylation of histone lysine residues
promotes open chromatin and increased gene expression (Li et al. 2007). In
mammalian cells, the acetyl donor for histone acetylation, acetyl-CoA, is
predominantly generated from glucose-derived citrate through the enzyme
ACL (Wellen et al. 2009). Through ACL activity, increased levels of glucose
availability and metabolism are thus able to facilitate open chromatin
formation via increased histone acetylation. Further evidence for the concept
that physiological variations in acetyl-CoA levels can alter the degree to
which histones are acetylated has been found in yeast, where the transcription
of cell growth genes is linked to increased histone acetylation that occurs
following a surge in acetyl-CoA production upon entry into a growth phase
(Cai and Tu 2011). In yeast, the acetylation of all proteins is not regulated by
variations in acetyl-CoA levels; this regulation appears limited to substrates
of the histone acetyltransferase Gcn5. Gcn5 has an in vitro Kd of 8.5 µM and
Km of 2.5 µM, within the range of yeast intracellular acetyl-CoA
concentrations that have been estimated to vary from 3 to 30 µM over the
metabolic cycle (Cai et al. 2011). As in yeast, in mammalian cells, not all
protein acetylation varies in response to the acetyl-CoA concentration.
Although the addition of acetyl groups to histone proteins has been shown to
vary with acetyl-CoA availability, the acetylation of tubulin remains
relatively constant (Wellen et al. 2009). The removal of acetylation marks
from histones can also be metabolically responsive. When glucose
availability and metabolism are diminished, cellular NAD+ levels and the
NAD+:NADH ratio are increased. This activates the sirtuin enzymes, class III
histone deacetylases that remove the acetylation mark from histone lysine
residues (Haigis and Sinclair 2010).
Note that cells use multiple other mechanisms to modify signaling
pathways and outputs based on the metabolic state of the cell. As previously
discussed, mTORC1 activity is regulated in part by the availability of
essential amino acids, and cells also sense their metabolic state through
AMPK (Ch. 14 [Hardie 2012]). AMPK generally functions to activate/up-
regulate ATP-producing pathways and to down-regulate ATP-consuming
activities when cellular free-energy levels are low. Other targets of AMPK
activity, in addition to mTORC1, include key enzymes for fatty acid
synthesis and cholesterol synthesis: acetyl-CoA carboxylase and HMG-CoA
reductase.

7 PERTURBATIONS OF CELLULAR METABOLISM IN

DISEASE
In disease, particularly in cancer, the control of cellular metabolism by
growth-factor-receptor-initiated signaling pathways often becomes
dysregulated. As discussed previously, the preferential expression of PKM2
over PKM1 has been found in all cancer cells examined to date, and this
selectivity is promoted by the Myc oncogene product. Loss-of-function
mutations in the PTEN tumor suppressor that antagonizes PI3K are also
common. These mutations impair negative regulation of PI3K/Akt signaling,
thereby enhancing glucose uptake and the flux of glucose into lipid synthesis.
Overexpression and/or constitutive activation of growth factor receptors, such
as the EGF receptor and Her2, are also found frequently and can promote
increased nutrient uptake and anabolic metabolism for cancer cells.
Not all disease-associated mutations affecting cellular metabolism are so
clearly linked to enhanced anabolic pathways. For example, recent
investigations have discovered that specific mutations in the active site of
NADP+-linked cytosolic isocitrate dehdyrogenase 1 (IDH1), or in its
mitochondrial relative IDH2, facilitate a neomorphic enzyme activity. This
neomorphic activity converts the Krebs cycle intermediate α-ketoglutarate to
a rare metabolite not found at high levels in mammalian cells under normal
conditions, 2-hydroxyglutarate (2HG) (Dang et al. 2009; Ward et al. 2010,
2011). IDH1 and IDH2 mutations occur in cancers including glioma, acute
myeloid leukemia, and chondrosarcoma, and in a large percentage of patients
with the inborn error of metabolism 2HG aciduria (Mardis et al. 2009; Yan et
al. 2009; Kranendijk et al. 2010; Ward et al. 2010; Amary et al. 2011).
Although the function of the “oncometabolite” 2HG in the context of various
cell types and tissues remains under active investigation, current evidence
suggests that its major role is to competitively inhibit α-ketoglutarate-
dependent enzymes that modify chromatin, particularly the TET family of
DNA 5-methylcytosine hydroxylases and Jumonji-C domain histone
demethylases (Figueroa et al. 2010; Chowdhury et al. 2011). Unlike other
cancer-associated mutations, direct evidence for IDH mutations promoting
cell-autonomous growth and activation of anabolic pathways is lacking,
which is consistent with IDH mutations predominantly arising in lower-
grade, more indolent lesions. This has led to the alternative proposal that the
effects of mutant IDH and 2HG in the tumor and its microenvironment
ultimately lead to a block in cellular differentiation (Ward et al. 2010).

8 CONCLUDING REMARKS
In multicellular organisms, cell growth and proliferation are normally not cell
autonomous. Receptor-mediated signal transduction, initiated by extracellular
growth factors, promotes entry into the cell cycle and reprograms cellular
metabolism to fulfill the biosynthetic needs of cell growth and division (Fig.
6). However, despite having become highly dependent on instruction from
extracellular growth factors, mammalian cells have retained the ability to
sense their internal metabolic reserves and adjust their growth and
biosynthetic activities accordingly. Much of this feedback control occurs at
the level of posttranslational modifications of signal transduction proteins by
key cellular metabolites. Moreover, intracellular metabolites can also regulate
chromatin accessibility to control gene expression.
Figure 6. Growth factor signaling reprograms cellular metabolism to promote biosynthesis but is
sensitive to feedback control by metabolically sensitive protein modifications. Signaling downstream
from PI3K/Akt enhances glucose uptake, glycolysis, and the flux of glucose carbon into cytosolic
acetyl-CoA and lipids. HIF1 signaling further promotes glycolysis while also enhancing the flux of
pyruvate into lactate under conditions of O2 limitation or nutrient excess. The Myc transcription factor,
activated downstream from Ras, enhances glutamine uptake and metabolism as well as nucleotide
biosynthesis. mTORC1 signaling responds to both upstream PI3K/Akt signaling and the levels of
essential amino acids to promote protein synthesis. PKM2 is a pyruvate kinase isoform specific to
proliferating cells that is uniquely sensitive to inhibition by tyrosine kinase signaling downstream from
growth factors. Metabolically sensitive protein modifications, such as receptor glycosylation and
nuclear histone acetylation, provide a way for the cell to exert feedback control on the output of growth
factor signaling.
 The evolution of the ability to regulate chromatin accessibility by specific
metabolites may have preceded the ability of growth factor signaling to
reprogram metabolism in multicellular organisms. Nonetheless, many aspects
of how variations in cellular metabolism influence chromatin accessibility
remain to be fully characterized. In addition to intracellular glucose
metabolism directly altering the acetylation state of histones, the methylation
state of histone lysine and DNA cytosine may also be metabolically
responsive. These methylation states were once thought to be irreversible, but
recent work has described demethylating enzymes for both histones and
DNA that are sustained by α-ketoglutarate production downstream from
glutamine metabolism. As discussed above, these α-ketoglutarate-dependent
enzymes can be inhibited by the 2HG produced by IDH1/IDH2 mutations.
Another area of continuing investigation is how minimizing ATP
production and enhancing ATP consumption can facilitate the rapid nutrient
metabolism of proliferating cells (Israelsen and Vander Heiden 2010).
Minimizing cellular ATP accumulation runs counter to the metabolic strategy
of quiescent cells, which completely oxidize the majority of glucose carbon
in the mitochondria to maximize ATP production. But proliferating cells with
activated growth factor signaling pathways usually take up nutrients far in
excess of the level required to maintain ATP levels and avoid AMPK
activation. Moreover, glycolysis in proliferating cells is limited by the rate of
ATP consumption, not ATP production (Scholnick et al. 1973). Increasing
cellular ATP consumption and the ADP:ATP ratio may be critical for
relieving the inhibition of glycolytic enzymes that can occur when ATP
levels are high, inhibition that would otherwise prevent high glycolytic flux
and enhanced macromolecular biosynthesis from glycolytic intermediates.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006783
CHAPTER 8

Signaling Networks that Regulate Cell

Migration

Peter Devreotes1 and Alan Rick Horwitz2

1Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland
21205
2Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia
22908
Correspondence: pnd@jhmi.edu a005959

SUMMARY

Stimuli that promote cell migration, such as chemokines, cytokines, and

growth factors in metazoans and cyclic AMP in Dictyostelium, activate
signaling pathways that control organization of the actin cytoskeleton
and adhesion complexes. The Rho-family GTPases are a key
convergence point of these pathways. Their effectors include actin
regulators such as formins, members of the WASP/WAVE family and
the Arp2/3 complex, and the myosin II motor protein. Pathways that link
to the Rho GTPases include Ras GTPases, TorC2, and PI3K. Many of
the molecules involved form gradients within cells, which define the
front and rear of migrating cells, and are also established in related
cellular behaviors such as neuronal growth cone extension and
cytokinesis. The signaling molecules that regulate migration can be
integrated to provide a model of network function. The network displays
biochemical excitability seen as spontaneous waves of activation that
 propagate along the cell cortex. These events coordinate cell movement
and can be biased by external cues to bring about directed migration.

Outline
1 Introduction
2 The migration machinery
3 Migration signaling networks
4 Biased excitable biochemical networks in chemotaxis
5 Concluding remarks
References

1 INTRODUCTION
Cell migration plays a pivotal role in a wide variety of phenomena
throughout phylogeny (Trinkaus 1969; Ridley et al. 2003; Lee et al. 2005;
Cai et al. 2010). In the amoeba Dictyostelium, it functions in nutrient seeking,
cell–cell aggregation, and the morphogenesis of multicellular structures. In
metazoans, cells migrate both throughout embryogenesis and in the adult. In
early developmental events, such as gastrulation or dorsal closure, large cell
sheets migrate and fold; at later stages, precursor cells that reside in the
neural crest, somites, brain ventricles, and other stem cell regions leave
epithelial sheets and migrate to their target destinations. In the adult, cell
migrations are critical for immune cell trafficking, wound healing, and stem
cell homing, among other processes. A closely related phenomenon is the
directed growth of specialized cellular extensions (e.g., yeast mating
“shmoos” [Slessareva and Dohlman 2006], pollen tubes [Takeuchi and
Higashiyama 2011], and the dendrites, axons, and spines of neurons [Tada
and Sheng 2006; Geraldo and Gordon-Weeks 2009]).
Most migrating cells and cellular extensions have an internal compass that
enables them to sense and move along gradients of soluble attractants and
repellents, a process referred to as chemotaxis (Devreotes and Janetopoulos
2003). Many chemoattractants act through G-protein-coupled receptors
(GPCRs). Examples include cAMP acting on cAMP receptors in
Dictyostelium and chemokines such as SDF1 acting on chemokine receptors
in metazoans. Growth factors acting on receptor tyrosine kinases and
cytokines such as transforming growth factor β (TGFβ) also function as
chemoattractants. Like migrating cells, the growth of axons can be guided by
a series of extracellular protein attractants and repellents (e.g., nephrins).
Cells can also be guided by gradients of immobilized signaling molecules
(haplotaxis), substrate rigidity (durotaxis), electric fields (galvanotaxis), and
shear force (mechanotaxis). Importantly, many diseases involve defective or
unregulated cell migration or protrusive growth (Ridley et al. 2003). For
example, tumor invasion and metastasis occur as a consequence of the
movement of both individual cells and large collectives (Friedl and Gilmour
2009; Friedl and Alexander 2011), arthritis and asthma result from excessive
migration of inflammatory cells (Montoya et al. 2002; Vicente-Manzanares et
al. 2002), and several cognitive disorders are accompanied by abnormal
neuronal extensions (Newey et al. 2005; van Galen and Ramakers 2005).
Cell migration requires coordination of cytoskeletal dynamics and
reorganization, cell adhesion, and signal transduction, and takes a variety of
forms (see Box 1) (Lauffenburger and Horwitz 1996; Mitchison and Cramer
1996; Ridley et al. 2003). Here, we first examine the machinery that drives
migration—the actin cytoskeleton, cell adhesions, and their regulators. We
then discuss signaling networks that control the migration machinery, starting
with those closest to the cytoskeleton then adding upstream components.
Finally, we address how chemotactic cues regulate motility. There are, of
course, other kinds of motility, such as sperm and cilial motility, but they use
microtubule-based mechanisms and are not addressed here.

BOX 1. THE SPECTRUM OF CELL MIGRATION BEHAVIORS

Cells can move in a variety of different ways, depending on the

 differentiated cell type, the surrounding environment, and the organism.
The “mesenchymal” migration of fibroblasts, which have large actin
filament bundles and prominent adhesions, is slow, for example.
Similarly, keratocytes have an actin-rich lamellipodium, but these move
more rapidly than fibroblasts. The amoeboid movements of neutrophils
and Dictyostelium are instead characterized by the presence of rapid,
efficient pseudopodial extensions and low adhesion. Cells such as
primordial germ cells and some leukocytes and tumor cells can move by
“blebbing,” a contraction-mediated squeezing from the rear that
produces a protrusion in regions lacking highly organized actomyosin
filaments (Charras and Paluch 2008; Friedl and Wolf 2010; Schmidt and
Friedl 2010). These migration modes are related, residing along a
continuum, and can interconvert depending on cell state, the
extracellular environment, and the relative activation of different
pathways; but they are distinct from the “swimming” driven by beating
of flagella or cilia that is observed in some cells. Migration can result in
the movement of single cells, small collectives, or large sheets. It can
also occur over a variety of substrata that include other cells and
extracellular matrix components. Tumor cells can adapt to their
environment by using diverse migration modes that include
mesenchymal, amoeboid, and blebbing modes. They can also use
specialized adhesion structures like invadopodia, which localize
proteolytic activity that degrades the local matrix (Linder et al. 2011).

2 THE MIGRATION MACHINERY

2.1 Actin Polymerization and Myosin-Mediated Contraction
Polymerization of globular (G) actin monomers to form filamentous (F) actin
is critical for cell migration (Pollard and Borisy 2003; Ridley 2011). It
produces oriented filaments that grow at the so-called barbed end and push
the front (the leading edge) of the cell forward, driving cell migration. In cells
that migrate by blebbing, actin stabilizes the blebs following their protrusion
(Charras and Paluch 2008; Fackler and Grosse 2008). Actin filaments arise
and grow through a complex but well-understood process (Fig. 1). Actin
nucleation and polymerization are regulated by formins (e.g., mDia1 and
mDia2) and the Arp2/3 complex (Insall and Machesky 2009; Chesarone et al.
2010; Ridley 2011). The formins nucleate and regulate the growth of linear
actin filaments (Goode and Eck 2007; Paul and Pollard 2009). These
processive capping proteins sequentially add actin monomers while
remaining weakly bound to the rapidly growing (barbed) end of the
filaments, a process termed processive elongation. The Arp2/3 complex
nucleates branches from existing actin filaments at a 70° angle and thereby
produces the dendritic actin network that is prominent near the leading edge
of broad protrusions and appears to stabilize them (Insall and Machesky
2009).
Figure 1. Regulation of actin dynamics by formins and Arp2/3 in cellular protrusions. The Rho
GTPases Rac, RhoA, and Cdc42 regulate actin dynamics at the leading edge via their effects on the
activities of formins (mDia), Arp2/3 complex, and LIM kinase (LIMK). Arp2/3 nucleates actin
branches that are seen in broad protrusions. Its activity is regulated by Cdc42 and Rac1, which act on
WASP/WAVE-containing protein complexes. Rac and Cdc42 also act on PAK, which phosphorylates
LIM kinase, which in turn regulates cofilin, a severing protein. Finally, RhoA acts on mDia1 and
Cdc42 acts on mDia2 to promote actin polymerization using a processive capping mechanism. RhoA
also activates profilin, which binds to actin monomers and increases the rate of polymerization. These
GTPases are activated in a clear temporal sequence near the leading edge (Machacek et al. 2009). AID,
autoinhibitory domain; FH, formin homology domains; RBD, Rho-GTPase-binding domain.

These mediators of actin branching and polymerization are highly

regulated. In fibroblasts and many other cells, mDia1 and mDia2 are
regulated by the Rho-family small GTPases RhoA and Cdc42, respectively,
which relieve an autoinhibitory state. Two other proteins, ABI1 and Gα12/13,
appear to direct mDia1 to actin filaments and adhesions. The Arp2/3 complex
contains six subunits, including two actin-related proteins, Arp2 and Arp3,
which nucleate new actin filaments by binding to the side of existing
filaments. WASP family members (WAVE [also known as Scar] and the
WASPs) are targets of the Rho-family GTPases Rac1 (WAVE) and Cdc42
(WASP) and in turn interact with the Arp2/3 complex and regulate its activity
(Pollitt and Insall 2009; Padrick and Rosen 2010).
Actin filaments are capped at the barbed end by capping proteins, which
inhibits depolymerization and thereby stabilizes them. Anticapping proteins
of the Mena/Vasp family, in turn, antagonize capping proteins and thereby
regulate capping (Krause et al. 2003; Bear and Gertler 2009). Cofilin is
another major regulator of actin filament stability that severs actin filaments;
it also binds to G-actin, increasing the off-rate of actin monomers at the
pointed (nonpolymerizing) end (Condeelis 2001; Bamburg and Bernstein
2008). LIM kinase, which is activated by Cdc42 and Rac1, stimulates cofilin
activity by phosphorylation. LIM kinase is itself regulated by Cdc42 and
Rac1, which act via the kinase PAK (Yamaguchi and Condeelis 2007;
Bamburg and Bernstein 2008). Finally, profilin binds to actin monomers and
increases the polymerization rate.
Contraction forces generated by myosin II motor proteins (Bugyi and
Carlier 2010) are coordinated with actin polymerization at the leading edge
and have several roles in migration (Small and Resch 2005; Vicente-
Manzanares et al. 2009). First, in fibroblasts and epithelial cells, myosin II
promotes retrograde movement of actin filaments away from the zone of
active actin polymerization in the lamellipodium. This retrograde flow
essentially subtracts from actin polymerization at the leading edge and can
reduce the net protrusion rate (Ponti et al. 2004). The forces from both
retrograde flow and actin polymerization can be “shunted” to the substratum
via integrin-based adhesions linked to actin filaments (Mitchison and
Kirschner 1988; Jay 2000). This shunting inhibits retrograde flow and
enhances the protrusion rate, because the full force of actin polymerization
acts at adhesions and the membrane at the leading edge. However, the
transmission of force from actin through adhesions to the substratum is not
always complete and can lead to varying rates of retrograde flow (Brown et
al. 2006; Hu et al. 2007; Wang 2007; Chen et al. 2012). Second, the pressure
from myosin-mediated contractions in the rear and sides can produce blebs in
regions depleted of actomyosin filaments (Charras and Paluch 2008). In cells
that move by blebbing, myosin-based contraction alone drives migration;
however, the blebs are stabilized by the formation of a dendritic actin
meshwork.
Myosin II activity is regulated by phosphorylation of myosin’s regulatory
light chain (RLC), and its assembly into filaments is regulated by
phosphorylation in the tail region of the heavy chain (Vicente-Manzanares et
al. 2009). A number of kinases can phosphorylate the RLC. Among the best
studied are myosin light-chain kinase (MLCK) and Rho-associated protein
kinase (ROCK); myosin phosphatase hydrolyzes the phosphate. In contrast to
RLC regulation, the kinases regulating filament assembly are not well
understood (Vicente-Manzanares et al. 2009).

2.2 Adhesion
Adhesion to the substrate is common to most migrating cells. Integrin-
mediated adhesions are the best studied (Hynes 2002). The integrins are a
large family of heterodimeric transmembrane receptors that link to actin via a
specialized set of molecules that include talin, vinculin, and α-actinin. The
adhesions in which these components reside are large assemblies containing
>150 different molecules that mediate intracellular signaling in addition to
adhesion to proteins in the extracellular matrix, such as fibronectin and
laminin (Zaidel-Bar et al. 2007; Parsons et al. 2010; Zaidel-Bar and Geiger
2010). The affinity of integrins is regulated by the binding of talin and
kindlin, cytoplasmic proteins that bind directly to the cytoplasmic domain of
the integrin β subunit, and also by phosphatidylinositol 4,5-bisphosphate
(PIP2) and other adhesion-associated molecules (Moser et al. 2009; Shattil et
al. 2010).
Adhesions serve both as traction points and as signaling centers during
cell migration (Parsons et al. 2010). As traction points, they transmit forces to
the substrate so that actin polymerization causes protrusion at the cell front.
These traction points are released at the cell rear as it retracts and the cell
moves forward. Although this release is efficient in some cells and substrates,
it is not in others and can be rate limiting for migration (Lauffenburger and
Horwitz 1996). Thus, there is an optimum strength of attachment that allows
sufficient adhesion for traction at the cell front and yet allows for efficient
release at the rear (Palecek et al. 1997). As signaling centers, adhesions in
protrusions regulate actin polymerization and myosin II activity through Rho-
family GTPases (Parsons et al. 2010). Although adhesions can vary
considerably in size, location, and presumably function, they have not yet
been classified clearly and meaningfully based on differences in composition
and function. Adhesions in vivo tend to be small and dynamic in migrating
cells; however, highly elongated adhesions have also been observed
(Harunaga and Yamada 2011; Kubow and Horwitz 2011).
The cytoskeleton in turn regulates adhesions via an incompletely
understood feedback loop involving actin polymerization and myosin-II-
mediated contraction (Fig. 2). Nascent adhesions form in the region of
dendritic actin, and their formation is coupled to actin polymerization
(Alexandrova et al. 2008; Choi et al. 2008). At the interface of dendritic actin
in the lamellipodium and the actin bundles in the adjacent lamellum,
adhesions elongate along actin filament bundles (Small et al. 2002; Choi et
al. 2008; Geiger and Yamada 2011; Oakes et al. 2012). The fraction of
adhesions that grow, as well as the extent of maturation, is determined at least
in part by myosin II activity (Vicente-Manzanares et al. 2009; Oakes et al.
2012). Proteases such as calpain, a calcium-activated protease, mediate
adhesion disassembly by acting on adhesion proteins such as talin, which link
actin and integrins (Franco et al. 2004; Chan et al. 2010; Cortesio et al.
2011). The repeated direct contact between microtubule tips and adhesions
and endocytosis of integrins driven by the GTPase dynamin also contribute to
disassembly (Kaverina et al. 1999; Broussard et al. 2008; Ezratty et al. 2009;
Gerisch et al. 2011).
Figure 2. Adhesions serve as contact points and signaling centers. Integrin-based adhesions are large,
complex assemblies that link the substratum to actin and generate signals that regulate Rho GTPases
and cell migration. The structural linkage to actin is thought to be mediated by talin, vinculin, and
perhaps α-actinin. The signaling is mediated by adhesion-associated complexes. The paxillin/FAK
module and its link to some Rac GEFs and Rho GEFs is shown as an example. The Arp2/3 complex
and myosin II, whose activity is regulated by Rho and Rac, are also shown.

The formation, dynamics, and function of adhesions are highly regulated.

In migrating cells, they form in protrusions near the leading edge. In rapidly
migrating, amoeboid-like cells, adhesions in protrusions are small and tend to
form and turnover rapidly, making them difficult to visualize. Few, if any
undergo significant maturation into large, elongated structures. Cells
undergoing slower, mesenchymal migration tend to have larger adhesions
with a significant number maturing to large, elongate adhesions (Parsons et
al. 2010). These large adhesions do not appear to generate signals that drive
actin polymerization.

2.3 Polarization
The presence of a distinct front and rear is a key feature of cell migration.
Some cells can polarize spontaneously and migrate in a directionally
persistent manner. The machinery that establishes polarity is incompletely
understood but microtubules, vesicle cycling, and actomyosin filaments
appear to be the drivers. In epithelial cells and astrocytes, polarity is
established through a signaling pathway involving Cdc42, Par3/6, and
atypical protein kinase C (aPKC) that targets microtubules (Etienne-
Manneville and Hall 2002; Etienne-Manneville et al. 2005). This pathway
orients the microtubule-organizing center (MTOC) and Golgi apparatus (Ch.
9 [McCaffrey and Macara 2012]). In fibroblasts, activated myosin II creates a
region of actomyosin filament bundles that terminate in adhesions that do not
contain guanine nucleotide exchange factors (GEFs) and therefore do not
support Rac or Cdc42 signaling and actin polymerization (Vicente-
Manzanares et al. 2008, 2011). This region becomes the rear and sides, and
zones of active Rac generate protrusions that elongate the cell to form the
front; the actomyosin system appears to set up the initial polarity, which is
then refined by the microtubule system (Vicente-Manzanares et al. 2008).

3 MIGRATION SIGNALING NETWORKS

A complex signaling network regulates the cytoskeleton and adhesion in the
context of migration. Below, we focus on Rho GTPases, integrins, and
phosphoinositides, which have been extensively investigated, although it is
clear that Ras proteins, calcium, cyclic nucleotides, numerous kinases, and
other components are also involved. These networks contain positive- and
negative-feedback loops, redundancies, and points of cross talk often
involving synergy between adhesions, chemotactic receptors, and growth
factor receptors. We speculate below on the different roles of these signaling
events, which are integrated to bring about migration.

3.1 Rho-Family GTPases Regulate Cytoskeletal Activity

Rho-family small GTPases are a major convergence point of migration-
associated signaling (Heasman and Ridley 2008). Protrusion, adhesion, and
polarization are all regulated by Rho-family GTPases, and many receptor-
initiated signaling pathways link to the Rho GTPases (Ridley et al. 2003;
Zaidel-Bar et al. 2007; Parsons et al. 2010). These include chemokine
receptors and growth factor receptors, such as the epidermal growth factor
(EGF) receptor. The response depends on the spatial and temporal
segregation of the activities of the different GTPases and involves multiple,
parallel, redundant, and synergistic pathways that form a complex network.
The pathways involved regulate Rho GTPase activity by acting on the
many GEFs and GTPase-activating proteins (GAPs) that control their activity
(Etienne-Manneville and Hall 2002; DerMardirossian and Bokoch 2005). The
GTPases are also regulated by Rho GDP dissociation inhibitors (GDIs),
which remove them from the membrane (Garcia-Mata et al. 2011). The Rho
family has several members, whose functions, in the context of migration, are
represented by Rac, RhoA, and Cdc42. These act on a number of effectors
that control the cytoskeletal machinery (see above) (Ridley 2006, 2011;
Heasman and Ridley 2008). For example, Rac and Cdc42 regulate actin
polymerization by acting on the WASP family and consequently Arp2/3:
Cdc42 regulates mDia2, and Rac regulates cofilin through LIMK (Ridley
2011). RhoA also regulates actin polymerization by acting on mDia1. In
addition, it regulates adhesion and actin organization via myosin II activity:
RhoA activates ROCK, which phosphorylates the myosin RLC and inhibits
myosin phosphatase, both of which stimulate myosin II. Cdc42 also regulates
microtubule dynamics, which in turn affects the turnover of some adhesions
(Kraynov et al. 2000; Nalbant et al. 2004; Machacek et al. 2009). The front-
back polarity required for migration requires that actin polymerization
localizes to specific cellular regions (i.e., the leading edge of migrating cells).
This is reflected in the polarized activity of Rac and Cdc42 and the intricate
relative kinetics of their activation.

3.2 The Paxillin/FAK Signaling Module

In mesenchymal cells at least, Rho-family GTPases are regulated by
signaling complexes that reside in adhesions; they are activated by ligation of
integrins to matrix proteins such as fibronectin, whose signaling synergizes
with growth factor and chemokine receptor pathways (Ridley et al. 2003).
The focal adhesion kinase (FAK)/paxillin signaling module is the best-
studied example. Paxillin is among the earliest molecules to enter adhesions
as they form and remains present until they disassemble (Webb et al. 2004).
It functions as a signaling adapter that binds to numerous molecules involved
in Rho-family GTPase signaling (Brown and Turner 2004; Deakin and
Turner 2008).
The amino terminus of paxillin has a series of LD regions. The first two
of these include two SH2-domain-binding sites (around Y31 and Y118).
They are phosphorylated by FAK and Src and bind to a number of molecules,
including Crk/p130Cas, FAK/Src, and Ras GAP. The phosphatase PTP PEST
binds near the carboxyl terminus of paxillin and dephosphorylates these sites.
p130Cas recruits a Cas-Crk-Dock180-Elmo complex, in which Dock180
functions as a GEF that activates Rac. Two other LD regions of paxillin bind
GIT1 and GIT2 (Turner et al. 2001; Hoefen and Berk 2006). These adapters
bind to Pix, a GEF for Cdc42 and Rac (Deakin and Turner 2008). Pix also
binds to the kinase PAK, a Rac effector, creating a Rac and Cdc42 activator-
effector signaling module (Bokoch 2003). The p85 subunit of
phosphoinositide 3-kinase (PI3K) also binds to this region of paxillin and is
involved in signaling to Vav, another Rac GEF (see below) (Tybulewicz et
al. 2003). This list of interactions is not exhaustive but serves to illustrate the
central role of paxillin as a regulator of protrusion through its action on Rac
and Cdc42. Vinculin, a tension-sensitive structural molecule implicated in the
integrin-actin linkage, also binds to the LD region of paxillin (Deakin and
Turner 2008).
Nearly all of these binding interactions are regulated by phosphorylation
of paxillin on Y31 and Y118, which creates the two SH2-binding sites as
well as inducing a major conformational change. Conformational regulation
is a general theme in adhesion signaling (Parsons et al. 2010; Zaidel-Bar and
Geiger 2010). Src, FAK, paxillin, and p130 Cas are all conformationally
activated, at least in part, by phosphorylation events, which often serve to
release an autoinhibitory state (Cohen et al. 2006; Sawada et al. 2006;
Parsons et al. 2010). Tension can also activate or regulate the activities of
adhesion molecules. For example, p130Cas, talin, and vinculin are all tension
sensitive (Sawada et al. 2006; del Rio et al. 2009; Grashoff et al. 2010).
These sensitivities are thought to regulate adhesion-generated signals
(Bershadsky et al. 2003; Schwartz 2010).
FAK binds to paxillin following tyrosine phosphorylation of paxillin Y31
and Y118 (Choi et al. 2011). The phosphorylation occurs after both
molecules are in nascent adhesions and appears to result from a
conformational activation of paxillin, because FAK binds to the carboxyl end
of paxillin. FAK also recruits molecules that regulate Rho-family GTPases,
functions as a tyrosine kinase, and possesses tyrosine phosphorylation sites,
most of which are phosphorylated by Src (Parsons 2003; Mitra et al. 2005;
Frame et al. 2010). Activated FAK binds to Src (via its SH2 domain), to
p130Cas (via an SH3 domain), and to a p120RasGAP-p190RhoGAP
complex, which negatively regulates RhoA activity. FAK binds to two Rho
GEFs, p190RhoGEF and PDZRhoGEF, which activate RhoA (Tomar and
Schlaepfer 2009). p190RhoGEF and p190RhoGAP do not appear to bind to
FAK at the same time, and in spreading cells, binding of p190RhoGAP
precedes that of p190RhoGEF. This provides a potential mechanism for the
transient, local and sequential activation of RhoA seen at the leading edge of
migrating cells. Thus, RhoA activity appears to be controlled by antagonistic
regulators that interact with FAK. In addition, the activity of p190RhoGAP,
for example, depends on phosphorylation, which suggests that the GAPs (and
probably the GEFs) are regulated by phosphorylation (Tomar and Schlaepfer
2009).

3.3 Other Rho-Regulating Modules

The Pax/FAK model reveals the importance and function of signaling
complexes that localize the activity of Rho-family GTPases; but other
complexes do this as well. For example, the ILK-pinch-parvin complex is
another adhesion-associated system that signals to Rac. The pseudokinase
scaffold protein ILK binds to parvin, which binds to Pix (Sepulveda et al.
2005; Legate et al. 2006). The SH2/SH3 adapter NCK is also implicated in
Rac signaling in adhesions (Ruusala and Aspenstrom 2008). Finally, note that
Rho-family GTPases are regulated by their association with the plasma
membrane. They are targeted to lipid rafts and regulated by phosphorylation-
dependent interactions with Rho GDI and endocytic events, which thereby
regulate their activity (Grande-Garcia et al. 2005).

3.4 The PI3K Signaling Module

The PI3K signaling module (p. 87 [Hemmings and Restuccia 2012]) acts
dynamically at the leading edge of the cell to regulate the accumulation of
phosphatidylinositol 3,4,5-trisphosphate (PIP3) and in turn cytoskeletal
activities. The links between PIP3 and the cytoskeleton are a subject of
intense investigation. PIP3 targets include substrates of Akt, including PAK,
as well as a series of PH-domain-containing proteins (Kamimura et al. 2008;
Tang et al. 2011). In randomly migrating cells, patches of PIP3 are generated
spontaneously and appear at the tips of protrusions. In cells migrating in
gradients of chemoattractants such as chemokines or growth factors,
receptors and G proteins are distributed uniformly around the cell perimeter.
However, PIP3 accumulation as well as other signaling events are
dynamically localized at the tips of pseudopodia at the front (Parent and
Devreotes 1999). PIP3 accumulates at the leading edge of the pseudopodia
because, in Dictyostelium at least, PI3K is recruited to, and PTEN (a 3′-
specific phosphatase that hydrolyzes PIP3) is lost from, these regions
(Funamoto et al. 2002; Iijima and Devreotes 2002; Arai et al. 2010). In
neutrophils, the 5′-phosphatase Ship1 also degrades PIP3 (Nishio et al. 2007;
Mondal et al. 2012).
Asymmetric PIP3 accumulation has been conserved throughout evolution
in migrating cells and also operates where cells undergo related
morphological changes (Fig. 3)—for example, in the extending membranes
of neuronal growth cones (Wang et al. 2002; Lacalle et al. 2004; Chadborn et
al. 2006; Evans and Falke 2007; Yoo et al. 2010), during cytokinesis (a
process which resembles two cells migrating away from each other)
(Janetopoulos et al. 2005; Janetopoulos and Devreotes 2006), in phagocytosis
(Clarke et al. 2006), and in mammary and prostate epithelia (in which the
basal lateral and apical portions of stationary cells are akin to the front and
rear of a migrating cell, PIP3 being localized to the basal–lateral region and
PTEN being localized to the apical region [Shewan et al. 2011]).

Figure 3. Asymmetric accumulation of PIP3 is a feature of a spectrum of cell morphological changes.

Panels show snapshots of the dynamic distribution of PIP3 in cells undergoing various morphological
changes. (A) Human neutrophils expressing a biosensor for PIP3 (PHakt-GFP). The cells have been
exposed to a gradient formed by a micropipette filled with the chemoattractant C5a (position indicted
by *). The arrow shows recruitment of PHakt-GFP to the membrane, indicating an elevated level of
PIP3. (B) A dividing Dictyostelium cell expressing PHCrac-GFP as a biosensor for PIP3. Arrows point
to the accumulation of PIP3 at the poles of the dividing cell. (C) Prostate epithelial cells expressing
PHakt-GFP. (Image courtesy of Tamara Lotan.) Arrows point to the accumulation of PIP3 on the
basal–lateral membranes. (D) Dictyostelium cell expressing PHCrac-GFP phagocytizing latex beads.
The arrow indicates accumulation of PIP3 around two beads; arrowheads point to PIP3-labeled
pseudopods in the same cell. (Image courtesy of Margaret Clarke.) (E) The growth cone of rat dorsal
root ganglion expressing PHakt-GFP. Arrows indicate the accumulation of PIP3 at the leading edge.
(Image courtesy of Britta Eickholt.)

It is now clear that the asymmetrical accumulation of PIP3 is sufficient to

promote actin polymerization and produce cellular projections; but it is one
of a number of parallel pathways. Alterations in PIP3 levels lead to defects in
cell migration, cytokinesis, phagocytosis, and epithelial architecture. In
Dictyostelium cells lacking PTEN or neutrophils lacking Ship1, for example,
excessive amounts of PIP3 are generated at the front of the cell and this
diffuses along most of the cell perimeter (Funamoto et al. 2002; Iijima and
Devreotes 2002; Nishio et al. 2007). This additional PIP3 elicits ectopic
pseudopodia at lateral regions outside the leading edge. If the PTEN-deficient
cells are treated with inhibitors of PI3K, the morphological defects are
suppressed, and the cells again display a single anterior pseudopod (Chen et
al. 2003). Using a synthetic PI3K activation system in neutrophils, Inoue and
Meyers showed that elevation of PIP3 alone is sufficient to initiate
pseudopodial extensions (Inoue and Meyer 2008). However, because
asymmetrical generation of PIP3 is only one of several parallel pathways, it is
not essential for a directional response. Inhibition of PI3K blocks migration
in zebrafish neutrophils and fibroblasts and random migration in
Dictyostelium but, under certain conditions, does not block chemoattractant-
driven migration in amoebae or human neutrophils (Chen et al. 2003;
Ferguson et al. 2007; Hoeller and Kay 2007). Furthermore, primordial germ
cells in zebrafish have persistent, uniformly distributed membrane PIP3 levels
even as they migrate directionally (Dumstrei et al. 2004). These observations
confirm that there are parallel pathways that allow cells to receive directional
cues from chemoattractant receptors in the absence of PIP3.

3.5 Genetic Analysis of a Signaling Network

The multiple parallel pathways that drive migration form a complex network
of coordinated events that are triggered by chemoattractants but can also
occur spontaneously as cells migrate. A successful explanation of cell
migration has to integrate the information from initially independent studies
of pathways believed to directly regulate the cytoskeleton, such as those
involving Rho GTPases, with others thought to transduce signals from
receptors, such as PI3K signaling. Interestingly, an emerging theme is that
downstream and upstream events are probably linked through multiple
feedback loops.
Genetic analyses in Dictyostelium have implicated about 95 nonlethal
genes in chemotaxis and about 40 can be organized into an internally
consistent “wiring diagram” (Swaney et al. 2010). Some of the major features
of the network include the presence of parallel pathways defined by cyclic
GMP, myosin heavy-chain kinase (MHCK), the kinase Tor complex 2
(TorC2), PIP3, and phospholipase A2 (PLA2) (Veltman et al. 2008). Four
isoforms of the small G protein Ras are activated by chemoattractant and
seem to act early in these pathways (Kae et al. 2004; Sasaki and Firtel 2009).
A portion of the network involving PIP3 and TorC2 is examined in more
detail below. Interestingly TorC2 is defined by subunits Pianissimo and Rip3.
These highly conserved genes were first identified as causing chemotaxis
defects in Dictyostelium and later renamed as Rictor and Sin1, respectively
(see p. 91 [Laplante and Sabatini 2012]). Remarkably, the basic elements of
this network appear to be conserved in human neutrophils, although further
analysis of each pathway is needed. Similarities include the rapid activation
of K-, H-, and N-Ras by chemoattractants, localization of PIP3 at the leading
edge of the cell, and the critical role for mTorC2 (Bokoch 2003; Van
Keymeulen et al. 2006; Liu and Parent 2011). One apparent difference is that
a role for cyclic GMP has not been described in neutrophils.
There is a spatiotemporal pattern to many elements of the network.
Biosensors for activation/formation/recruitment of Ras, PI3K, PIP3,
HSPC300 (a subunit of the WAVE complex), and LimE (an actin-binding
protein) serve as dynamic markers for the front of the cell whereas others,
such as PTEN and myosin, define the back. The front markers reside in the
cytosol, but as protrusions form they are recruited to the tips of pseudopodia
(Van Haastert and Devreotes 2004). In contrast, the back markers reside
uniformly in the cortex and dissociate from regions where protrusions form
(Funamoto et al. 2002; Iijima and Devreotes 2002; Robinson and Spudich
2004). For example, a biosensor for the collective activation of Ras proteins
(the Ras-binding domain of the kinase Raf fused to GFP) moves to
protrusions at the leading edge of a migrating cell, whereas PTEN-GFP falls
off. When cells are stimulated with a uniform chemoattractant, all of the front
components are recruited from the cytosol to the cell periphery, whereas the
back components fall off and move to the cytosol (Swaney et al. 2010). These
changes are transient and the components reestablish their original locations
within a few minutes.
Examining a small portion of the network involving PIP3 and TorC2 in
detail reveals how genetic analyses have helped delineate signaling
mechanisms involved in cell migration (Fig. 4). As outlined above, cells with
elevated PIP3 levels owing to loss of PTEN display a “migration” phenotype
in which ectopic protrusions form over most of the cell perimeter. This can
be reversed by disrupting the Akt ortholog PKBA (Tang et al. 2011).
Interestingly, the cells lacking both PTEN and PKBA have elevated PIP3
levels around the perimeter but still respond effectively to chemotactic cAMP
gradients. Parallel pathways must therefore mediate the directional response
despite the uniform PIP3 distribution. One of these involves a second PKB
isoform, PKBR1, which can be activated independently of PIP3; unlike Akt
and PKBA, PKBR1 lacks a PH domain and is instead tethered to the
membrane by myristoylation (Kamimura et al. 2008). The activation of
PKBR1 is mediated by phosphorylation of a hydrophobic motif by TorC2.
Phosphorylation of serines/threonines within conserved hydrophobic motifs
in the carboxy-terminal region of many ACG-family kinases (which included
PKA, PKC, and PKG) can be required for their action. The phosphorylation
of PKBR1 is part of a pathway that leads from chemoattractant-mediated
activation of Ras through TorC2 to regulation of the cytoskeleton (Chen et al.
1997; Lee et al. 2005; Cai et al. 2010). In cells lacking RasC or Aimless, a
Ras GEF for RasC, TorC2 activation is greatly reduced, whereas in cells
expressing a constitutively active RasC (Q62L), TorC2 activation is elevated
and ectopic sites of actin polymerization appear around the cell perimeter.
However, in cells lacking Pianissimo, a key subunit of TorC2, there is no
effect of expressing RasC Q62L and no phosphorylation of PKBR1. These
studies focus attention on PKB substrates. In Dictyostelium, there are at least
nine substrates that are rapidly, transiently phosphorylated in response to
chemoattractant. These include signaling and cytoskeletal proteins, such as
Ras GEFs and Rac GAPs, talin, PakA, and phosphatidylinositol 5-kinase
(PI5K) (Kamimura et al. 2008).

Figure 4. A portion of the Dictyostelium migration signaling network involving PIP3 and TorC2.
Colored blocks delineate modules. The overlapping of blocks indicates that some components belong to
several modules. CARE, cystic AMP receptors.

The PIP3-independent role of TorC2 is another example of an element of

the network that is conserved in neutrophils. Neutrophils lacking PI3K or
exposed to PI3K inhibitors migrate along chemoattractant gradients when
plated on certain extracellular matrices (Ferguson et al. 2007). Knocking
down the Rictor subunit of mTorC2 causes a severe defect in neutrophil
chemotaxis (Liu et al. 2010; Wang, pers. comm.). Although Akt is a substrate
of mTorC2 in neutrophils, other targets are probably more important.
Because PKC and other ACG kinases are phosphorylated on their
hydrophobic motifs, it is possible that the mTorC2 targets are these kinases in
neutrophils and other cells.

4 BIASED EXCITABLE BIOCHEMICAL NETWORKS IN

CHEMOTAXIS
4.1 Excitability of Signaling Networks Linked to Migration
The signaling network controlling cell migration displays behavior, including
oscillations and cortical wave propagation, which suggest the system is
excitable. Excitability typically arises when a system contains opposing
positive- and negative-feedback loops. These systems are in a resting state
until a threshold is crossed and an all-or-nothing response ensues. Total
internal reflection microscopy (TIRF) reveals that subunits of the WAVE
complex and actin-binding proteins participate in wavelike phenomena that
propagate along the basal surface of the cell. When these waves reach the
edge of the cell, they appear to push the perimeter outward. Essentially
similar phenomena are observed in human neutrophils and Dictyostelium
amoebae (Gerisch et al. 2004, 2011; Weiner et al. 2007; Bretschneider et al.
2009; Gerisch 2010). Actin and integrin waves have also been observed in
fibroblasts (Giannone et al. 2004; Döbereiner et al. 2006; Case and Waterman
2011). The waves form in the absence of stimulation and in mutants lacking
G proteins, indicating that this is an intrinsic behavior of motile cells.
Furthermore, activation of Ras and accumulation of PIP3 also occur in flashes
and bursting waves, which are propagated across the cortex (Arai et al. 2010;
Xiong et al. 2010).
These waves can be modeled mathematically, like action potentials in
neurons (Levine et al. 2006; Insall and Machesky 2009; Xiong et al. 2010;
Hecht et al. 2011), by linking components of a hypothetical network in
positive- and negative-feedback loops. Some feedback loops have been
described in the real network. For example, a positive-feedback loop appears
to link cytoskeletal events and PIP3 because inhibition of either reduces the
spontaneous activation of the other (Weiner et al. 2002; Inoue and Meyer
2008). Second, a negative-feedback loop involving the phosphorylation of
upstream Ras GEF by downstream PKB has been described in Dictyostelium
(Charest et al. 2010).
Cell migration may thus involve a mechanism in which guidance cues
differentially alter the excitability of the network on one side of the cell. That
is, if a cue decreases the threshold for excitability on the side of the cell
closer to the source and increases the threshold on the distal side, the cell will
be attracted to it. Such theoretical models are referred to as biased excitable
networks (BENs). Because they are typically excitable within very narrow
parameter ranges, BENs can provide extreme sensitivity to external signals
(Iglesias and Devreotes 2012). This mechanism may explain how
professional chemotactic cells such as leukocytes and Dictyostelium are able
to move up gradients of chemoattractant that differ by <2% over their body
length.

4.2 Adaptation to Chemotactic Signaling and Local

Excitation–Global Inhibition Models
The influence of an external signal on a BEN can depend on the absolute or
relative amount of the signal. The chemotactic response of 3T3 fibroblasts to
platelet-derived growth factor (PDGF), for example, depends on the absolute
concentration and diminishes as the concentration increases and the fractional
difference in receptor occupancy across the cell decreases (Schneider and
Haugh 2006). In contrast, responses to chemoattractants that signal via
GPCRs, such as FMLP in human leukocytes or cAMP in Dictyostelium,
depend primarily on the relative steepness rather than the absolute
concentration of the gradient (Devreotes and Zigmond 1988). The “relative”
systems can maintain sensitivity over a wide range of concentrations.
Studies of cells treated with inhibitors of the cytoskeleton, which allow
the direction-sensing system to be examined in isolation, have shown these
receptors produce rapid but transient signaling events, such as PIP3
accumulation and Ras activation. Further responses can only be elicited if the
stimulus is increased or removed and then reapplied (i.e., the cells adapt
when receptor occupancy is held constant). In contrast, when cells are
exposed to a chemotactic gradient, the biosensors form a crescent toward the
high side. The crescent is maintained persistently at steady state but can
immediately adjust if the chemoattractant gradient is shifted to a new
direction. How do they adapt to uniform stimuli yet respond persistently to a
gradient? The differential response to uniform versus gradient stimuli can be
explained by local excitation–global inhibition (LEGI) models (Fig. 5)
(Parent and Devreotes 1999; Janetopoulos et al. 2004; Levine et al. 2006). In
a LEGI model, an increase in receptor occupancy triggers a rapid excitatory
process, such as the dissociation of G-protein subunits, and a slower
inhibitory process that balances excitation. Whenever excitation exceeds
inhibition, a response regulator is generated; when inhibition catches up with
excitation, the response regulator returns to its basal level. Because excitation
is local, whereas the inhibitor is more global, in a gradient there is a persistent
deflection of the response regulator above and below its basal level at the
front and back of the cell, respectively.
Figure 5. Responses to uniform increases and gradients of chemoattractants in a LEGI model. (A)
Micrographs show translocation of a biosensor for PIP3 (PHCrac-GFP) to the membrane. PIP3 levels
rise transiently during persistent stimulation with a uniform chemoattractant. The schematic depicts the
response of a “front” marker such as PIP3 to uniform stimulation. A LEGI model assumes that the level
of a response regulator is controlled by the difference between rapid excitatory and slower inhibitory
processes. The response regulator (RR, blue line) rises when excitation (green line) is higher than
inhibition (red line) and then falls as inhibition catches up. (B) The micrograph shows that the steady-
state accumulation of PIP3 forms a crescent facing the high side of the gradient produced by a
micropipette releasing chemoattractant. The schematic depicts the behavior of a “front” marker such as
PIP3 in response to a gradient of chemoattractant. In the LEGI model, the response regulator (blue line)
rises when excitation (green line) is higher than inhibition (red line) and then falls to a new steady state.
Because inhibition is more global than the excitation the differences generate a response regulator that
has a higher concentration than basal at the front and a lower concentration than basal at the back.

The LEGI model is a useful conceptual device that allows one to predict
the response to any combination of applied temporal and spatial stimuli, but
further studies are needed to define the underlying biochemical events and to
link the model to cell migration. First, the excitatory process likely
corresponds to G-protein activation. When cells are exposed to
chemoattractant, the G-protein subunits dissociate within a few seconds and
all of the biochemical responses in the network are triggered. During the next
several minutes, the responses gradually subside even though the G protein
does not reassociate. The mechanism that offsets the activity of the G-protein
and causes the responses to subside remains to be determined. Second, LEGI
schemes can account for all of the behavior of immobilized cells but fail to
explain migration or polarity. However, the output of LEGI could enhance
excitability at the front and suppress it at the rear (Xiong et al. 2010). This
would ensure that the system responds to the steepness of a gradient but is
independent of its midpoint concentration. LEGI-BEN schemes are capable
of extraordinary sensitivity, and computer simulations show that this model
can produce realistic temporal and spatial chemotactic responses.

5 CONCLUDING REMARKS
Many of the principles described in this chapter appear to be general and
apply to cellular behaviors analogous to migration. For example, dendritic
spines, small extensions along dendrites of neurons in the central nervous
system, contain a highly organized postsynaptic density that receives
excitatory signals. Like protrusions in migrating cells, dendritic spines are
highly dynamic, they undergo complex morphologic changes, and they
contain a highly organized adhesion associated with the postsynaptic density.
Actin polymerization and actomyosin activity play a major role in spine and
postsynaptic density (PSD) organization (Oertner and Matus 2005; Hodges et
al. 2011). Rho-family GTPases have emerged as major regulators of spine
organization and dynamics and are implicated in human cognitive diseases.
Indeed, mutations in regulators of Rho-family GTPases are implicated in
spine-related diseases including autism, schizophrenia, and nonsyndromic
mental retardation. With respect to the latter, α-Pix (a Rac GEF), PAK3 (a
Rac/Cd42 effector), and oligphrenin 1 (a Rho GAP) are all associated with
nonsyndromic mental retardation in humans—a disease characterized by
spine defects (van Galen and Ramakers 2005). Pix and PAK are localized to
dendritic spines via GIT1 and thought to spatially restrict their formation
(Zhang et al. 2005). Asymmetric localization of PIP3 is observed in many
migrating cells. Oncogenic mutations leading to overproduction of PIP3 are
typically assumed to increase growth rates; but it is likely that many of the
cancer-causing effects can also be attributed to alterations in the cytoskeleton
(Kim et al. 2011). Distortion of cell migration signaling networks plays a
critical role in migration-related diseases such as invasive and metastatic
cancer (Ch. 21 [Sever and Brugge 2014]). The plethora of signaling pathways
that converge on Rho GTPases means there is a very large potential set of
loci for the misregulation of migration. It also suggests that drugs directed
against any particular pathway may not be effective for long given the
selection that occurs in the tumor environment. However, the convergence on
Rho-family GTPases and the limited migration machinery on which it acts
hold promise for therapeutic strategies targeting these GTPases and their
downstream effectors or diagnostic routes to identifying cells with invasive
potential.
Another emerging area of research is the study of migration in 3D. Until
recently, most migration studies focused on migration on planar substrates
using integrin-mediated adhesion. Analogous mechanisms are probably used
by cells migrating in 3D, or using other receptors (e.g., in the central nervous
system). However, different pathways might play more prominent roles in
each case. For example, in 3D, cell protrusions, adhesion, and cell
morphology all appear to differ from that generally seen on rigid planar 2D
substrates (Even-Ram and Yamada 2005; Provenzano et al. 2009; Friedl and
Wolf 2010; Sanz-Moreno and Marshall 2010). In 3D, the cells are more
elongated and possess narrower protrusions and smaller adhesions (Harunaga
and Yamada 2011). Moreover, there is evidence that different signaling
pathways are indeed involved. For example, depleting paxillin produces a
mesenchymal phenotype in 3D environments, whereas depleting the paxillin
relative Hic5 produces an amoeboid morphology (Deakin and Turner 2008,
2011). The particular signaling pathway used seems to depend on the cellular
microenvironment, which can differ between normal and tumor cells.
Research into cell migration has clearly made enormous progress. The
basic machines that drive migration have been described, and many of the
pathways that regulate them have been identified. However, we have only
scratched the surface and much remains to be understood. The interactions
and regulation of the complex signaling networks that orchestrate migration
and the mechanism by which extracellular forces affect these networks are
not understood. Furthermore, new modes of migration are being uncovered,
including blebbing-mediated migration and the newly described lobopodia
migration (Petrie et al. 2012). In addition, migration in complex in vivo
environments differs from that seen on rigid planar substrates and its study
presents unexpected challenges. Finally, integrative, quantitative models of
migration that conjoin the plethora of regulatory networks are only now
beginning to be developed.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005959
CHAPTER 9

Signaling Pathways in Cell Polarity

Luke Martin McCaffrey1 and Ian G. Macara2

1Department of Oncology, Rosalind and Morris Goodman Cancer Research Centre, McGill University,
Montreal, Quebec, Canada
2Department of Microbiology, Center for Cell Signaling, University of Virginia School of Medicine,
Charlottesville, Virginia 22908
Correspondence: Igm9c@virginia.edu

SUMMARY

A key function of signal transduction during cell polarization is the

creation of spatially segregated regions of the cell cortex that possess
different lipid and protein compositions and have distinct functions.
Polarity can be initiated spontaneously or in response to signaling inputs
from adjacent cells or soluble factors and is stabilized by positive-
feedback loops. A conserved group of proteins, the Par proteins, plays a
central role in polarity establishment and maintenance in many contexts.
These proteins generate and maintain their distinct locations in cells by
actively excluding one another from specific regions of the plasma
membrane. The Par signaling pathway intersects with multiple other
pathways that control cell growth, death, and organization.

Outline
 1 Introduction
2 The polarization machinery
3 Factors that control polarity protein localization
4 Polarity signaling through PAR3–PAR6–APKC
5 Conclusion
References

1 INTRODUCTION
The asymmetric distribution of proteins, lipids, and RNAs is necessary for
cell fate determination, differentiation, and a multitude of specialized cell
functions that underlie morphogenesis (St Johnston 2005; Gonczy 2008;
Knoblich 2008; Macara and Mili 2008; Martin-Belmonte and Mostov 2008).
The establishment of cell polarity can be dissected into three primary
processes: (1) breaking symmetry, either through extrinsic cues or
stochastically; (2) establishing spatial organization through signal
transduction; and (3) amplifying and maintaining the polarized state through
feedback loops (Fig. 1). Even single-celled organisms such as budding yeast
are polarized and engage sophisticated signaling mechanisms to initiate and
organize asymmetric cell divisions. Higher organisms use polarity to build
diverse cell types, such as neurons and epithelial cells in animals or stomatal
cells in plants. Polarity spatially segregates important cellular functions from
one another—for instance, in neurons, it separates synaptic inputs (along
dendrites) from signaling outputs (along the axons). Epithelial cell polarity
separates the apical membrane, which is specialized for interactions with the
external environment, from the baso-lateral membrane, which contacts
extracellular matrix or other cell types. In some epithelia a barrier called the
tight junction separates the two membrane regions and prevents the
intercellular diffusion of material across the epithelial sheet. Once
established, cell polarity is often stable for the lifetime of the cell, as in
neurons, but it can also be dynamic, for example, during development, when
neural crest cells lose their epithelial character and become mesenchymal
(this is termed the epithelial mesenchymal transition, EMT).

Figure 1. (A) Extrinsic signals normally are responsible for driving cell polarization, although it can
also occur spontaneously under certain conditions. (B) Cell polarization is established by signal
transduction pathways that spatially segregate different regions of the cell, especially the cell cortex,
and this organization is reinforced and maintained by positive-feedback loops.

A conserved set of proteins called the Par polarity proteins is used in

many contexts throughout the animal kingdom—for example, to polarize
epithelia, to specify axons versus dendrites in a neuron, and to drive the
asymmetric division of a nematode zygote (Fig. 2). These proteins are
components of signal transduction pathways and include kinases, GTPases,
adaptor proteins, and scaffolds. Additional pathways have evolved that play
more specific roles—for instance, in epithelial apical/basal polarization or in
planar polarity of epithelial sheets. However, our knowledge of the inputs to
and outputs from these pathways and their intersection with other signaling
networks remains incomplete. Moreover, despite the high level of
conservation at the sequence level, the regulation and cross talk between the
polarity proteins and other signaling components vary from one context to
another and from one species to another, which complicates the task of
dissecting polarity protein function. Nonetheless, rapid progress is being
made in our understanding of polarity signaling, which we outline here, with
an emphasis on Cdc42 and the Par proteins.

Figure 2. (A) Schematic showing domain structures of Par polarity proteins and their interactions. Phox
and Bem1 domain (PB1), forms homodimers and heterodimers; zinc finger domain (Zn); PSD95, Dlg1,
ZO-1 domain (PDZ), binds other PDZ domains and carboxy-terminal peptide motifs; conserved region
domain (CR1), forms homo-oligomers; atypical protein kinase C binding domain (aPKCBD);
ubiquitin-binding-associated domain (UBA); kinase-associated domain (KA). (B) The different
distributions of these polarity proteins in an epithelial cell and a neuroblast stem cell, together with the
localization of other interacting proteins. Note that whereas in neuroblasts all of the polarity proteins
form a complex (the “Par complex”) at the apical cortex, this is not the case in epithelial cells, in which
Par3 is not associated with Par6 and aPKC but is associated instead with the tight junction complex.
The orientation of the mitotic spindle is controlled by the Par proteins and is different in neuroblasts
(vertical) versus epithelial cells (horizontal). This difference reflects the distinct functions of polarity in
the two cell types: segregation of cell fate determinants into only one daughter cell in the neuroblast
versus formation of a polarized sheet of cells by the epithelium.

2 THE POLARIZATION MACHINERY

2.1 Symmetry Breaking and Positive-Feedback Loops
Symmetry breaking has been studied most intensively in the budding yeast
Saccharomyces cerevisiae, which during the cell cycle switches from isotopic
growth as a spherical cell to the polarized growth of bud formation before
cell division, or to the “schmoo” formation necessary for mating (Slaughter et
al. 2009). The key signaling pathway involves the small GTP-binding protein
Cdc42 and its various regulators. A positive-feedback loop generates a high
local enrichment of GTP-bound Cdc42 at the cell cortex that nucleates actin
cables, which recruit more Cdc42 to the site, which, in turn, leads to further
actin nucleation (Fig. 3). The initial local enrichment of Cdc42 relies on a
separate feedback loop involving a complex of a guanine nucleotide
exchange factor (GEF) for Cdc42, called Cdc24, and an adaptor protein
(Bem1) (Fig. 3). Cdc42-GTP recruits the adaptor, which, in turn, recruits the
GEF, which generates more Cdc42-GTP locally (Butty et al. 2002). Note that
wild-type haploid yeast cells are never entirely unpolarized, and the new bud
always forms adjacent to the bud scar left over from the previous cell cycle.
Figure 3. Positive-feedback loops that drive polarization of budding yeast during cell division. Cdc42
can cycle between GDP- and GTP-bound states, catalyzed by guanine nucleotide exchange factors
(GEFs) that load GTP onto the protein, and GTPase-activating proteins (GAPs) that stimulate
hydrolysis of the bound GTP. In the actin/Cdc42 loop, local enrichment of Cdc42-GTP at the cell
cortex triggers nucleation of actin cables, along which vesicles recruit more Cdc42, which nucleates
more actin cables. In the Cdc42/adaptor/GEF loop, local enrichment of Cdc42-GTP recruits an adaptor
protein (Bem1), which, in turn, recruits a GEF for Cdc42 (Cdc24), which produces more Cdc42-GTP,
which can, in turn, recruit more Bem1 and Cdc24.

A pair of membrane-associated proteins near the scar function as the

landmark for the new bud and recruit the GEF for a different GTPase, Rsr1,
which then recruits Bem1 and the Cdc42-specific GEF Cdc24. This GTPase
cascade is thought to amplify and stabilize the initial local cue. Interestingly,
a GTPase-activating protein (GAP) that inactivates Cdc42 is localized to the
old bud site and prevents its reuse in the next cycle (Tong et al. 2007). Other
GAPs, which are not localized, inactivate any Cdc42-GTP that diffuses away
from the bud site, thereby helping to maintain a focused spot of active Cdc42
at the correct cortical location. Therefore, local activation and global
inactivation of a GTPase, plus cortical landmarks and organization of the
actin cytoskeleton, are all used by budding yeast to ensure correct
polarization for bud formation.
Do other organisms or cell types use the same mechanisms to drive
polarization? Although the details differ, the general concept of reinforcing
initial polarity cues with positive-feedback loops is widespread. Perhaps the
best understood examples are the migration of cells up gradients of a
chemical attractant (chemotaxis) in Dictyostelium and in mammalian
neutrophils (see Ch. 8 [Devreotes and Horwitz 2012]). These cells use
positive feedback to reinforce polarization in the direction of the gradient, but
instead of Cdc42, the loop involves PI-3 kinase, the phosphatase PTEN, and a
protein kinase, Akt (Charest and Firtel 2006). As in yeast, local activation
coupled with global inactivation seems to play an important role in stabilizing
polarity (Xiong et al. 2010). Interestingly, Dictyostelium spontaneously and
transiently polarizes in random directions even in the absence of any external
gradient. This suggests that the detection of the chemotactic signal functions
primarily to reinforce and stabilize a preexisting polarity rather than to break
symmetry.

2.2 Par Proteins

Perhaps the clearest example of symmetry breaking and cell polarization is
the fertilization of the Caenorhabditis elegans oocyte. Here the entry of the
sperm into the egg breaks symmetry, driving a wave of acto-myosin
contractions across the cell cortex and the establishment of an
anterior/posterior polarity, exemplified by the distribution of the Par proteins.
The par (for “partition-defective”) genes were identified in an elegant screen
by Jim Priess and Ken Kemphues for maternal-effect genes that are
embryonically lethal in C. elegans (Kemphues et al. 1988). Seven genes were
identified in the screen, and they are all essential for the first asymmetric cell
division of the zygote. Par1 and Par4 (also known as LKB1) are
serine/threonine kinases (Guo and Kemphues 1995; Watts et al. 2000); Par2
is a RING-finger domain protein that may function as an E3 ubiquitin ligase
(Levitan et al. 1994); Par3 and Par6 are PDZ-domain-containing proteins that
have scaffolding or adaptor functions (Etemad-Moghadam et al. 1995; Hung
and Kemphues 1999); Par5 is a 14-3-3 protein that binds to phosphorylated
serine and threonine residues (Morton et al. 2002); and PKC-3 is an atypical
protein kinase C (aPKC) (Fig. 2). With the exception of Par2, all of the Par
proteins and aPKC are conserved throughout the Metazoa.
Strikingly, most of these polarity proteins show a polarized distribution
within the zygote (Tabuse et al. 1998). Par1 and Par2 are restricted to the
posterior of the zygote cortex, whereas Par3, Par6, and aPKC are restricted to
the anterior cortex (although they are also present in the cytoplasm)
(Schneider and Bowerman 2003; Munro 2006). The segregation of the two
cortical groups of Par proteins depends on their mutual antagonism, and loss
of one Par protein results in escape of the others from their respective
domains. In addition, the worm homolog of Lethal Giant Larvae (Lgl), a
polarity protein originally discovered in Drosophila, localizes to the posterior
cortex of the worm zygote and helps exclude the Par3–Par6–aPKC complex
(Beatty et al. 2010; Hoege et al. 2010). Only Par4 and Par5 are non-polarized,
and they are distributed diffusely throughout the cytoplasm, but they are
required for the asymmetric distribution of the cortical Par proteins. The
mechanisms that underlie this mutual antagonism are described below.
Par3, Par6, and aPKC can form a physical complex (Fig. 2), sometimes
called the Par complex (Joberty et al. 2000; Lin et al. 2000; Wodarz et al.
2000), that has been identified in all animal cells that have been examined
(Goldstein and Macara 2007). Par6 acts as a regulatory subunit for aPKC.
The two proteins are attached to one another through their amino-terminal
PB1 domains (Hirano et al. 2005), and this association inhibits the basal
activity of aPKC. Par6 can also recruit substrates for phosphorylation
(Yamanaka et al. 2001). Interaction of Par6 with Cdc42-GTP induces a
conformational switch that relieves the inhibition, enabling the kinase to
phosphorylate its substrates. One of these substrates is Par3. Atypical PKC
binds through its kinase domain directly to a small region in the carboxy-
terminal half of Par3 and phosphorylates S827 within this region (Nagai-
Tamai et al. 2002), which causes the disassociation of the kinase from Par3.
However, an additional interaction, between the PDZ domains of Par6 and
Par3, can indirectly tether aPKC to Par3 even after S827 phosphorylation.
The function of this rather complicated set of interactions is necessary for
delivery of aPKC and Par6 to the apical surface of epithelial cells.
Importantly, Par3, Par6, and aPKC do not form a constitutive complex.
Their interactions are regulated by multiple protein kinases, by small
GTPases, and by competition for other binding partners, including other
polarity proteins. These regulators determine the subcellular distribution of
the Par proteins. Two striking examples are Drosophila neuroblasts and
epithelial cells (Fig. 2). In the neuroblasts, Par3, Par6, and aPKC all localize
together at the apical crescent, in a complex with two other proteins,
Inscuteable and Partner of Inscuteable (Pins), which control spindle
orientation during mitosis. This clustering of polarity proteins is independent
of the phosphorylation of Par3 by aPKC. In contrast, only aPKC and Par6 are
apical in epithelial cells, whereas Par3 segregates to the lateral/apical
boundary (or to tight junctions in mammalian epithelial cells) (Fig. 2) (Izumi
et al. 1998; Joberty et al. 2000). Moreover, the localization of aPKC and Par6
to the apical cortex depends on the ability of aPKC to phosphorylate Par3. As
described above, the phosphorylation partially disengages aPKC from Par3;
but the two proteins remain attached through Par6. An epithelium-specific
polarity protein at the apical membrane, called Crumbs, outcompetes Par6,
displacing Par3 (Morais-de-Sa et al. 2010). In this way, Par3 is completely
disengaged, and the Par6–aPKC complex is retained at the apical cortex.

2.3 Intercellular Junctions

Intercellular junctions are a universal feature of multicellular organisms.
They provide the glue that binds cells together into tissues and organs, but
also provide for the transmission of signals between adjacent cells. Many
types of adhesive proteins have evolved, but the most widespread are the
cadherins—transmembrane proteins that form calcium-dependent homophilic
interactions between adjacent cells. The intracellular domains of cadherins
bind to catenins, which perform multiple functions, including stabilizing
adhesive clusters at cell–cell interfaces, interacting with the actin
cytoskeleton, and serving as signaling platforms that are coupled to many of
the known signal transduction networks within the cell, including the Par
polarity proteins. Vertebrate epithelia and endothelia also possess tight
junctions, which form both a barrier between cells and a fence between the
apical and lateral domains of the plasma membrane within each cell. The
fence prevents the free diffusion of membrane proteins and lipids between
these two domains, thereby helping to maintain apical/baso-lateral polarity.
Tight junctions are composed principally of transmembrane proteins called
claudins, but—as is true for adhesive junctions—there are numerous
additional proteins that associate with the claudins to form the junctional
structures.
Despite the important role of intercellular junctions in polarity, cell
polarization is not dependent on their existence. Baas et al. (2004) have
shown that single intestinal cells can be induced to polarize simply by the
activation of Par4, in the complete absence of attachment to any neighboring
cell. Moreover, Drosophila epithelial cells do not possess tight junctions, yet
are able to segregate apical from lateral proteins as efficiently as do their
mammalian counterparts.

3 FACTORS THAT CONTROL POLARITY PROTEIN

LOCALIZATION
The Par proteins provide critical spatial information during polarization, to
identify different regions of the cell cortex. The localization of polarity
proteins is therefore central to their biological functions. Protein localization
often involves distinct steps that can include transport, delivery, anchoring at
the destination, and active exclusion from other areas of the cell. Transport
can simply involve passive diffusion, or directed movement along the
cytoskeleton. Alternatively, the mRNA encoding the protein might be
transported to the destination, where it is locally translated. Anchors can
include phospholipids, cytoskeletal elements, or more specific protein
complexes.

3.1 Membrane Attachment via Phospholipids

All of the Par proteins except for Par4 and Par5, plus other polarity proteins
including Lgl, Scribble, Dlg, Pals1, Patj, and Crumbs, are found
predominantly at the cell cortex. Crumbs is a transmembrane protein that
tethers Patj, Pals1, and Lin7 to the apical cortex. Par3 contains a conserved
basic amino acid motif in the carboxy-terminal half of the protein that can
bind directly to phosphoinositides (Krahn et al. 2010), which is both
necessary and sufficient for association with membranes. An additional
phosphoinositide-binding mechanism has been proposed for this protein,
through phosphoinositide binding to its PDZ2 domain (Wu et al. 2007). No
other polarity protein is known to contain lipid-binding motifs, but Par4 is
farnesylated at its carboxyl terminus, and this hydrophobic posttranslational
modification could facilitate association with membranes.

3.2 Oligomerization
The amino-terminal conserved region 1 (CR1) is necessary for self-
association of Par3 into higher-order complexes (Fig. 2) (Benton and
Johnston 2003a; Mizuno et al. 2003; Feng et al. 2007). It is essential but not
sufficient for membrane attachment. How the oligomerization of Par3
maintains the protein at the plasma membrane is unclear, but oligomerization
might complement weak phosphoinositide binding by increasing avidity (Fig.
4).
Figure 4. Mechanisms for the transport, cortical association, and anchoring of Par3 in mammalian
cells. It is not yet known if all of these mechanisms operate in any one cell type, and additional
processes, such as RNA localization, might play roles in certain circumstances. Junctional adhesion
molecule (JAM) is shown as an example of a transmembrane protein to which Par3 can be anchored,
but others exist, such as the neurotrophin receptor, p75NTR, in mammalian Schwann cells (Chan et al.
2006). PP1α is a phosphatase.

3.3 Anchoring to Membrane Proteins

Enrichment and retention in a specific region of the cell cortex is frequently
mediated by direct interactions with transmembrane proteins. Pals1 and Par6
can both bind directly to the carboxy-terminal sequence of Crumbs, via their
PDZ domains. Par6 and Patj can also associate indirectly with Crumbs
through Pals1. The recruitment of Par3 to tight junctions in mammalian
epithelial cells is mediated, at least in part, through association between the
first PDZ domain of Par3 and the carboxyl terminus of junction adhesion
molecules (JAMs) (Fig. 4) (Ebnet et al. 2000; Itoh et al. 2001). Because Par6
also binds to the first PDZ domain of Par3, the associations with JAM and
Par6 are mutually exclusive. A similar mechanism occurs in the mouse
neuroepithelium, where Par3 is recruited to newly formed intercellular
junctions through a direct interaction with nectin 1 or nectin 3 (Takekuni et
al. 2003). In the Drosophila wing disc epithelium, Par3 is also recruited to
junctions through its PDZ domains, by interaction with the adherens junction
proteins β-catenin and Echinoid, an immunoglobulin-domain transmembrane
protein (Wei et al. 2005). In embryonic epithelium, Par3 instead associates
with E-cadherin (Harris and Peifer 2005). Thus, different tissues use distinct
mechanisms to organize the spatial distribution of Par3 and other polarity
proteins.

3.4 Localized mRNA Translation

RNA localization and local translation are important drivers of cell
polarization in several situations and have been intensively studied in the
Drosophila oocyte, in budding yeast, and in vertebrate neurons. The mRNAs
for two epithelial-specific polarity proteins, Crumbs and Pals1 (also called
Stardust), are enriched near the apical surface in Drosophila epithelial cells
(Horne-Badovinac and Bilder 2008; Li et al. 2008). Why localize RNAs?
One potential function is to regulate translation, perhaps in response to
extracellular signals, or to cell density changes. As an example, the Par3
mRNA—but not that of Par6 or aPKC—is transported out along the axons of
mammalian motor neurons, where it can be locally translated in response to
stimulation of the neurons by nerve growth factor (NGF), a process that is
essential for NGF-dependent axon outgrowth (Hengst et al. 2009).

3.5 Active Exclusion

None of the mechanisms described above stably anchors Par proteins at the
plasma membrane. Cortical Par proteins are highly dynamic in the C. elegans
zygote, rapidly exchanging with cytoplasmic pools and undergoing lateral
diffusion along the plasma membrane (Goehring et al. 2011). Lgl protein is
also highly dynamic in Drosophila embryonic cells (Mayer et al. 2005).
Therefore, the targeting and retention mechanisms described above are
insufficient to maintain their polarized distribution within the cell. Active
mechanisms drive the segregation of polarity proteins into different cortical
domains and maintain their asymmetry. As mentioned above, the Par3–Par6–
aPKC complex is often localized in a complementary pattern to that of Par1,
and through mutual phosphorylation reactions, the two kinases, aPKC and
Par1, exclude each other from their respective regions of the cortex. Par1
directly phosphorylates Par3 on S144 (equivalent to S151 in Drosophila)
(Benton and Johnston 2003b; Hurd et al. 2003a) near CR1, the region
necessary for oligomerization of Par3. The phosphorylated S144/151 residue
acts as a docking site for Par5 (14-3-3), which might reduce oligomerization
and thereby destabilize membrane association of Par3 (Fig. 4). This
mechanism can therefore exclude Par3 from cortical regions that contain
Par1. The phosphorylation and binding of Par5 (14-3-3) to Par3 can be
reversed by protein phosphatase 1α (PP1α), allowing recycling of Par3 to
appropriate cortical sites.
Conversely, aPKC can phosphorylate Par1, which both inhibits Par1
kinase activity and disassociates it from the plasma membrane (Fig. 5A).
Two distinct mechanisms have been identified, one in which aPKC directly
phosphorylates Par1 on T595 (Hurov et al. 2004) and an indirect mechanism
by which aPKC activates protein kinase D (PKD), which then phosphorylates
Par1 on S400 (Watkins et al. 2008). Phosphorylation of T595 reduces the
kinase activity and displaces Par1 from the membranes. Furthermore,
phosphorylation of S400 recruits Par5 (14-3-3), which displaces Par1 from
the membrane.
Figure 5. Active exclusion from cortical domains. A common mechanism for the establishment of
discrete cortical regions of the cell occurs through the active removal of unwanted proteins from within
a particular region. A kinase within the region phosphorylates the protein, resulting in the association of
Par5 (14-3-3), which displaces the protein from the cell cortex. (A) Par1 is restricted to the lateral
membranes in epithelial cells. Any Par1 that strays onto the apical surface is phosphorylated by aPKC,
which recruits Par5 (14-3-3), causing its disassociation from the membrane. (B) aPKC at the apical
surface of an epithelial cell phosphorylates any Pins (also known as LGN) protein that binds to Gαi
within this domain. Association of the phosphorylated Pins with 14-3-3 triggers its dissociation from
Gαi. Because phosphorylation does not occur at the basolateral membrane, Pins can remain associated
with this region of the cortex. Pins is diffuse in the cytoplasm in interphase cells and can only associate
with Gαi at the cell cortex after it has undergone a conformational switch triggered by the binding of
NuMA, a nuclear protein that is released in mitosis. Also shown in this schematic is the recruitment by
Par3 of aPKC to the apical surface, where the aPKC is disengaged and binds to the Crumbs–Pals1–Par6
complex. The apical aPKC is activated by the binding of Cdc42-GTP to Par6.
 The C. elegans aPKC can also phosphorylate Par2 and exclude it from the
anterior cortical domain of the zygote, whereas Par2 in the posterior domain
recruits Par1 to phosphorylate and exclude Par3 (Hao et al. 2006).
The spatial distributions of many downstream effectors of the Par
signaling pathway are also controlled by phosphorylation. For example, the
cell fate determinants Numb and Miranda, the polarity protein Lgl, and the
spindle orientation factor Pins (called LGN in mammals), are all removed
from the plasma membrane by aPKC-dependent phosphorylation (Fig. 5B)
(Betschinger et al. 2003; Hao et al. 2006; Smith et al. 2007; Atwood and
Prehoda 2009). Pins associates with the cell cortex in mitosis, by binding to
Gαi subunits, where it functions to attach astral microtubules, which orient
the mitotic spindle. In epithelial tissues, normal organization is often dictated
by the ability of cells to divide in the plane of the epithelial sheet, but not
perpendicular to the sheet. To this end, apical aPKC phosphorylates any Pins
that diffuses into the apical region, resulting in the recruitment of Par5 (14-3-
3), which disengages Pins from Gαi. In this way, Pins is excluded from the
apical cortex, preventing astral microtubule attachment and perpendicular
orientation of mitosis. Pins also orients the mitotic spindles in Drosophila
stem cells and in some mammalian progenitors. Similar mechanisms,
involving not only aPKC and Par1 but other protein kinases that target Par5
(14-3-3) consensus sites, probably also exist.
The mechanism underlying exclusion of Numb from the apical region of
progenitor cells in Drosophila has been worked out in considerable detail. At
the onset of mitosis, Aurora-A phosphorylates Par6 within the PB1 domain
that binds to aPKC (Fig. 2), releasing Par6 and relieving its inhibition of
aPKC (Wirtz-Peitz et al. 2008). The activated aPKC phosphorylates Lgl,
which causes it to dissociate from the Par6–aPKC complex and allows the
complex to interact with Par3. Par3 then acts to recruit Numb as a substrate
for aPKC. Phosphorylation of Numb by aPKC causes it to be released from
the cell cortex (Smith et al. 2007; Wirtz-Peitz et al. 2008). Because the Par
complex is initially asymmetrically distributed, the loss of Numb occurs at
only one side of the mitotic cell; thus, one daughter will inherit Numb while
the other does not. Interestingly, Lgl seems here to function as a buffer, to
suppress the phosphorylation of Numb until the appropriate time in the cell
cycle. The recruitment of Numb by Par3 seems to be an evolutionarily
conserved function, because in migrating mammalian fibroblasts, the same
mechanism is used to release Numb from the cell cortex and regulates the
internalization of integrins (Nishimura and Kaibuchi 2007).

4 POLARITY SIGNALING THROUGH PAR3–PAR6–

APKC
4.1 Polarity Signaling through Small GTPases
Actin filaments and microtubules are the two major asymmetric components
of cells. Both are vectorial polymers, and their organization is, therefore, of
fundamental importance to cell polarization. This organization is dynamic
and is highly regulated by signaling networks that respond to external and
internal cues. Central to these signaling networks are the Rho family
GTPases (Fig. 3), which regulate and are regulated by polarity proteins.
Cdc42 is a pivotal component of the polarity machinery in yeast and is
conserved throughout the metazoa. Dominant-negative Cdc42 mutants
disrupt polarized migration in mammalian fibroblasts (Nobes and Hall 1999),
and Cdc42-GTP binds to Par6, providing a mechanism by which the GTPase
can control cell polarization. Par6 contains a partial CRIB domain, a motif
conserved among most Cdc42 effectors. This domain interacts with a region
of the GTPase that undergoes a GTP-dependent switch in conformation
(Garrard et al. 2003). Binding of Cdc42-GTP to Par6 relieves the inhibition
of aPKC activity by Par6 (Yamanaka et al. 2001). In addition, the
cytoplasmic tail of the receptor tyrosine kinase ephrin B1 competes with
Cdc42 for binding to Par6, blocking tight junction formation (Lee et al.
2008). Tyrosine phosphorylation of ephrin B1 releases it from Par6. In this
way, signaling through tyrosine kinase receptors could affect aPKC activity
and consequently cell polarity decisions.
In addition to counteracting the inhibition of aPKC by Par6, Cdc42 can
also recruit the Par6–aPKC complex to specific regions of the cell cortex
where Cdc42 is activated. For example, in Drosophila neuroblasts mutant for
Cdc42, Par6–aPKC is mislocalized to the cytoplasm (Atwood et al. 2007),
and depletion of Cdc42 from mammalian epithelial cells can partially
mislocalize aPKC from the apical cortex (Martin-Belmonte et al. 2007). A
positive-feedback loop probably reinforces the positioning of these proteins,
because robust Cdc42 localization in the neuroblasts also requires Par6. In
the C. elegans zygote, Cdc42 is not essential for the initial anterior
enrichment of Par6, although the asymmetry is lost later, during the first cell
division, which suggests that other factors set up the polarity (Aceto et al.
2006). In mammalian epithelial cells, Par6 localization to the apical surface
probably requires its association with Pals1 and/or Crumbs, rather than
Cdc42 (Gao et al. 2002; Hurd et al. 2003b).
Another small GTPase, Rho1, helps organize the polarity of the C.
elegans zygote. The RhoGEF Ect1 is excluded from the posterior cortex,
which restricts Rho-GTP production to the anterior end of the zygote (Motegi
and Sugimoto 2006), where it stimulates myosin contractility. This
contraction generates a cortical actin flow, translocating Par6, aPKC, Par3,
and Cdc42 to the anterior end of the zygote. Cdc42-GTP then maintains this
distribution of the Par proteins.
The Rho GTPase may play a distinct role in mammalian cells by
controlling the association of Par3 with Par6–aPKC (Fig. 6). ROCK, a
protein kinase downstream from RhoA, can phosphorylate Par3 on T833,
adjacent to the aPKC-binding site in the carboxyl terminus of Par3, and this
phosphorylation blocks the association with aPKC (Nakayama et al. 2008).
However, aPKC can also phosphorylate ROCK, which suppresses its
association with epithelial junctions. Because ROCK phosphorylates the
myosin light chain and activates actomyosin contractility, this represents an
additional polarity mechanism (Ishiuchi and Takeichi 2011).
Figure 6. Interaction map showing potential links between the Par proteins and components of the
Hippo pathway. A signaling cascade involving Salvador, Hippo, and Warts controls phosphorylation
and nuclear localization of the transcription factors Yorkie (YKI) and TAZ to regulate epithelial
growth. Epithelial integrity is monitored by cell-adhesion complexes (E-cadherin, α-catenin) and by the
Par and Crumbs polarity complexes through the adaptor Kibra. The extracellular matrix (ECM) also has
an impact on YKI nuclear localization through the Rho GTPase, independently of the Hippo pathway.

4.2 Rho GTPases as Downstream Effectors of Par3–Par6–

aPKC
The carboxy-terminal region of Par3 can bind to a Rac GEF called Tiam1
(Chen and Macara 2005; Mertens et al. 2005; Nishimura et al. 2005; Zhang
and Macara 2006). Par3 sequesters Tiam1 to prevent inappropriate activation
of Rac (Chen and Macara 2005; Zhang and Macara 2006). Loss of Par3
causes an increase in Rac-GTP levels, which results in a misorganization of
actin filaments at the cell cortex. A similar mechanism operates in the notum
epithelium of Drosophila, where Par3 inhibits Sif, the fly homolog of Tiam1,
to restrict filopodium formation to basal regions of the cell (Georgiou and
Baum 2010). In other cell types, Par3 might recruit Tiam1 to sites within the
cell where Rac needs to be activated. In such cases, loss of Par3 might reduce
Rac activation at these sites (Pegtel et al. 2007).
Par6 can also regulate RhoA activity. By associating with aPKC, Par6 can
activate a RhoGAP called p190, thereby reducing Rho-GTP levels (Zhang
and Macara 2008). The link between Par6–aPKC and the p190 RhoGAP is
unknown, but the pathway is important for controlling synapse density in
hippocampal neurons. Elevated expression of Par6 increases dendritic spine
density, whereas silencing of Par6 reduces spine density.
A second, different mechanism by which Par6 can affect Rho is through
association with Smad-ubiquitin regulatory factor 1 (Smurf1), an E3 ligase
that down-regulates the TGFβ signaling pathway (Fig. 4). In addition to
targeting Smads for degradation, Smurf1 can also ubiquitylate RhoA (Wang
et al. 2003). TGFβ type II receptor can bind to and phosphorylate Par6 on a
conserved carboxy-terminal residue (S345) (Ozdamar et al. 2005). This
stimulates the association of Par6 (or aPKC) with Smurf1, which mediates
the localized ubiquitylation and destruction of RhoA. In mammary NMuMG
cells, loss of RhoA can cause the dissolution of tight junctions and an EMT
(Wang et al. 2003). However, these effects might be cell type specific
because, for example, in MDCK cells, dominant-negative RhoA expression
had no effect on tight junctions (Bruewer et al. 2004).

4.3 Cross Talk between Wnt Signaling and Polarity Proteins

Planar cell polarity (PCP) signaling establishes directional asymmetry at the
tissue level rather than at the cellular level (as occurs in apical/basal
polarization or in the asymmetric divisions of some stem cells). PCP
signaling establishes the directionality of wing hair orientation in Drosophila,
for example, and the orientation of cilia in the outer hair cells of the
mammalian cochlea. It is also essential for the oriented migration of cell
sheets during gastrulation. PCP is stimulated by Wnt ligands that activate
seven-transmembrane-span receptors of the Frizzled family (Simons and
Mlodzik 2008). Other transmembrane proteins, including Vangl and
Flamingo (also called Celsr), are also essential for PCP signaling.
Downstream from these proteins, an adaptor called Dishevelled (Dvl) can
recruit a GEF to activate RhoA (Tsuji et al. 2010), which stimulates the
downstream kinase ROCK. As described above, ROCK phosphorylates Par3,
in addition to the myosin light chain and other targets. Dvl is itself a target
for phosphorylation by the Par1 polarity protein and can directly associate
with aPKC (Sun et al. 2001; Ossipova et al. 2005). The association with
aPKC stabilizes and activates the kinase and has been implicated in axonal
differentiation and polarized migration of mammalian cells (Schlessinger et
al. 2007; Zhang et al. 2007). Moreover, the Drosophila Frizzled receptor is
phosphorylated and inhibited by aPKC, which is recruited to Frizzled through
another polarity protein, Patj (Djiane et al. 2005). Thus, aPKC mediates
multiple signaling connections between PCP components. Whether its
binding partners Cdc42 and Par6 participate in PCP regulation remains to be
determined.
Another apical/basal polarity protein, Scribble, has also been implicated
in PCP. The mechanism remains obscure, but it interacts genetically and
physically with Vangl, which, in turn, can bind to the extracellular domain of
Frizzled, perhaps enabling non-cell-autonomous signaling between
neighboring cells (Courbard et al. 2009). Signaling downstream from
Scribble is poorly understood and requires the carboxyl terminus of the
protein, including the third and fourth PDZ domains, which bind to Vangl.
Control of apical/basal polarity by Scribble in Drosophila, in contrast, does
not require any of the PDZ domains (Zeitler et al. 2004).

4.4 Connections between the Hippo Pathway and Polarity

Signaling
The Hippo pathway was first discovered in Drosophila as a central regulator
of organ size (Grusche et al. 2010). It controls the balance between
proliferation and apoptosis in flies and mammals and involves a protein
kinase cascade that ultimately phosphorylates and inactivates the
transcriptional coactivators Yorkie (YKI, also known as Yap1) and TAZ
(Fig. 6) (p. 133 [Harvey and Hariharan 2012]).
Phospho-Yap1 is sequestered at adherens junctions through association
with α-catenin. This catenin can function as a tumor suppressor, and in the
epidermis, loss of α-catenin results in the nuclear accumulation of YKI and
increased cell proliferation (Schlegelmilch et al. 2011; Silvis et al. 2011).
How cell density controls the α-catenin–YKI interaction remains unclear, but
another Hippo pathway component, Merlin, also binds directly to α-catenin
and is required for stable adherens junction formation (Gladden et al. 2010).
Merlin couples α-catenin to Par3 during junction maturation in keratinocytes,
which might be important for the apical positioning of tight junctions relative
to the adherens junctions. In addition, Par5 (14-3-3) is required for the
association of α-catenin with YKI. Merlin might in some way regulate the
association of YKI–Par5 with α-catenin.
Remarkably, mechanical forces also regulate YKI (Fig. 6). Increasing the
stiffness of the extracellular matrix activates Rho and its downstream kinase
ROCK—through an unknown mechanism—which drives the nuclear
accumulation and activation of YKI (Dupont et al. 2011). This pathway
seems to be entirely separate from the canonical Hippo pathway but provides
a direct mechanism through which the physical environment can reprogram
gene expression. Whether α-catenin is involved in this pathway, through
interactions with actin, remains to be determined.
Apical/basal polarity proteins also interact with components of the Hippo
pathway (Fig. 6). For example, the Drosophila protein Expanded binds to the
intracellular domain of Crumbs (Chen et al. 2010; Grzeschik et al. 2010; Ling
et al. 2010; Robinson et al. 2010). Expanded is a FERM-domain protein
distantly related to Merlin. Changes in the level of Crumbs expression cause
a mislocalization of Expanded and are sufficient to induce hyperproliferation
of epithelial cells in Drosophila (Lu and Bilder 2005). The vertebrate
homolog of Expanded has not yet been unambiguously identified, however,
and it remains unknown whether a similar signaling pathway operates in
mammalian epithelial cells. The wiring might be somewhat different, because
in mouse epithelial cell lines, Crumbs3-dependent apico–basal polarization
leads to sequestration of SMADs in the cytoplasm through the Hippo
pathway, which inhibits TGFβ signaling and EMT (Varelas et al. 2010), but
there is no evidence of a link to TGF-β in Drosophila.
An important component of the Hippo pathway is Kibra, which binds to
many pathway components, including Expanded, Merlin, Warts, Hippo, and
Salvador, but how these interactions are regulated remains unclear. Kibra also
associates with the Par complex and localizes to tight junctions and the apical
membrane (Yoshihama et al. 2010). Kibra is a substrate for aPKC
phosphorylation (Büther et al. 2004), but Kibra can also negatively regulate
aPKC activity to control exocytosis of apical membrane components
(Yoshihama et al. 2010). A similar mechanism might function in cell
migration, during which an aPKC–Kibra–exocyst complex regulates focal
adhesion dynamics at the leading edge by recruiting the MAP kinase ERK
(Rosse et al. 2009). It seems likely that these connections could play
important roles in the misorganization, overgrowth, and invasion of epithelial
cancers.

4.5 Polarity Signaling and Cancer

The majority of human cancers arise from epithelial cells, and tumorigenesis
is commonly believed to involve a loss of apical/basal polarity (Ch. 21 [Sever
and Brugge 2014]). In particular, cells at the invasive fronts of carcinomas
often express proteins associated with a mesenchymal phenotype, such as
vimentin and Snail, whereas epithelial proteins such as E-cadherin and
Crumbs are either lost or mislocalized away from the cell cortex. Some of
these changes in expression are caused by reduced expression of micro-
RNAs in the MiR-200 family, which suppress the expression of a
transcription factor, ZEB1. ZEB1, in turn, suppresses the expression of
epithelial genes, including E-cadherin and the polarity genes Crb3 (a Crumbs
homolog), Lgl, and Patj (Brabletz and Brabletz 2010). Because only the
outermost layers of tumor cells in a carcinoma usually show mesenchymal
characteristics, they are probably induced by interactions of the tumor cells
with the surrounding stroma, but the mechanisms involved remain unclear
(Vidal et al. 2010). Cells that have undergone an EMT are more motile, more
invasive, and less adherent, and might contribute to dissemination of tumors
throughout the body and establishment of metastases. However, it is
important to note that there is very little evidence of a role for the polarity
machinery in human cancer. Although several polarity proteins, including
Scribble and Lgl, have tumor suppressor behavior in Drosophila, their role in
human cancers remains ill defined (Wu et al. 2010). Moreover, it remains to
be established whether loss of polarity is an essential aspect of malignancy.
Epithelial cells could escape from their tissue of origin by mechanisms
independent of classical EMT, including changes in spindle orientation
during mitosis and collective migration of clusters of cells (Rorth 2009).

5 CONCLUSION
Understanding cell polarization is one of the major goals of cell biology and
will inevitably have a broad impact on research into diseases such as cancer
and neurological degeneration. A complicated web of signaling systems
surrounds and intersects with the polarity machinery, yet we still understand
very little about what the Par proteins do, how they are localized, how their
various interactions are regulated, and which signaling components operate in
which contexts. After all, the organization of a polarized cell is a formidably
complicated process that involves cytoskeletal remodeling, membrane traffic,
RNA localization, and protein complex assembly and disassembly, with
feedback to gene expression and protein turnover. It is conceivable that the
Par proteins participate in all of these processes, either directly or indirectly.
It is worth remembering, however, that although the polarity machinery can
work in a cell-autonomous fashion, the Par genes do not exist in any
unicellular organism, which suggests that a key role for the Par proteins is to
facilitate, mediate, or interpret cell–cell and cell–matrix interactions. The
tissue context might, therefore, be expected to modulate Par protein
functions. It will be interesting to determine whether regulation of these
interactions controls morphogenesis and to what extent differential Par
function contributes to phenotypic variation both between different cell types
in one organism and between species.
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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a009654
CHAPTER 10

Signaling Mechanisms Controlling Cell

Fate and Embryonic Patterning

Norbert Perrimon1,2, Chrysoula Pitsouli1,3, and Ben-Zion

Shilo4
1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115
2Howard Hughes Medical Institute, Boston, Massachusetts 02115
3Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus
4Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Correspondence: perrimon@receptor.med.harvard.edu

SUMMARY

During development, signaling pathways specify cell fates by activating

transcriptional programs in response to extracellular signals. Extensive
studies in the past 30 years have revealed that surprisingly few pathways
exist to regulate developmental programs and that dysregulation of these
can lead to human diseases, including cancer. Although these pathways
use distinct signaling components and signaling strategies, a number of
common themes have emerged regarding their organization and
regulation in time and space. Examples from Drosophila, such as Notch,
Hedgehog, Wingless/WNT, BMP (bone morphogenetic proteins), EGF
(epidermal growth factor), and FGF (fibroblast growth factor) signaling,
illustrate their abilities to act either at a short range or over a long
distance, and in some instances to generate morphogen gradients that
 pattern fields of cells in a concentration-dependent manner. They also
show how feedback loops and transcriptional cascades are part of the
logic of developmental regulation.

Outline
1 Introduction
2 Embryonic patterning: Interplay between transcriptional cascades and
signaling
3 Juxtacrine signaling: Notch as an example
4 Patterning by secreted paracrine factors
5 Controlling the signaling range of secreted factors
6 The logic of signaling
7 Integrating signaling pathways
8 Concluding remarks: Developmental versus physiological signaling
References

1 INTRODUCTION
Key to multicellularity is the coordinated interaction of the various cells that
make up the body. Indeed, patterning of embryos, establishment of cell type
diversity, and formation of tissues and organs all rely on cell-to-cell
communication during development. Thus, arguably one of the most
important principles of developmental biology involves “one group of cells
changing the behavior of an adjacent set of cells, causing them to change
their shape, mitotic rate, or fate” (Gilbert 2000).
Classically, the ability of one group of cells to affect the fate of another is
called “induction.” The cells that produce the signals are referred to as
“inducing cells,” whereas the receiving cells are termed “responders”
(Spemann and Mangold 1924). The ability of cells to respond to the inducers,
referred to as “competence” (Waddington 1940), usually reflects the presence
of a receptor at the top of a pathway that regulates the expression of specific
transcription factors in the responding cells. The responding cells, in turn, can
become inductive and change the fate of their neighbors by producing new
signals, thus generating sequential inductive events that increase cell-fate
diversity in tissues.
Identification and characterization of the signaling pathways involved in
development has led to the surprising realization that only a few exist
(Gerhart 1999; Gilbert 2000; Barolo and Posakony 2002). These fall into 11
main classes, defined by the ligand or signal transducers involved: Notch,
FGF, EGF, Wnt/Wingless (Wg), Hedgehog (Hh), transforming growth factor
β (TGFβ)/BMPs, cytokine (nonreceptor tyrosine kinase JAK-STAT [signal
transducers and activators of transcription] pathway), Hippo, Jun kinase
(JNK), NF-κB, and retinoic acid receptor (RAR). These pathways involve
either cell-to-cell contact via surface proteins (juxtacrine signaling), or
secreted diffusible growth and differentiation factors (paracrine signaling).
Among the pathways mentioned above, only two of them, Notch and Hippo,
are juxtacrine, whereas the others are paracrine.
With the exception of those that release steroid hormones and retinoic
acid, which cross the membrane and activate gene expression by binding
directly to receptor proteins that act as transcriptional regulators, inducing
cells generally produce secreted or transmembrane ligands, which in some
cases require complex processing in the producing cells or the extracellular
matrix. When these ligands bind to transmembrane receptors on target cells
they activate a cascade of events that ultimately regulate the activity of a
small number of transcription factors and/or cofactors, triggering gene-
expression programs that drive the cellular changes. For example, Notch
signaling (p. 109 [Kopan 2012]) regulates CSL (for CBF1, Suppressor of
Hairless, and Lag1) proteins that possess an integrase domain, receptor
tyrosine kinases (RTKs) regulate ETS (erythroblast transformation-specific)
transcription factors, Wnt ligands (p. 103 [Nusse 2012]) mostly regulate the
high-mobility group (HMG) box-containing TCF (T-cell factor) transcription
factor, Hh proteins (p. 107 [Ingham 2012]) regulate Gli (glioblastoma)
transcription factors that have DNA-binding zinc-finger domains, and BMPs
(p. 113 [Wrana 2013]) regulate Smads (Sma- and Mad-related proteins)
transcription factors. Cytokine pathways (p. 117 [Harrison 2012]) regulate
STATs, and Hippo (p. 133 [Harvey and Hariharan 2012]) regulates TAZ (for
transcriptional coactivator with PDZ-binding motif) proteins that contain a
WW domain and a carboxy-terminal PDZ-binding motif (Table 1). In
addition, many pathways activate feedback loops that modulate or terminate
the incoming signal (Perrimon and McMahon 1999; Freeman 2000).

Table 1. Key signaling pathways that orchestrate development—receptors,

ligands, transcription factors, and outputs are shown for each
Signaling Receptor Ligand Transcriptional Output
pathway effector
Wnt/Wg Frizzled, Wg/Wnt Armadillo/β-catenin Patterning, growth, PCP (β-
dFrizzled2 with TCF/LEF catenin independent)
Hh Patched Hh Ci/Gli Patterning, growth
TGFβ Thickveins Dpp/TGFβ Smad (Mad/Medea) Patterning, growth
RTK EGFR Spitz, Gurken, Pointed/Yan Patterning, morphogenesis
Keren, Vein
FGFR Branchless, Pointed/Yan Patterning, morphogenesis,
(Breathless, Thisbe, migration
Heartless) Pyramus
InR dIlp1-dIlp7 Pointed/Yan, Foxo Growth, metabolism, aging
PDGF/VEGF Pvf1-3 Pointed/Yan Morphogenesis, migration
receptor
(PVR)
Torso Trunk, PTTH Pointed/Yan Patterning, metamorphosis
dALK Jelly belly Pointed/Yan Growth on starvation (CNS)
Sevenless Boss Pointed/Yan Patterning, cell-fate
specification
Notch Notch Delta, Serrate NICD with Su(H) Patterning, lateral inhibition,
cell-fate specification
Hippo Fat Dachsous Yorkie with Growth, PCP
Scalloped
NF-κB Toll Spatzle Dorsal/Dif Patterning, innate immunity
JAK/STAT Domeless Unpaired1-3 STAT92E Patterning, innate immunity
JNK Eiger/TNF Wengen Jun and Fos Migration, patterning, innate
immunity
Nuclear EcRA, EcRB Ecdysone EcRA, EcRB with Patterning, growth,
receptors USP metabolism
Abbreviations: TCF, T-cell factor; LEF, lymphoid enhancer-binding factor; PCP, planar cell
polarity; TGF, transforming growth factor; RTK, receptor-tyrosine kinase; EGFR, epidermal growth
factor receptor; FGFR, fibroblast growth factor receptor; PVDF, polyvinylidene difluoride; VEGFR,
vascular endothelial growth factor; PTTH, prothoracicotropic hormone; CNS, central nervous system;
NICD, Notch intracellular domain; STAT, signal transducer and activator of transcription; JNK, JUN
kinase; TNF, tumor necrosis factor; USP, ubiquitin-specific protease.

The response to signaling-pathway activation is usually complex and

involves the regulation of many processes, such as control of cell fate,
apoptosis, cell proliferation, cytoskeletal reorganization, cell polarity,
adhesion, and cell migration. Importantly, each pathway does not specifically
regulate a single biological process but can elicit diverse effects, depending
on the state of the cell at the time the pathway is activated. Furthermore,
because few pathways exist, there are no unique signals for induction of each
cell type. Instead, the response of a given cell to a signal depends on its
amplitude, duration, interactions between pathways, and integration of
transcription factor effectors at promoters and enhancers of target genes. It
may also be predetermined by the set of transcription factors expressed in the
cell at the time the signal is received.
Here, we use specific examples, mostly taken from Drosophila, to
illustrate general principles and mechanisms by which signaling pathways
operate in development to specify cell fates. Thus, this is not a
comprehensive review of the structures and roles of all the pathways that
have been implicated in developmental processes. A number of excellent
reviews elsewhere describe in detail the roles of individual pathways in
development (Notch [Artavanis-Tsakonas et al. 1999; Lai 2004; Fortini
2009], FGF [Ghabrial et al. 2003; Pownall and Isaacs 2010], EGF [Shilo
2005], Wnt/Wg [Logan and Nusse 2004; MacDonald et al. 2009], Hh
[Ingham and McMahon 2001; Jiang and Hui 2008], TGFβ [Feng and
Derynck 2005; Wu and Hill 2009], JAK/STAT [Hou et al. 2002; Arbouzova
and Zeidler 2006], and Hippo [Pan 2007; Saucedo and Edgar 2007]).
Note also that a number of other pathways, such as those involving
cadherins and integrins, are not discussed here as they are involved in
permissive interactions whereby a tissue is made competent to respond and
requires the proper environment to trigger the appropriate cellular changes
(Gilbert 2000). We also do not discuss signaling pathways that control
cellular behavior and cytoskeletal reorganization—for example, cell
migration and axonal pathfinding.

2 EMBRYONIC PATTERNING: INTERPLAY BETWEEN

TRANSCRIPTIONAL CASCADES AND SIGNALING
Following fertilization, as embryonic development proceeds, different cell
types are formed progressively. With time, cells become more and more
restricted in their developmental potential, and become determined to a
specific fixed fate that represents a stable change in the internal state of the
cell as a result of alterations in gene expression. The gradual increase in
complexity occurring during determination and subsequent differentiation
involves complex combinations of transcription factors. Some of these
factors are common to many cell types, whereas others are present in only
specific cell types. The changes in gene expression rely in part on the
activation of signaling pathways by cell–cell communication. In the context
of development, signaling pathways dictate developmental switches and as
such are usually irreversible, pushing forward the developmental program in
a ratchetlike mechanism by regulating the activity of transcription factors.
For example, patterning along the anteroposterior axis of the Drosophila
embryo is initially set up by graded activity of the Bicoid transcription factor,
which acts in a concentration-dependent manner to control the expression of
gap genes (Fig. 1A) (St Johnston and Nusslein-Volhard 1992). These gap
genes, in turn, coordinately define the domain of expression of pair-rule
genes, which then define the expression of segment-polarity genes (Nusslein-
Volhard and Wieschaus 1980). Although both gap and pair-rule genes encode
diverse types of transcription factors, some of the segment-polarity genes,
such as hh and wg, encode signaling molecules that activate pathways that
operate in positive regulatory loops, to maintain each other’s expression and
the induced cell fates within the embryonic segmental unit (Heemskerk et al.
1991). In addition to the Bicoid patterning system, the Torso RTK pathway
activates the Ras/ MAP kinase (MAPK) pathway to control the spatial
expression of the Tailless and Huckebein transcription factors at the
embryonic termini (Fig. 1C) (Duffy and Perrimon 1994). Finally, along the
dorso–ventral axis of the embryo activation of the Toll receptor by the
Spätzle ligand activates Dorsal (the fly homolog of NF-κB), which regulates
the expression of Twist and Snail, two transcription factors that control
mesoderm development, while repressing other genes, such as rhomboid and
sog (Fig. 1B) (Levine 2008).

Figure 1. Patterning of the early Drosophila embryo. (A) Anterior–posterior patterning and
segmentation of the embryo is initiated by maternally deposited gene products that regulate the
expression of gap genes. Gap genes in turn control the expression of pair-rule genes, which themselves
regulate segment-polarity genes. The gene hierarchy and activation/repression interactions between
different transcription factors that coordinate patterning of the anterior–posterior axis of the early
Drosophila embryo are shown to the right. (B) Dorsal–ventral patterning is initiated by a Dorsal nuclear
gradient regulated by the Toll/NF-κB pathway. Graded nuclear localization of Dorsal subdivides the
dorso–ventral axis into distinct domains expressing twist (twi) and snail (sna), rhomboid (rho) and
decapendaplegic (dpp), zerknullt (zen) and tolloid (tld), which will form the prospective mesoderm,
neurogenic ectoderm, and dorsal ectoderm, respectively. Dorsal activates the zygotic transcription
program in the dorsoventral axis. (C) Terminal patterning is initiated in the germline by the localized
expression of Torsolike in the space outside the poles of the embryo. Torsolike activates the Torso
ligand (Trunk) locally and this is followed by Torso activation at the poles of the embryo, which will
lead to induction of the terminal patterning genes tailless and huckebein. Torso is an RTK and its action
is propagated through the MAPK pathway.

Hierarchies of transcription factor expression that progressively dictate

distinct cell fates are common at later developmental stages in a variety of
tissues. For example, in response to Dorsal signaling, Twist is activated to
define the mesoderm and in turn activates MEF2 and Tinman in different
cells to induce the skeletal muscle and cardiac muscle fates, respectively (Fig.
2A) (Sandmann et al. 2007). Another example of progressive specification
owing to hierarchical expression of transcription factors and activity of
signaling pathways is the specification of Drosophila blood cell types (Jung
et al. 2005). In the Drosophila hemocyte (blood cell) lineage the blood cell
precursors are specified in the embryo by expression of the transcription
factors Serpent (SRP) and Odd paired (ODD) and progressively express
Hemese (HE) and activate the RTK PDGF/VEGF receptor (PVR), as well as
the cytokine receptor Dome, to finally reach the prohemocyte stage. Then,
cell-type-specific transcription factors are activated in response to signaling
by the Notch, PVR or Notch and JAK/STAT pathways, which specify the
different populations of mature hemocytes, namely, the plasmatocytes, the
crystal cells, and lamellocytes, respectively, that are destined to perform
specialized functions (Fig. 2B).
Figure 2. Cell-fate hierarchies in the mesodermal lineage. (A) Muscle cell differentiation. Muscle
progenitors are specified in the embryonic mesoderm by Dorsal and activation of the transcription
factor Twist. Further subdivision of Twist-positive cells to skeletal and cardiac muscle lineages
depends on the expression of the transcription factors MEF2 and Tinman, respectively. (B) Hemocyte
maturation in the Drosophila lymph gland. The earliest lymph gland cells, the hemocyte precursors,
express SRP and ODD. As these cells transition into preprohemocyte fate, they initiate the expression
of HE and PVR. Prohemocytes initiate Dome expression. Maturation to the various hemocyte fates
requires down-regulation of Dome, up-regulation of different maturation markers, and the involvement
of the indicated signaling pathways. Srp, Serpent; ODD, Odd Skipped; He, Hemese; PVR,
PDGF/VEGF receptor; Dome, Domeless; PXN, Peroxidasin; P1, P1 antigen; LZ, Lozenge; ProPOA1,
Prophenoloxidase A1; L1, L1 antigen; MSN, Misshapen.
3 JUXTACRINE SIGNALING: NOTCH AS AN EXAMPLE
The Notch signaling pathway is a highly conserved mechanism for cell
communication between adjacent cells. Both the receptor Notch and its
ligands, which belong to the Delta/Serrate/Lag2 (DSL) family, are
transmembrane proteins. The requirement for direct cell–cell contact between
the signal-sending and signal-receiving cells is necessitated by the
membrane-anchored nature of the ligands. Interestingly, studies of the
specification of sensory organs in the Drosophila thorax indicate that in some
instances DSL ligands can activate Notch signaling beyond directly adjacent
cells, because the signal-sending Delta cells extend filopodia that can reach
cells a few cell diameters away (de Joussineau et al. 2003; Cohen et al. 2010).
Notch signaling is a simple linear pathway with no amplification step.
Interaction of Notch receptors with DSL ligands presented by neighboring
cells triggers two proteolytic cleavages within the receptor. The first one is
extramembrane, executed by ADAM-family metalloproteases; this generates
the substrate for the second cleavage, which is intramembrane, secretase-
dependent (like amyloid generation), and releases the intracellular domain of
Notch (NICD). NICD is subsequently transported to the nucleus and acts as a
transcriptional coactivator that associates with a member of the CSL DNA-
binding transcription factor family and turns on target gene expression.
Among the targets of the NICD-CSL complex are the E(spl)/HES family
genes, which are transcriptional repressors and account for many of the
downstream effects of the pathway (reviews by Artavanis-Tsakonas et al.
1999; Bray 2006).
Notch signaling regulates a broad range of cellular processes in organisms
ranging from sea urchins to humans, including cell-fate specification,
formation of growth-organizing boundaries, stem cell maintenance,
proliferation, apoptosis, and migration. Therefore, it is not surprising that its
dysfunction has been implicated in many heritable developmental diseases,
including Allagille and CADASIL syndromes, as well as cancer, where it
promotes tumor growth in some contexts but can prevent it in others. How
Notch signaling, especially considering the simplicity of the pathway,
specifies so many different biological outcomes, depending on the cell
context, is a major question in the field (reviews by Artavanis-Tsakonas et al.
1999; Lai 2004; Fortini 2009). Below we provide just a few examples of
developmental processes regulated by different Notch modes of action:
lateral inhibition, lineage decisions, and inductive signaling.
One of the best-characterized roles of Notch signaling is lateral inhibition,
in which a specific cell fate is defined for a single cell within a group of
equivalent cells (Fig. 3). For example, in the Drosophila embryonic
neuroepithelium, equivalent ectodermal cells differentiate into either
neuroblasts or epithelial cells through the action of Notch signaling. Initially,
all neuroepithelial cells express low levels of both the Delta ligand and the
Notch receptor. However, probably as the result of stochastic variations,
some cells begin to express higher levels of Delta. These small differences
are amplified through a positive-feedback loop that activates its transcription.
Because the cells expressing high levels of the ligand cannot activate
signaling because of cis-inhibitory interactions with the receptor (Heitzler
and Simpson 1993), the system quickly resolves into Delta-expressing signal-
sending cells and signal-receiving cells with low Delta levels that activate
Notch signaling, which differentiate into neuronal and epithelial cells,
respectively. This Notch-dependent lateral inhibition mechanism is used
widely in development to pattern tissues containing initially identical cells.
The same mechanism is used to select myoblast founder cells in the
mesoderm (Bate and Rushton 1993; Rushton et al. 1995) and R8
photoreceptor fate from neural preclusters during eye development (review
by Roignant and Treisman 2009). Another well-characterized example of
lateral inhibition between two cells is the AC/VU (anchor cell/ventral uterine
precursor cell) decision in Caenorhabditis elegans vulva, which is induced by
activation of the Notch ortholog Lin12 that specifies the VU fate (reviews by
Greenwald and Rubin 1992; Greenwald 1998).
Figure 3. Lateral inhibition. (A) The process is progressive and can be separated in three steps: (1)
Initially, all cells in the cluster express both Delta and Notch and are equivalent. (2) Stochastic changes
in gene expression change the balance of ligand and receptor molecules, such that the cell in the middle
expresses more Delta. (3) Asymmetry is established when Delta expression in the middle cell is
stabilized through a positive-feedback loop, resulting in lateral inhibition whereby the Delta-expressing
cell becomes the signal sender whereas its neighbors activate Notch signaling and adopt the receiving-
cell fate. (B) During Drosophila neurogenesis the cell that activates Delta in the proneural cluster
becomes a neuroblast, whereas its neighbors will be laterally inhibited and adopt an epidermal cell fate.
The asymmetry between the neighboring cells is established by a negative-feedback loop that inhibits
Delta in the signal-receiving cell mediated by repressors of the E(spl) complex. In addition, cis-
inhibitory interactions between Notch and Delta exist and contribute to asymmetry generation and
lateral inhibition. (C) The Notch receptor and its ligands are subject to a number of protein
modifications, such as glycosylation (Ofut1 and Fringe), proteolysis (Furin, ADAM10, and γ-
Secreatase), and ubiquitylation (Deltex plus NEDD4). These events are critical for maturation of the
receptor and its presentation on the cell membrane, for Notch activation on ligand binding, degradation,
and trafficking of ligand-receptor complexes.

Notch signaling also operates in control of lineage decisions and inductive

signaling between nonequivalent cells. In these cases, the cells are initially
distinct from each other either because they asymmetrically express
regulators of the Notch pathway or because the ligand and receptor are
differentially distributed in adjacent cells (review by Bray 2006). For
instance, asymmetric segregation of Numb, which down-regulates Notch
signaling through polarized receptor-mediated endocytosis, in the progeny of
sensory organ precursors (SOPs) makes the Numb-positive cell Notch
sending (Jan and Jan 1995). In contrast, during wing vein specification in
Drosophila, expression of Delta in the vein regions induces Notch signaling
in the intervein cells to inhibit vein fate, and patterning is established through
a positive-feedback loop (Huppert et al. 1997). Often a combination of these
mechanisms can account for the developmental outcomes. For example, in
the Drosophila wing disc, both restricted expression of the
glycosyltransferase Fringe, which increases the ability of Notch to bind to
Delta, as well as restricted expression of ligands, lead to the specification of
the wing margin (Panin et al. 1997).
As a rule of thumb, Notch represents a signaling modality that provides an
on/off switch. How is this switch modulated and how is precise signaling
ensured? First, multiple levels of regulation of both the receptor and ligands
are deployed. These include posttranslational modifications such as
ubiquitylation that leads to proteasomal degradation, glycosylation, and
phosphorylation, as well as trafficking into specific cellular compartments
(Shilo and Schejter 2011). Second, when the pathway is used iteratively with
a specific duration (e.g., Drosophila and vertebrate neurogenesis and
vertebrate somitogenesis), then oscillatory activation/termination mechanisms
are utilized. This is achieved not only because the NICD is a very short-lived
transcription cofactor, but also because the pathway targets, the HES/E(spl)
family, have very unstable messenger RNAs (mRNAs) and proteins, and
exert autoinhibitory effects on their own transcription (reviewed by Fior and
Henrique 2009).

4 PATTERNING BY SECRETED PARACRINE FACTORS

In the case of signaling pathways that are triggered by secreted ligands, a
different set of rules applies. First and foremost, the range of signaling
elicited by the ligand-producing cell can span tens of cell diameters. Different
ligands have diverse distribution ranges, which are used in distinct contexts.
The distribution of ligands over a distance of several cell diameters generates
a graded signaling profile, which is used in many cases to generate several
distinct responses, rather than a single on/off switch.
The observation that a single diffusible molecule could specify and
pattern different cell fates in a concentration-dependent manner led to the
concept of morphogen gradients (Turing 1952; Wolpert 1969; Meinhardt
1978). Morphogen gradients provide spatial information and generate
different cell types in a distinct spatial order. The concentration gradient of
the diffusing morphogen subdivides a field of cells by inducing or
maintaining the expression of different target genes at distinct concentration
thresholds. Accordingly, cells close to the morphogen source receive high
levels of morphogen and express both low- and high-threshold target genes.
Cells far from the source of the morphogen receive low levels of morphogen
and express only low-threshold target genes. As a result, distinct cell types
emerge.
The physical properties of the ligand, as well as its diffusion capacity,
mode of transport, endocytosis, and interactions with heparan-sulfate
proteoglycans (HSPGs), all affect the final distribution of the ligand and
hence the resulting signaling profile. A clear hierarchy of ranges is evident in
paracrine signaling in Drosophila tissues. In the case of RTK ligands,
including Spitz (which activates the Drosophila EGF receptor), Branchless
(which triggers the Drosophila FGF receptor Breathless), and the ligands that
activate the Drosophila FGF receptor Heartless, the signaling range is
restricted to a small number of cell diameters, typically two to eight. HH
displays a similarly limited range. In contrast, the ligands for the BMP and
Wnt/Wg pathways have a longer range, which can extend up to 30 cell
diameters. Interestingly, as discussed below, lipid modifications of both HH
and Wnts are important for distribution of these molecules in tissues. Below,
we provide several examples illustrating the mechanisms underlying the
regulation of ligand distribution for each of these major signaling pathways.

5 CONTROLLING THE SIGNALING RANGE OF

SECRETED FACTORS
5.1 EGF and FGF
The range of EGFR signaling in Drosophila is regulated primarily by the
amount of secreted ligand provided to the receiving cells. Three of the four
EGFR ligands (Spitz, Keren, and Gurken) are produced as inactive
transmembrane precursors that are sequestered in the endoplasmic reticulum
(ER). The fourth ligand, Vein, is produced from the outset as a secreted
molecule. Trafficking of ligand to a secretory compartment where processing
takes place is facilitated by a transmembrane chaperone named Star (Lee et
al. 2001; Tsruya et al. 2002). Within the secretory compartment, cleavage of
the precursor is performed by intramembrane proteases of the Rhomboid
family, and the cleaved extracellular ligand portion is subsequently secreted
(Urban et al. 2001). The chaperone Star is also cleaved by Rhomboid proteins
but this cleavage generates an inactive molecule (Tsruya et al. 2007). Some
of the Rhomboid proteins localize not only to the secretory compartment but
also to the ER. When Star encounters Rhomboid in the ER, it is inactivated
before it can promote trafficking of the ligand precursors to the secretory
compartment where ligand cleavage should take place (Yogev et al. 2008).
Thus, only a fraction of the chaperone molecules escape inactivating cleavage
in the ER, and hence the level of ligand precursor that is trafficked and
secreted is significantly reduced. This leads to a corresponding reduction in
the range of signaling. In tissues where a restricted range of EGFR activation
is required, such as the eye disc or the germline, Rhomboid proteins are
present in both the ER and secretory compartment.
Once ligand is secreted, another tier of regulation is used. High levels of
EGFR activation induce the expression of the target gene argos, which
encodes a secreted molecule that neutralizes the ligand (Golembo et al. 1996;
Klein et al. 2004). Induction of Argos thus reduces the levels of active ligand
that can diffuse from the source, and hence the range of signaling.
In responding cells, additional mechanisms restrict the signaling range,
functioning in a cell-autonomous manner. Two inhibitor-encoding genes
(kekkon1 [Ghiglione et al. 1999] and sprouty [Casci et al. 1999; Kramer et al.
1999; Reich et al. 1999]), in particular, are induced in a classical negative-
feedback loop. In both cases, the induction of the inhibitors in a fairly broad
range of the receiving cells results in productive signaling only in cells that
are closer to the ligand source, and receive enough input to overcome the
inhibitory effects. Kekkon1 encodes a transmembrane protein that generates
inactive heterodimers with EGFR. Sprouty is an inhibitor of ERK/MAPK
signaling whose mechanism of inhibition of RTK signaling remains
incompletely understood. It interacts with several proteins impinging on
signaling, including Grb2, Raf, Cbl, and PP2A, and undergoes
phosphorylation that alters its binding properties and stability (Edwin et al.
2009; Reddi et al. 2010). Because Sprouty operates downstream from the
receptor, by interacting with components common to multiple RTK
pathways, it attenuates signaling by both FGF- and EGF-induced pathways
(Hacohen et al. 1998). Both Kekkon1 and Sprouty are conserved in
vertebrates. Sprouty, in particular, is an essential component that modulates
RTK pathways in normal development and disease (Edwin et al. 2009).
The transcriptional output of RTK signaling is mediated by members of
the ETS family of transcription factors. Most prominent is the ETS-domain
protein Pointed, which has two isoforms generated by alternative splicing
(Klambt 1993; O’Neill et al. 1994). ERK activates each of the two forms in a
different manner. Phosphorylation of PointedP2 converts an inactive protein
to the active form. The mechanistic basis for activation by phosphorylation is
not known but may involve stabilization, nuclear translocation, and exposure
of the transcriptional activation or DNA-binding domains. The second
isoform, PointedP1, is constitutively active even in the absence of ERK
signaling. However, its expression is dependent on ERK activity (Gabay et
al. 1996). The transcription factor that responds to ERK activity to trigger
PointedP1 expression is not known.
The YAN protein contains an ETS DNA-binding domain but is devoid of
a transcriptional activation domain. YAN is also a target for ERK
phosphorylation, but in this case phosphorylation leads to its inactivation by
promoting nuclear exit and degradation (Rebay and Rubin 1995). The dual
and opposite effects of ERK on the activators and inhibitor may make the
induction of ETS-target genes more robust (Fig. 4).
Figure 4. The EGFR pathway in Drosophila. Three membrane-anchored ligands—Spitz (SPI), Gurken
(GRK), and Keren (KRN)—are retained in the ER, and are processed following trafficking by the
chaperone protein Star, which is dedicated to these molecules, to the Rab4/14 compartment in the
secretory pathway. In this compartment, the ligands encounter Rhomboid proteins, seven-
transmembrane-span intramembrane serine proteases, which cleave the ligand precursors within the
transmembrane domain, to release the active, secreted form. Rhomboids also reside in the ER cleave
and inactivate Star, thus attenuating the level of ligand precursor that is trafficked to the Rab4/14
compartment. Within the receiving cells, the ligands encounter the EGF receptor, which on
dimerization triggers the canonical SOS/Ras/Raf/MEK/MAPK pathway. The cardinal transcriptional
output of the pathway is mediated by the ETS protein Pointed (PNT). In addition, the ETS protein
YAN provides a constitutive repressor, which competes for Pointed binding sites, and can be removed
from the nucleus and degraded upon phosphorylation by ERK. Several negative regulators keep the
pathway in check. Especially important is a group of inducible repressive elements, which constitute a
negative-feedback loop. Argos is a secreted molecule, which sequesters the ligand SPI, whereas
Sprouty and Kekkon1 attenuate signaling within the receiving cell.

An interesting variation occurs in the case of the Breathless FGF receptor.

The receptor itself restricts diffusion of the ligand Branchless. The role of
Breathless is to guide migration of tracheal cells toward the ligand source. To
increase the sharpness of the attracting ligand gradient, expression of the
receptor is induced by high levels of signaling, generating a trap that restricts
the diffusion of the Branchless ligand (Oshiro et al. 2002).

5.2 Hedgehog
HH transmits information over several cell diameters, but its range is
restricted. The distribution of HH has been studied most intensively in the
wing imaginal disc, where it defines a zone of activation in the boundary
between the posterior and anterior compartments of the disc. All posterior
cells produce HH but do not respond to it, whereas the anterior cells do not
produce it but can respond to it (review by Ingham and McMahon 2001). The
range of HH diffusion from the posterior compartment determines the
signaling range, and the region of the anterior compartment that receives HH
subsequently becomes the domain that produces the BMP family ligand DPP,
which directs long-range patterning of the wing.
HH is unusual as it undergoes dual lipid modification and autoproteolytic
cleavage (Porter et al. 1996; Pepinsky et al. 1998; Chen et al. 2004). The
cholesterol moiety that is added limits HH trafficking within and between
cells and palmitoylation is required for the production of a soluble multimeric
HH protein. Binding of HH to its receptor Patched (PTC) leads to its
endocytosis and degradation. Because PTC functions by inhibiting the next
step in the pathway (the transmembrane protein Smoothened [SMO]), this
leads to pathway activation. Interestingly, ptc itself is a transcriptional target
gene for HH signaling (reviewed in Wilson and Chuang 2010). As in the case
of Branchless, this leads to more effective trapping of HH by the first rows of
cells receiving the signal, and hence to a restriction of the signaling range.
Recently, studies in mammalian cells have shown that mammalian Hh
signaling depends on the primary cilium, a small cellular projection found on
most vertebrate cells (Goetz et al. 2009). In particular, Smo proteins
participate in the transduction of Hh signals, moving into the cilium in
response to Hh ligand. Interestingly, the absence of cilia in Drosophila
suggests that a fundamental difference exists between the organization of the
Hh pathway between invertebrates and mammals.

5.3 BMPs/TGFβ and Wnt/Wg Ligands

The BMP and Wnt/Wg family ligands act over a long range, especially in the
wing disc, to pattern not only the cells close to the ligand source but also
those positioned many cell diameters away. In these cases the regulation is
more intricate as it involves shaping the distribution of the ligand over a long
range, restricting signaling close to the source while facilitating signaling
further away. This is important for maintaining the robustness of the resulting
gradient to changes in the level of ligand produced (Eldar et al. 2003).
As in the case of Hh signaling, signaling by BMP regulates the expression
level of the receptors; however, in this case the expression of the BMP
receptor is inhibited by signaling (Lecuit et al. 1996). This generates a
situation where less ligand trapping takes place close to the source,
facilitating long-range diffusion. In addition, the elevated receptor levels
further from the source make these cells more responsive to the low levels of
ligand they encounter. Finally, the induction of inhibitors that block
intracellular signaling, such as DAD (Tsuneizumi et al. 1997), which
competes with the Smad proteins that transduce BMP signals, further restricts
signaling close to the ligand source. The range of this response is dictated by
the sensitivity of the promoter/enhancer of the inhibitory molecules to
induction by signaling.
Another set of extracellular molecules that shape the distribution of BMPs
and Wnts/Wg are HSPGs, which comprise a transmembrane protein core and
long chains of sugars that emanate from this core (Perrimon and Bernfield
2000). The versatility of covalent links that can be formed between the sugar
molecules has the potential to generate enormous complexity and hence
specificity. Although the association between the ligands and HSPGs
represents a low-affinity interaction, the sheer number of HSPGs may
compensate for this low affinity. It is estimated that the number of HSPG
molecules per cell is at least two orders of magnitude higher than that of the
specific ligand receptors. Hence, the ligands travel in a “forest” of HSPGs,
where they rarely encounter their specific receptors. HSPGs can have
opposing effects on activation, and hence analysis of their function is
complicated. They may facilitate signaling locally by trapping ligands and
functioning as coreceptors that present the ligand to receptors such as FGFR.
However, they may also facilitate signaling at a distance by either stabilizing
the ligand or functioning as long-range carriers after cleavage of their
extracellular protein stem. This latter activity also reduces the level of
signaling close to the ligand source (reviewed in Yan and Lin 2009).
Wnt/Wg proteins, in addition to interacting with HSPGs, are modified by
palmitoylation, a hydrophobic modification on a conserved cysteine residue
that affects their distribution, as well as a second lipid modification by
palmitoleic acid esterification of a serine residue. Studies in Drosophila and
vertebrates have provided evidence that Wnt palmitoylation is controlled by
Porcupine, predicted to be a membrane-bound O-acyl transferase, and that
this modification is important for the generation of Wnt gradients. In
Drosophila lack of palmitoylation in porcupine mutants affects WG secretion
(Kadowaki et al. 1996). Similarly, in the chick neural tube porcupine-
mediated lipid modification reduces the range of activity of Wnt1 and Wnt3a
(Gali et al. 2007).

5.4 Long-Range Ligand Distribution

Studies of DPP and Wnt signaling in cell clones, in which the receptor is
eliminated or a constitutively active receptor is expressed, have shown that
the original signals are transmitted even to the most distant cells (Lecuit and
Cohen 1996; Nellen and Basler 1996; Neumann and Cohen 1997), rather than
being relayed by inducing secondary signals. Several models have been
proposed for the long-range distribution of ligands, which may use multiple
strategies. Although all of the proposed models are supported by several lines
of compelling evidence, critical experiments directly eliminating one mode of
trafficking and monitoring the outcome have not been performed for
technical reasons. We therefore present the prevailing models below.
The simplest mechanism for ligand distribution is diffusion in the
extracellular milieu. Reduction in ligand levels over a distance may be driven
by endocytosis or extracellular degradation. HSPGs may enhance or reduce
diffusion and keep the ligand in the plane of the epithelium by low-affinity
interactions (Strigini and Cohen 2000). The association of some ligands with
hydrophobic moieties, most notably Hh and cholesterol and palmitoylate, as
well as Wnt/Wg proteins and palmitoylate, have raised the possibility of
another mode of extracellular ligand trafficking, in which membrane
fragments bearing these named argosomes are dispersed over large distances
(Greco et al. 2001; Panáková et al. 2005). Exovesicles like these have been
characterized in Drosophila imaginal discs. They contain Wnt/Wg proteins,
are derived from basolateral membranes, and travel through tissues, where
they are found predominantly in endosomes.
Another option is transcytosis of the ligand. In this scenario the ligand
travels most of its journey in vesicles within cells. Its dilution over a distance
is affected by the fraction of ligand that is endocytosed and by efficiency of
ligand transfer between cells versus its intracellular degradation. Experiments
have shown that elimination of the receptor in a group of cells adjacent to the
ligand source reduces signaling in more distant cells that have a normal
receptor. This suggests that the receptor is required in the proximal cells for
incorporating the ligand into the cells and transferring it distally (reviewed in
Wartlik and Gonzalez-Gaitan 2009).
One of the most provocative suggestions for the transfer of ligands over
tens of cell diameters involves very thin cellular protrusions termed
cytonemes. These have been proposed to serve as conduits in which
morphogens move between producing and target cells (reviewed in Kornberg
and Guha 2007). These structures have been identified in the epithelium of
the wing disc and point toward cells that produce ligands. They contain the
relevant ligand receptors, and their polarity is disrupted by uniform
presentation of the respective ligand (Roy et al. 2011). Cytonemes have been
proposed to serve to traffic the ligand to the cell body where signaling may
take place. Interestingly, cytonemes are also found in vertebrate cells and
thus may play a general role in long-range cell–cell communication.

6 THE LOGIC OF SIGNALING

Although pathways use distinct signaling components and signaling
strategies, a number of common universal themes have emerged regarding
their structures and regulation in time and space.

6.1 Linear Signaling Pathways

Developmental signaling elicited by ligand-receptor binding appears to be
transmitted in a linear fashion within the cell, leading to induction of target
genes. This is very different from typical signaling schemes in which
multiple converging and diverging links are observed. The most compelling
evidence for such linearity is that mutations in different components along a
pathway give rise to very similar phenotypes (reviewed by Friedman and
Perrimon 2007). This linearity of developmental signaling stems from the
need to transmit a clear signal, in view of the irreversibility of the resulting
decisions. This holds true both for cases where an on/off switch is induced
and for situations where graded signaling elicits diverse responses, according
to the level of signaling. Each pathway regulates the activity of one or more
transcription factors, which bind to specific signaling pathway response
elements in the enhancers and promoters of target genes (Barolo and
Posakony 2002).

6.2 Negative-Feedback Switches

Tight regulation of signaling is essential for generation of reproducible
patterns during development. In the case of pathways that function as
switches that induce a particular cell fate within a zone of competent cells,
this will determine the spatial boundaries of signaling. For ligands that
function as morphogens to induce several distinct cell fates, this regulation
will determine the overall spatial profile of resulting patterns. Another
important consideration in signaling is the need to buffer against noise
stemming from heterozygosity, unequal distribution of components between
dividing cells and environmental fluctuations. Negative feedback provides a
way of fine-tuning the signal over a range of signaling levels and sharpening
boundaries between regions that respond differently.
Many examples of transcriptional induction of negative regulators exist.
In some cases, these regulators function extracellularly to restrict the level or
distribution of active ligand. For example, Noggin inhibits TGFβ signaling by
binding to TGFβ family ligands and preventing them from binding to their
receptors (Smith 1999). Similarly, the Argos molecule binds to EGFR
ligands, thus effectively reducing their levels (Klein et al. 2004). In other
cases, the inhibitor induced is acting only in the receiving cells. Examples of
transmembrane molecules that compromise receptor activity include
Kekkon1, which inhibits EGFR (Ghiglione et al. 1999). Inducible molecules
that interfere with signaling intracellularly include Sprouty, which is a
general repressor of ERK kinase signaling (Casci et al. 1999; Kramer et al.
1999; Reich et al. 1999), Dad, which competes with Smad proteins
(Tsuneizumi et al. 1997), and axin, which negatively regulates Wnt/Wg
signaling (Ikeda et al. 1998). For the cell-autonomous inhibitors, a relatively
broad range of induction by signaling is required, such that only the cells that
receive a signal above a certain threshold level experience productive
signaling.

6.3 Generating a Threshold

In the case of Notch signaling, in which the ligand is membrane anchored, the
boundaries of signaling are dictated by the contact zones between the sending
and receiving cells. However, in cases where the ligand is diffusible, a graded
signaling profile will be generated. Regardless of the range, this graded
activation pattern is converted to sharp borders of induction of gene
expression.
The underlying mechanisms for generating transcriptional thresholds are
crucial for proper patterning. Several mechanisms have been identified.
During early dorsoventral patterning in the Drosophila embryo, graded
nuclear localization of the transcription factor Dorsal (an NF-κB homolog) is
converted to sharp borders of zygotic gene expression (e.g., twist and snail in
the ventralmost cells, which define the future mesoderm). The snail
regulatory region contains multiple adjacent binding sites for Dorsal. Binding
of one Dorsal molecule to DNA may facilitate the binding of additional
molecules by protein–protein interactions, generating a sharper response
(Rusch and Levine 1996).
In the case of BMP target genes in the wing disc, different stringencies of
regulation may apply depending on the position of the responding cell
relative to the ligand source. Of particular interest is the potential
transcription factor Brinker (BRK), which negatively regulates DPP target
genes in both the Drosophila wing disc and embryo. BRK antagonizes
transcription of target genes, and forms a gradient that opposes the BMP
activation gradient. Genes that are expressed closer to the ligand source
require simultaneous suppression of brk expression and activation of
transcription by Smads (along with binding of accessory transcription
factors). For genes that are expressed in a broader pattern and hence require
lower signaling levels for their induction, suppression of brk expression is
sufficient (Affolter and Basler 2007).
Finally, a mechanism for generating transcriptional thresholds termed
zero-order hypersensitivity has been proposed. In cases where a transcription
factor, or transcriptional repressor, undergoes reversible phosphorylation and
is in excess, even small differences in the rates of the reversible
phosphorylation and dephosphorylation reactions will lead to the complete
accumulation of the protein in one form or another. This generates a sharp
threshold response (Melen et al. 2005).

7 INTEGRATING SIGNALING PATHWAYS

Many of the mechanisms underlying cell-type specification and formation of
distinct tissues rely on interactions between signaling pathways. Often the
activation of one pathway leads to activation of a second, the two pathways
acting in a sequential or relay mode. In addition, two signaling pathways can
act in parallel and converge to regulate the activity of the same target.
Finally, cross talk can result from “pathway interference,” in which one
pathway modulates the activity of a canonical component of another.
A striking example of how the spatial and temporal interaction of
signaling pathways can produce complex patterns during development is
somitogenesis, the process that generates the spine through the periodic
establishment of the embryonic segments from the paraxial mesoderm in
vertebrates (Dequeant and Pourquie 2008). Somites are masses of mesoderm
distributed along the two sides of the neural tube that give rise to the dermis,
skeletal muscles, and vertebrae. During somitogenesis an oscillating
mechanism, called the segmentation clock, drives pulses of expression of a
limited number of genes repeatedly in the presomitic mesoderm (PSM) every
time a new somite is formed. The first evidence that cyclic gene expression
drives somitogenesis came from the observation that the HES1 (Hairy and
Enhancer of split 1) mRNA is expressed in a dynamic cyclic pattern
coinciding with the formation of each somite (Fig. 5A). Subsequently, several
other genes with similar cyclic behavior were identified, the vast majority of
which have been shown to be components of the Notch, FGFR, and Wnt
signaling pathways (Fig. 5B). In particular, in the mouse, Notch-FGF-
regulated genes oscillate out of phase with Wnt-regulated genes and their
activation in the PSM is mutually exclusive. This suggests tight, coordinated
regulation of signaling (Dequeant et al. 2006), which is achieved by a large
number of negative-feedback loops (Fig. 5B) and the presence of a
pacemaker that triggers the rhythmic coordinated activation of these signaling
pathways.
Figure 5. The segmentation clock oscillator. (A) Evidence of an oscillator underlying vertebrate
segmentation comes from the transcriptional expression of the hairy1 gene (dark green) in periodic
waves in the presomitic mesoderm (PSM). These waves are associated with the timely formation of
pairs of somites that are added sequentially. Experiments in zebrafish have shown that a remarkable
property among neighboring PSM cells is that they undergo synchronized gene-expression oscillations
(as shown in boxed area), which are coordinated by the Notch signaling pathway. (B) The FGF, Notch,
and Wnt signaling pathways underlie the mouse oscillator. Cyclic genes belonging to the FGF (left) and
Notch (middle) pathways oscillate in opposite phase to cyclic genes of the Wnt pathway (right). Several
feedback loops are indicated. These are involved in reinforcing activity or shutting down a pathway.
Some instances of pathway cross talk have also been observed. APC, adenomatous polyposis coli;
DACT1, dapper homolog 1; DKK1, dickkopf homolog 1; FGFR, fibroblast growth factor receptor;
Grb2, growth factor receptor bound protein; Dll1, Delta-like 1; DSH, dishevelled; DUSP6, dual
specificity phosphatase 6; ERK, mitogen-activated protein kinase 1; GSK3, glycogen synthase kinase
3; HES, hairy enhancer of split-related; LFng, lunatic fringe; LPR6, low-density lipoprotein receptor-
related protein 6; MEK, mitogen-activated protein kinase 1; NICD, Notch intracellular domain; NKD1,
naked cuticle 1 homolog; Nrarp, Notch-regulated ankyrin repeat protein; SHP2, Src homology region
2-containing protein tyrosine phosphatase 2; SOS, son of sevenless.

FGF and Wnt signaling are regulated temporally and spatially. The
ligands are expressed in gradients in the precursor tissue of the segments
where they regulate the progressive maturation/differentiation of the tissue
and define the domain of the clock activities. The Wnt pathway, for example,
is activated in the PSM before segmentation, plays a role upstream of both
the Wnt and Notch oscillations, and is thought to entrain the Notch feedback
loop. As a result, the spatial and highly dynamic temporal regulations of
these signaling activities guarantee the robust segmentation patterning of the
vertebrate axis and are evolutionarily conserved in vertebrates (Dequeant and
Pourquie 2008). In addition, experiments in zebrafish have indicated that the
Notch pathway is required for synchrony of the oscillations at the cellular
level and the coordinated expression of the correct targets within neighboring
cells, because lack of Notch leads to a “salt and pepper” pattern of
oscillations (Fig. 5A) (Dequeant and Pourquie 2008).
Determination of mesodermal progenitors in the Drosophila embryo
(Carmena et al. 1998) exemplifies the complex interplay and integration of
signaling pathways at the promoter level (Fig. 6). Using the regulation of the
even-skipped (eve) promoter, Halfon et al. (2000) have illustrated how the
synergistic integration of transcription factors, regulated by the Wnt/Wg,
DPP/BMP, and EGF/FGF/ERK pathways, generates a specific developmental
transcriptional response at a single defined enhancer. Because some of the
pathways are activated earlier than others and in a broader domain, they
determine the “competence group” of cells (expressing markers like Lethal of
scute, L’sc) and lead to subsequent activation of additional pathways within a
more restricted cell population (Fig. 6A). These later pathways are regarded
as inductive, and it is the final integration of the transcriptional signals from
all pathways, within a single enhancer, that induces the relevant target gene.
In this system, the WG and DPP signals are orthogonal to each other and
define the intersection zone as the competence group, through signal-
responsive transcription factors (MAD and TCF) that induce two tissue-
specific transcription factors (Tinman and Twist). In addition, TCF also
contributes to the expression of essential elements for ERK signaling (i.e.,
Rhomboid, Heartless, and Heartbroken). Once activated, ERK provides the
inductive signal, by activating the transcription factor Pointed and
inactivating the YAN repressor (Fig. 6B). Finally, singling out of
mesodermal Eve progenitors is achieved through the process of lateral
inhibition mediated by Notch/Delta signaling (Carmena et al. 2002).

Figure 6. The WG/DPP/FGF interplay during specification of Drosophila mesodermal progenitors. (A)
A model for patterning of the embryonic Drosophila mesoderm through the combinatorial actions of
WG, DPP, and RAS/ERK signals. This model applies to both somatic muscle and pericardial muscle.
The intersection between WG (red) and DPP (blue) delineates a prepattern (purple) in which Lethal of
scute (L’SC) is initially activated in a precluster (orange). The entire L’SC precluster is competent to
respond to RAS1. However, the spatially restricted activation of Heartless (HTL) and EGFR restricts
L’SC to a subset of precluster cells that correspond to an equivalence group. RAS1 signaling activates
EVE expression in all cells of the L’sc cluster (green) and subsequently a single EVE-expressing
progenitor (red) is determined by lateral inhibition mediated by the Notch/Delta pathway. (B) WG,
DPP, and RAS1 signal integration during specification of mesodermal EVE progenitors. WG and DPP
provide developmental competence by regulating tissue-specific transcription factors (Tinman [TIN]
and Twist [TWI]), signal-responsive transcription factors (MAD, TCF), and proximal components of
the RTK/ERK pathway (FGFR/HTL, Heartbroken [HBR]/DOF and Rhomboid [RHO]). The RAS
pathway leads to activation of the ETS-binding transcription factor Pointed (PNT) and inactivation of
the ETS-binding YAN repressor. The activities of all five transcriptional activators (TIN, TWI, MAD,
TCF, and PNT) are integrated at the MHE (Muscle and Heart Enhancer) of eve, which is located 6 kb
downstream from its transcription start site, and synergistically promote eve expression. In the absence
of inductive RAS signaling, YAN represses eve by binding to ETS sites. In addition, RAS/PNT
signaling in the EVE progenitor promotes Delta and Argos (AOS) expression, which in combination
activate Notch and shut down EGFR signaling in the nonprogenitor cells, to ensure lateral inhibition.

Such integration of signaling pathways at the promoter/enhancer level

allows each gene to define its “rules” of regulation, according to the tissue
setting in which it is activated. Two given pathways can act synergistically in
one setting and antagonistically in another. Thus, whereas only a small
number of signaling pathways are used during development, the
combinatorial flexibility at the promoter level generates a vast array of
possible responses. This would not be possible if more stringent and
hardwired interactions between pathways operated more broadly at the
cytoplasmic level.
Studies of cell-fate specification in the Drosophila eye have illustrated
how two pathways, EGFR and Notch, can be utilized both sequentially and in
parallel (Flores et al. 2000). Specifically, cone cell differentiation, visualized
by the expression of the transcription factor PAX2, requires inputs from the
EGFR and Notch pathways by neighboring R photoreceptor cells that
produce both EGF and Delta ligands. In addition, the expression of the Delta
ligand in R photoreceptor cells requires high levels of EGFR signaling
(Flores et al. 2000; Tsuda et al. 2002; see reviews by Nagaraj and Banerjee
2004; Doroquez and Rebay 2006). Interestingly, differentiation of the R7
photoreceptor cell requires input from another RTK, Sevenless (SEV),
activated by the transmembrane protein Bride of Sevenless (BOSS)
(Perrimon and Perkins 1997).
 Finally, a number of pathway interference mechanisms operating
upstream of transcription have been documented. For example, studies in
mammals and Drosophila have identified a mechanism by which the Hippo
pathway coordinates Wnt/Wg morphogenetic signaling with growth control.
Signaling via the Hippo pathway is critical for the precise control of organ
size. Activation of the Hippo serine/threonine kinase leads to inhibition of the
transcriptional coactivator TAZ and YAP (also known as Yorkie [YKI] in
Drosophila) through phosphorylation and nuclear exclusion dependent on
binding to 14-3-3 proteins. Varelas et al. (2010) showed that the Hippo
pathway restricts Wnt/β-catenin signaling by promoting interactions between
TAZ and Dishevelled, a cytoplasmic component of the canonical Wnt/Wg
pathway. Similarly, Xia et al. (2010) have reported that the Fused (FU)
serine/threonine kinase, a component of the canonical Hh pathway, functions
together with the E3 ligase Smurf to regulate the ubiquitylation and
subsequent degradation of Thick veins (TKV), a BMP receptor, during
oogenesis. This mechanism ensures the generation of a steep gradient of
BMP activity between the germline stem cell and its progeny. The
degradation of TKV then permits expression of differentiation genes in the
daughter cell.
Such examples may represent special cases rather than the norm in the
context of developmental processes. Indeed, as stated above, during
developmental signaling, cytoplasmic cross talk between pathways appears to
be kept to a minimum to ensure that cells integrate signals quickly and
effectively (Noselli and Perrimon 2000). An example of the simplicity more
commonly seen is ERK regulation during Drosophila embryogenesis. When
the active form of MAPK is monitored immunohistochemically, for every
pattern that is observed, a single RTK has been shown to be responsible
(Gabay et al. 1997). This reveals the striking absence of overlaps between
RTK pathways in time and space.

8 CONCLUDING REMARKS: DEVELOPMENTAL

VERSUS PHYSIOLOGICAL SIGNALING
In addition to their developmental roles, the signaling pathways discussed
here play central roles in animal physiology. However, in contrast to their
roles in development, where they act in ratchetlike mechanisms pushing
forward developmental programs by regulating the activity of transcription
factors, in physiological contexts the pathways are used to gauge the
environment and fine-tune the physiological state of the cell, and as such are
reversible.

ACKNOWLEDGMENTS
We thank Mary-Lee Dequeant for helpful comments. Work in the Perrimon
laboratory is supported by the Howard Hughes Medical Institute and NIH,
and work in the Shilo laboratory by a grant from the Minerva Foundation.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005975
CHAPTER 11

Signaling by Sensory Receptors

David Julius1 and Jeremy Nathans2

1Department of Physiology, University of California School of Medicine, San Francisco, California
94158
2Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, Maryland
21205
Correspondence: David.Julius@ucsf.edu and jnathans@jhmi.edu

SUMMARY

Sensory systems detect small molecules, mechanical perturbations, or

radiation via the activation of receptor proteins and downstream
signaling cascades in specialized sensory cells. In vertebrates, the two
principal categories of sensory receptors are ion channels, which
mediate mechanosensation, thermosensation, and acid and salt taste; and
G-protein-coupled receptors (GPCRs), which mediate vision, olfaction,
and sweet, bitter, and umami tastes. GPCR-based signaling in rods and
cones illustrates the fundamental principles of rapid activation and
inactivation, signal amplification, and gain control. Channel-based
sensory systems illustrate the integration of diverse modulatory signals
at the receptor, as seen in the thermosensory/pain system, and the rapid
response kinetics that are possible with direct mechanical gating of a
channel. Comparisons of sensory receptor gene sequences reveal
numerous examples in which gene duplication and sequence divergence
have created novel sensory specificities. This is the evolutionary basis
 for the observed diversity in temperature- and ligand-dependent gating
among thermosensory channels, spectral tuning among visual pigments,
and odorant binding among olfactory receptors. The coding of complex
external stimuli by a limited number of sensory receptor types has led to
the evolution of modality-specific and species-specific patterns of
retention or loss of sensory information, a filtering operation that
selectively emphasizes features in the stimulus that enhance survival in a
particular ecological niche. The many specialized anatomic structures,
such as the eye and ear, that house primary sensory neurons further
enhance the detection of relevant stimuli.

Outline
1 Introduction
2 Receptors: Detection and transduction
3 Structural basis of sensory receptor activation
4 The logic of sensory coding
5 Evolution and variation
6 Concluding remarks
References

1 INTRODUCTION
An organism’s perception of the world is filtered through its sensory systems.
The properties of these systems dictate the types of stimuli that can be
detected and constrain the ways in which these stimuli are reconstructed,
integrated, and interpreted. Here we discuss how sensory signals are received
and transduced, focusing on the first steps in the complex process of
perceiving an external stimulus. A recurrent theme is the way in which the
biochemical and biophysical properties of sensory receptor molecules, and
the neurons in which they reside, have been sculpted by evolution to capture
those signals that are most salient for the survival and reproduction of the
organism. As a result, some classes of sensory receptors, such as the night
vision receptor rhodopsin, show great conservation, whereas others, such as
olfactory receptors, show great diversity.
Evolutionary comparisons are fascinating at many levels, not least of
which is their power to highlight the logic of the stimulus–response
relationship. For example, honeybees can see UV light, enabling them to
locate sources of nectar and pollen based on the UV reflectance of flower
petals (Kevan et al. 2001), whereas humans and Old World primates have
excellent sensitivity and chromatic discrimination at longer wavelengths,
permitting the identification of red, orange, and yellow fruit against a
background of green foliage (Mollon 1989). Star-nosed moles use a
specialized mechanoreceptive organ on their snout to locate meals and
navigate through lightless subterranean tunnels (Catania 2005), and pit vipers
have evolved thermoreceptive organs to detect the infrared radiation emitted
by their warm-blooded prey (Campbell et al. 2002). In each of these cases,
evolution has fine-tuned a sensory organ through anatomical and/or
molecular changes to enhance the detection of relevant stimuli.
For simplicity, we focus on eukaryotic sensory systems, in which G-
protein-coupled receptors (GPCRs) and ion channels predominate as sensory
receptors. The one exception is mechanosensation, in which the molecular
basis of membrane stretch detection has been beautifully delineated in
bacteria but remains less clear in eukaryotes. Thus, our discussion of
mechanosensation is focused largely on prokaryotic systems. We also
describe the diverse cast of downstream transduction pathways and the
manner in which receptors and transduction pathways are regulated to
terminate signaling and set receptor sensitivity.
Before discussing individual receptors, it is worth noting that the
physiological attributes of sensory systems are dictated not only by the
molecular properties of receptor molecules and their associated signal
transduction proteins, but also by the architecture of the sensory organs, cells,
and subcellular structures in which they reside. A notable example is the
retina, where visual pigments, together with other components of the
phototransduction pathway, are localized within outer segments of rod and
cone photoreceptor cells at near millimolar concentrations, thereby enhancing
light sensitivity and transduction efficiency (Yau and Hardie 2009). Another
example is seen in the cochlea, where sound pressure waves are transmitted
through the middle ear to induce a localized and frequency-dependent
distortion of the basilar membrane in the cochlea. Progressive changes in
both the mechanical properties of the basilar membrane and the electrical
properties of the auditory hair cells along the length of the cochlea generate a
tonotopic map in which the amplitudes of different frequency components in
a complex sound are reflected in the magnitudes of auditory receptor
activation at different locations within the cochlea (Roberts et al. 1988).

2 RECEPTORS: DETECTION AND TRANSDUCTION

Both GPCRs and ion channels contribute to sensory transduction pathways
by initiating or modulating stimulus-evoked responses (Fig. 1; Table 1). In
vertebrates, GPCRs predominate as stimulus detectors in vertebrate visual
and olfactory receptor cells. In contrast, recent studies suggest that fly
olfactory neurons use ionotropic glutamate receptor-like channels to detect
some classes of odorants (Benton et al. 2009), revealing a striking divergence
of signal transduction mechanisms between insect and vertebrate
chemosensory systems. Ion channels predominate in the detection of auditory
and somatosensory stimuli, and both GPCRs and ion channels serve as
stimulus detectors in the gustatory (taste) system.
Figure 1. Comparison of sensory signaling systems for vision, olfaction, hearing and balance, taste,
and pain/thermosensation. The events underlying signal transduction are shown schematically for (A)
rod and cone photoreception; (B) olfaction in the main olfactory epithelium; (C) salt (left) and sweet
(right) taste; (D) hearing and balance; and (E) pain/thermosensation. Schematics A–E refer to
vertebrates. The final step in olfactory signaling consists of the calcium-dependent opening of anion
channel TMEM16B, although recent work suggests that the resulting anion current plays only a minor
role in olfactory signal transduction (Billig et al. 2011). For pain/thermosensation mediated by TRPV1,
the figure shows inflammatory agents (extracellular protons, bioactive lipids, peptides, and
neurotrophins) acting to enhance channel opening either as direct allosteric modulators of TRPV1 or
through second-messenger signaling pathways. In auditory and vestibular hair cell bundles, it is not
known whether transduction channels reside at both ends or at only one end of the extracellular elastic
elements (tip links); in panel D the channels are shown at both ends.

Table 1. Classes of sensory receptors

Sensory modalitya Receptor(s) Transduction mechanism

Vision GPCRs G-protein/cGMP phospho-diesterase/cGMP-gated

ion channel
Olfactionb GPCRs G-protein/adenylyl cyclase/cAMP-gated ion
channel
Hearing/balance Nonselective cation Direct gating by mechanical force
channel
Taste (sweet and bitter) GPCRs G-protein/TRP channel
Taste (sour) Ion channel Direct sensing of ion flux
Mechanosensation (in Ion channel Membrane stretch
bacteria)
aData are for vertebrates unless otherwise noted.
bData are for the main olfactory epithelium. A minority of olfactory sensory neurons appear to use
transmembrane guanylate cyclases as receptors.

Because GPCRs transduce information through multicomponent second-

messenger-based “metabotropic” signaling pathways (Henrik-Heldin et al.
2012), they endow physiological systems with a tremendous capacity for
signal amplification (Fig. 2). In vertebrate chemosensory and visual systems,
GPCR-based signaling enables sensory cells to detect nanomolar
concentrations of ligands or single photons, respectively. Such high
sensitivity is possible because a single GPCR, during its active lifetime, can
activate dozens to hundreds of G proteins, and each activated G protein in
conjunction with its associated target enzyme can synthesize or destroy
thousands of second-messenger molecules. This type of signaling cascade has
been most thoroughly analyzed in vertebrate rod photoreceptors, where light-
evoked activation of a single rhodopsin molecule leads to the activation of
about 500 downstream effector proteins [the G protein transducin and its
associated cyclic (c)GMP phosphodiesterase], with the consequent hydrolysis
of about 100,000 molecules of cGMP per second (Fig. 1A) (Stryer 1986;
Arshavsky et al. 2002). The reduction in cytosolic cGMP leads to the closure
of cGMP-gated ion channels in the outer segment plasma membrane, thereby
hyperpolarizing the cell and decreasing the release of the neurotransmitter
glutamate onto bipolar cells, the second-order neurons within the retina.

Figure 2. Schematic representation of temporal response profiles for single or multicomponent

transduction systems. (A) Time course of stimulus-evoked responses for transduction systems
consisting of zero, one, or two enzymatic stages of amplification. (B) For each system, the schematic
diagram shows the flow of information in the transduction system, with R and R* representing the
sensory receptor molecule in the inactive and active states, respectively. (Left panel) Here, there are no
transduction components beyond the receptor itself, as in the case of a sensory ion channel, where
activation is nearly instantaneous. (Middle panel) Here, the active receptor functions as a catalyst and
converts inactive molecules of A to their active derivatives A*, which accumulate linearly with time
following receptor activation. (Right panel) Here, the active derivative A* functions as a catalyst to
convert inactive molecules of B to their active derivatives B*, resulting in second-order response
kinetics. The left panel represents a mechanosensory system, and the right panel represents vertebrate
phototransduction, as diagrammed below.

A similar biochemical logic governs signaling in vertebrate olfactory

sensory neurons, where activation of G-protein-coupled odorant receptors
increases the synthesis of cAMP, which binds directly to and thereby opens
cyclic-nucleotide-gated ion channels in the plasma membrane of olfactory
cilia (Fig. 1B). The resulting depolarization of the plasma membrane initiates
an action potential that is transmitted from the sensory neuron’s body in the
olfactory epithelium to its presynaptic terminal in the olfactory bulb. Thus, in
both visual and olfactory sensory neurons, temporal control of signaling
comes down to a balance between cyclic nucleotide synthesis and
degradation. Interestingly, the detection of tastants and pheromones by
GPCR-containing gustatory and vomeronasal sensory neurons, respectively,
proceeds through a somewhat different signaling pathway involving G-
protein-mediated activation of phospholipase C, which promotes hydrolysis
of membrane phospholipids to generate second messengers (such as
diacylglycerols, inositol phosphates, and polyunsaturated fatty acids) and
thereby triggers calcium release from intracellular stores (Fig. 1C). These
actions promote the opening of excitatory TRP ion channels, leading to
depolarization and neurotransmitter release (Chandrashekar et al. 2006).
The same principles that underlie signal amplification—namely, the
involvement of multiple sequential steps in a transduction pathway—endow
metabotropic (i.e., GPCR) systems with a great capacity for adaptation and
other forms of signal modulation. Here, again, the most detailed analysis has
been performed in the visual system. The retina faces the daunting task of
discriminating luminance changes on a timescale of tens of milliseconds and
under conditions as disparate as sunny afternoons and moonless nights, in
which background light intensity varies by more than six orders of
magnitude. These challenges are met, in part, through negative-feedback
mechanisms in the photoreceptor outer segment that terminate signaling
and/or reset the baseline in the presence of a persistent stimulus. Although
first delineated in studies of rod phototransduction, these feedback
mechanisms are now known to be more-or-less generic to many GPCR
signaling pathways (DeWire et al. 2007; Moore et al. 2007). The most
receptor- proximal signal termination mechanism involves phosphorylation
of activated receptors by specific serine/threonine kinases, such as rhodopsin
kinase, a member of the G-protein receptor kinase (GRK) family, at several
residues in the long carboxy-terminal tail of the receptor on a timescale of
tens of milliseconds (Arshavsky et al. 2002). Once phosphorylated, the
receptor is capped by the inhibitory protein arrestin, which blocks subsequent
G-protein activation. In hormone and neurotransmitter receptor systems,
arrestin binding also facilitates receptor endocytosis and recycling. In
contrast, in the photoreceptor outer segment, rhodopsin remains stably
localized to the disc membrane. Rhodopsin is recycled to its dark state by the
combination of dephosphorylation and exchange of the photoisomerized all-
trans retinal chromophore for a new molecule of 11-cis retinal, reactions that
occur on a timescale of minutes (Arshavsky et al. 2002; Yau and Hardie
2009).
The time course of photoreceptor signal termination is also shaped by the
accelerated hydrolysis of G-protein-bound GTP via an allosteric interaction
between the regulator of G-protein signaling (RGS) family member RGS9
and the α-subunit of the photoreceptor G protein, transducin (Krispel et al.
2006). RGS-modulated signal termination plays an analogous role in primary
olfactory sensory neurons in Caenorhabditis elegans (Ferkey et al. 2007).
RGS-dependent enhancement of GTP hydrolysis is an ancient and
evolutionarily conserved mechanism that accelerates signal termination
across a wide variety of heterotrimeric and small G-protein signaling systems
in organisms as diverse as yeast and man (Dohlman and Thorner 1997; Ross
and Wilkie 2000; Netzel and Hepler 2006). In vertebrate photoreceptors, a
third mechanism for activity-dependent feedback involves a light-dependent
decline in intracellular calcium levels, which leads to the allosteric activation
of guanylate cyclase, the enzyme that synthesizes cGMP, by small calcium-
binding proteins termed guanylate-cyclase-activating proteins (GCAPs) (Yau
1991; Yau and Hardie 2009). In other GPCR signaling systems, calcium
feedback acts on a wide variety of cellular effectors.
Ion channels are distinguished from multicomponent signaling cascades
by the rapidity of their response, enabling ionotropic receptors to convert
stimuli into neuronal depolarization on a millisecond timescale, as compared
with tens or hundreds of milliseconds for most metabotropic systems (Fig. 2).
This is especially relevant when the physiological timescale of stimulus
presentation is rapid, as in acoustic signals. In this case, sound pressure
waves with vibration frequencies on the order of several thousand cycles per
second (or, for bats, up to 100,000 cycles per second) are detected by
mechanically gated ion channels with open probabilities that are modulated
by the rapid back-and-forth movements of a tightly interconnected set of
microvilli, the stereociliary bundle (Fig. 1D). In this system, adaptation is
effected by dynamic changes in the tension in the elastic elements that gate
the channels.
Ion channels also have a great capacity for signal integration and gain
control. This phenomenon is nicely illustrated in the somatosensory system,
where the capsaicin- and heat-activated receptor TRPV1 is modulated by
multiple components of the inflammatory milieu, including bioactive lipids,
extracellular protons, neurotrophic factors, and inflammatory peptides
(Fig. 1E). These agents enhance the sensitivity of TRPV1 to heat by
functioning either directly as allosteric modulators of the channel or
indirectly through metabotropic pathways that modulate TRPV1 channel
function. These actions contribute to heightened pain sensitivity following
tissue injury, such as sunburn, and constitute an important part of the pain
pathway’s protective function (Caterina and Julius 2001).
One outstanding question in sensory transduction concerns the molecular
mechanisms used by cells to detect and respond to mechanical stimuli (focal
pressure, stretch, and osmotic challenge) (Gillespie and Walker 2001; Kung
2005). To date, this is best understood in prokaryotic systems, in which
potentially lethal hypotonic shock leads to the activation of both small- and
large-conductance cell surface mechanosensory channels, MscS and MscL,
respectively, that equalize solute gradients by conducting anions, cations, and
other small molecules relatively nonselectively. Reconstitution of purified
MscS and MscL proteins in synthetic lipid bilayers has shown that these
channels are intrinsically mechanosensitive, opening and closing in direct
response to changes in lateral membrane pressure (Sukharev et al. 1994;
Sukharev 2002; Vásquez et al. 2008; Kung et al. 2010).
A more complex model has been proposed for mechanosensory
transduction in metazoan systems, based on genetic studies in C. elegans.
Here, screens for touch-insensitive mutants have identified loci encoding
microtubule-associated proteins as well as members of the amiloride-
sensitive sodium channel family, arguing for the existence of a
mechanosensory complex in which membrane stretch promotes channel
opening via cytoskeletal changes (Chalfie 2009). In contrast to the single-
component bacterial Msc system, this model has not yet been fully validated
through functional reconstitution in heterologous systems. Genetic and
physiologic studies in flies and mammals have identified several additional
candidates for mechanotransducers, including members of the TRP and Piezo
channel families. Whether these channels respond to mechanical stimuli
directly, as in bacteria, or indirectly through membrane/cytoskeletal
attachment, as in nematodes, remains to be determined.

3 STRUCTURAL BASIS OF SENSORY RECEPTOR

ACTIVATION
Among eukaryotic sensory receptors, we currently know most about the
structure of GPCRs and how they interact with ligands, G proteins, and
arrestins. Much insight has been gleaned from studies of rhodopsin and the β-
adrenergic and adenosine receptors, for which three-dimensional (3D)
structures of active and inactive conformational states have recently been
determined (Rosenbaum et al. 2009; Choe et al. 2011; Rasmussen et al. 2011;
Standfuss et al. 2011; Xu et al. 2011). Crystallographic studies confirm that
these receptors contain a membrane-embedded core of seven transmembrane
α-helices with amino- and carboxy-terminal tails facing outside and inside the
cell, respectively. We note that that in the case of rhodopsin, the amino
terminus faces the lumen of the outer segment disc, which is topologically
equivalent to the outside of the cell.
Binding of low-molecular-weight agonists and antagonists to the β-
adrenergic and adenosine receptors, or light-induced isomerization of
covalently bound retinal in rhodopsin, occurs within a pocket that is formed
by four of the seven α-helices and is located below the surface of the plasma
membrane (Fig. 3A). Subtle agonist-induced structural changes within this
pocket promote rotational movements in hinge regions of helices 5 and 6 that
sit at the midpoint of the lipid bilayer. This motion, in turn, exposes a
hydrophobic G-protein-binding site formed by loops linking the cytoplasmic
ends of transmembrane domains 5, 6, and 7.
Figure 3. 3D structures of rhodopsin and a mechanically gated ion channel. (A) Ribbon diagram of
rhodopsin in the inactive state with bound 11-cis-retinal (red spheres) compared with the light-activated
Metarhodopsin II state containing all-trans-retinal (blue spheres) and Metarhodopsin II state in
complex with a peptide (pink) corresponding to the receptor-binding site on the Gα subunit of
transducin. Rotation and elongation of light-activated retinal lead to a slight rotational tilt of
transmembrane helices 5 and 6, thereby enlarging a crevice at the cytoplasmic side of the receptor in
which Gα can dock (Choe et al. 2011). (B) Bacterial mechanosensory ion channel, MscL, showing
transmembrane topology of a monomer (top left) and the assembled pentameric channel complex (top
middle). Membrane stretch and consequent changes in tension at the membrane–protein interface
produce structural rearrangements that result in compression of the channel and expansion of the
central ion permeation pore, as depicted in the side and top views (Kung 2005).

Crystallographic and molecular dynamics simulations suggest that

interactions with both ligand and G protein are required to fully stabilize the
activated conformation of some GPCRs. This is consistent with classic
pharmacological observations showing that agonist affinities decrease
substantially when the G-protein is released. Interestingly, the conformational
equilibrium between inactive and active states differs among GPCRs. Some
receptors, such as the β2-adrenergic receptor, make occasional transitions to
the activated state in the absence of ligand. In contrast, rhodopsin remains
almost completely inactive in the absence of retinal isomerization, thereby
maintaining the low level of dark noise that is characteristic of vertebrate rod
phototransduction.
Much less is currently known about the tertiary structure of most ion
channels that function as sensory receptors. Bacterial MscS and MscL
mechanosensory channels are the exception: their crystal structures have been
resolved to approximately 4 Å resolution (Perozo and Rees 2003; Kung et al.
2010). These stretch-activated channels assume two rather different overall
configurations. MscS is a homoheptameric channel in which the seven
subunits are arranged around a central axis. The third transmembrane helix of
each subunit lines the ion-conducting pore, whereas the other two helices
form a cone-shaped outer shell that interacts with membrane lipids. MscL is a
homopentameric channel in which each subunit has two transmembrane
helices, one facing the ion pore and the other facing the lipid bilayer. Despite
their distinct stoichiometries and architectures, MscS and MscL perform the
same function of opening and closing in response to changes in lateral
pressure or surface tension exerted by the surrounding lipid bilayer as it
undergoes deformation. Structural and molecular dynamics simulation
studies suggest that in response to membrane stretching, both channels
undergo rotational and kinking movements of their transmembrane helices to
open a central ion-conducting pore, much as an iris opens in an old-fashioned
camera (Fig. 3B).
At present, structural insights into metazoan sensory channel function are
limited. For TRP channels, progress is currently limited to high-resolution
structures of small soluble domains and low-resolution (∼20 Å) structures of
the intact channel derived from electron microscopic images (Malnic et al.
1999; Gaudet 2008; Li et al. 2011). How some TRP channels, such as
TRPV1 and TRPM8, detect and respond to changes in ambient temperature
remains an intriguing and unresolved question. Heterologous expression and
in vitro proteoliposome reconstitution studies suggest that TRPV1 and
TRPM8 are intrinsically temperature-sensitive, but whether gating in vivo
involves channel–lipid interactions or association with other cellular factors
remains uncertain. Mutational studies have implicated several channel
domains as being important in temperature detection or specification of
thermal activation thresholds, but the structural underpinnings of these
processes have yet to be elucidated.

4 THE LOGIC OF SENSORY CODING

Sensory systems create an internal representation of the external world
filtered through the molecular specificities of primary receptor proteins.
Thus, the act of sensory coding can be conceptualized as an act of remapping:
One multidimensional space (of sensory stimuli) is remapped onto another
multidimensional space (of receptor cell responses). We focus here only on
the representation of sensory stimuli at the level of the primary receptors, but
note that the remapping process continues at each stage of sensory processing
up to and including brain circuitry. For example, in the visual system, the
distinct attributes of form, color, motion, and depth are extracted from the
retinal image and processed in partially overlapping information streams in
the visual cortex (Livingstone and Hubel 1988).
In bacteria, plants, and animals, light-sensing systems are based on the
photoisomerization of a chromophore with a conjugated π-electron system:
retinal or one of its derivatives in bacterial and animal rhodopsins (Fig. 4),
and a tetrapyrrole in plant phytochromes. Although the energy difference
between ground and excited electronic states is quantized, the receptor’s
absorbance spectrum (i.e., absorbance as a function of wavelength) appears
as a relatively broad and approximately Gaussian curve. The breadth of the
absorbance band arises from the large number of closely spaced vibrational
energy states that are superimposed on the larger electronic energy gap
(Abrahamson and Japar 1972). These broad absorbance bands permit a
relatively small number of photoreceptors with partially overlapping spectral
sensitivities to cover the biologically relevant region of the electromagnetic
spectrum, the interval from near ultraviolet (∼350 nm) to far red (650 nm). In
humans, there are five classes of light receptors: cone photoreceptors have
sensitivity maxima of ∼440 nm, ∼530 nm, and ∼560 nm; rod photoreceptors
have sensitivity maxima of ∼500 nm; and intrinsically photosensitive retinal
ganglion cells—which mediate pupil constriction and light entrainment of
circadian rhythms—express a highly divergent photoreceptor protein,
melanopsin, that confers a sensitivity maximum of ∼480 nm. The differential
excitation of the three cone pigments provides information about the
wavelength composition of a stimulus, a process that we recognize as color
vision (Fig. 4).
Figure 4. Color vision in humans and honeybees. (A) Photoisomerization of retinal from 11-cis to all-
trans, the photochemical event that initiates receptor activation in vertebrate and invertebrate
photoreceptors. (B) Spectral sensitivities of human cone photoreceptors and honeybee rhabdomeric
photoreceptors. (Adapted from Osorio and Vorobyev 2008; reprinted, with permission, from Elsevier ©
2008.)

The strategy of overlapping spectral sensitivities was first suggested by

Thomas Young in a remarkably prescient passage in his 1801 Bakerian
Lecture before the Royal Society:
As it is almost impossible to conceive each sensitive point of the retina to contain an infinite
number of particles (receptors), each capable of vibrating in perfect unison with every
possible undulation (frequency), it becomes necessary to suppose the number limited … and
that each particle is capable of being put in motion more or less forcibly by undulations
differing less or more from perfect unison (Young 1802).
Following Young’s argument, we could measure light intensity as a function
of wavelength from a particular location in a scene, sampling from 400 nm to
650 nm in 1-nm steps, and then represent the data by a single point in a 250-
dimensional space. Young correctly surmised that the visual system remaps
this high-dimensional stimulus space onto a lower-dimensional space of
receptor activities.
One inevitable result of such remapping is that some pairs of points that
reside at distinct locations in the higher-dimensional stimulus space will
reside at indistinguishably close locations in the lower-dimensional receptor
space. For color vision based on only three or four classes of receptors,
surprisingly little information is lost relative to that which could be extracted
by a larger ensemble of receptors. The reason for this is that the biological
pigments that dominate natural scenes—like the chromophores of
photoreceptors—have broad and relatively smooth bell-shaped absorbance
curves. (Recall that the visual stimulus, i.e., the light reflected from an object,
is proportional to the reciprocal of the absorbance spectrum.) In other words,
the information content in the absorbance spectra of biomolecules is
contained largely in their low-frequency components, with the word
“frequency” referring not to a particular wavelength of light but to a Fourier
decomposition of the curve of reflectance versus wavelength (Maloney
1986).
A simpler transformation between stimulus and response spaces is
effected by taste receptors (Yarmolinsky et al. 2009). In mammals, sweet,
umami (amino acid), and bitter tastants stimulate GPCRs, whereas sodium
and hydrogen ions (i.e., salty and acidic modalities) are detected by members
of the amiloride-sensitive and TRP ion channel families, respectively. Two
classes of specialized taste cells express low-affinity GPCRs to detect
common nutrients: T1R1-T1R3 heterodimers detect L-amino acids, and
T1R2-T1R3 heterodimers detect sugars. The relatively low ligand–receptor
affinities (in the millimolar range) are appropriate given the organism’s
interest in identifying quantities of ligand sufficient for its nutritional needs.
The umami and sweet taste receptors are of additional interest because,
together with the gamma-aminobutyric acid (GABA)-B receptor, they
represent the best-validated examples in which receptor dimerization is
required for G-protein signaling (Milligan 2009).
A distinct class of taste receptors coexpress a mixture of about 30 high-
affinity GPCRs (T2Rs) that recognize a broad array of bitter compounds,
many of which are plant-derived toxins. The uniformly bitter sensation
elicited by diverse T2R ligands attests to the compression of a
multidimensional chemical stimulus space onto a single psychophysical
dimension of bitterness—a logical feature given that the only behavioral
output is aversion. This arrangement sacrifices discriminatory power, but it
enables an organism to determine whether a substance is nutritionally
beneficial (eliciting an attractive response) or potentially toxic (eliciting an
aversive response).
Another apparently simple transformation between stimulus and response
spaces is seen in mammalian thermosensation (Lumpkin and Caterina 2007).
In this case, a scalar quantity, temperature, is detected by largely distinct sets
of sensory neurons, each of which expresses one type of temperature-gated
TRP channel. The critical features of this system are (1) the polarity of the
response—channel opening that is either heat-activated (TRPV1) or cold-
activated (TRPM8); (2) the temperature at the midpoint of the S-shaped
stimulus–response curve; and (3) the steepness of the stimulus–response
curve. The last of these features may reflect cooperative interactions among
TRP channel subunits that line a central pore. For each class of
thermosensory neurons, the monotonic mapping of stimulus temperature to
response shows systematic compressions and expansions along the
temperature axis, with maximal sensitivity to small changes in temperature
occurring, as one would expect, in the steepest region of the receptor’s
stimulus–response curve. For mammals, the intervals of maximal sensitivity
flank normal peripheral body temperatures: ∼15°C to ∼25°C for cold
receptors and ∼35°C to ∼50°C for heat receptors (Iggo 1982). In keeping with
this pattern, the temperature of the half-maximal response of TRPM8
orthologs differs among species and correlates with core body temperature
(Myers et al. 2009).
In reality, the situation is more complex than the preceding paragraph
indicates because, as noted above, thermosensory neurons integrate
additional stimulus dimensions by virtue of the sensitivity of various TRP
channels to chemical ligands and by the sensitizing action of inflammatory
mediators at the cellular level (Basbaum et al. 2009). Many TRP channel
activators are plant-derived compounds that provoke a sensation of irritation
or burning pain. Primary afferent neurons coexpressing capsaicin (TRPV1)
and wasabi (TRPA1) receptors are dually sensitive to heat and electrophilic
irritants, a mixed-modality arrangement wherein chemical irritants and
inflammatory agents excite heat-sensitive fibers to produce thermal
hyperalgesia (increased sensitivity to pain). This phenomenon of cross-
modality signaling appears as a distinctive feature of somatosensation,
presumably reflecting the protective function of the pain pathway. Thus,
thermosensory neurons provide the organism with a sensory space in which
noxious chemical and thermal stimuli are integrated with tissue injury and
inflammation to yield gradations of two relatively simple sensations, pain and
temperature.
Olfactory systems illustrate the most complex stimulus–response
relationships. This complexity reflects the extraordinary diversity of chemical
space and the large number of distinct odorant receptors (∼100 to ∼1000)
expressed in vertebrates and invertebrates (Su et al. 2009). The most naive
mapping would assign each odorant an independent axis in stimulus space
and each receptor an independent axis in response space. Even if the two
spaces are compressed by combining the attributes of related compounds and
related receptors into a smaller number of axes—a method referred to as
principal component analysis—the dimensionality of each space remains
extremely large.
Systematic analyses in which a transgenic receptor is expressed in a single
neuron from which the endogenous receptor has been eliminated (the “empty
neuron” technique) have delineated odorant responses for the entire olfactory
repertoire in fruit flies and other insects (Hallem and Carlson 2006; Carey et
al. 2010). Similarly, taking advantage of the one-receptor–one-neuron
relationship in the main olfactory epithelium of mammals, researchers have
used calcium imaging during odorant exposure followed by single-cell PCR
to delineate basic patterns of odorant receptor specificities (Malnic et al.
1999). Several fundamental observations have emerged from these studies
(Malnic et al. 1999; Hallem et al. 2004; Hallem and Carlson 2006; Carey et
al. 2010; Wang et al. 2010): First, individual odorants typically activate more
than one receptor, and, conversely, individual receptors are typically
activated by more than one odorant. Second, receptors differ in the breadth of
their odorant responses: Some receptors are activated by a small number of
odorants, whereas others are activated by a larger number of odorants, often
with related chemical structures. Third, both the level of receptor activation
and the number of classes of activated receptors increase with increasing
odorant concentration. Organisms face the further challenge of interpreting
mixtures of odorants, and for this task, additional identifying information can
be obtained based on antagonistic interactions between pairs of odorants and
on odorant-specific temporal dynamics of receptor responses (Hallem et al.
2004; Oka et al. 2004, 2009; DasGupta and Wadell 2008; Su et al. 2011).

5 EVOLUTION AND VARIATION

The strongest selective pressure for optimal performance in sensory systems
occurs when the stimulus affects behaviors most directly related to Darwinian
selection: feeding, mating, and avoiding death from predation, poisoning, and
so on. Conversely, sensory systems that gather information that is of little or
no utility will disappear over time, a process recognizable by the loss or
inactivation of their associated gene sequences. As the following examples
illustrate, these considerations inform any comparisons among species of the
performance characteristics of sensory systems.
Variations among different types of photoreceptors, both within and
between species, beautifully illustrate the evolution of sensory system
performance to fit diverse ecological needs (Yau and Hardie 2009). For
example, different signal-to-noise ratios are apparent in rod-mediated night
vision (where the ratio must be high) compared with cone-mediated daytime
vision (where the ratio can be lower). The rod visual pigment, rhodopsin, has
a half-life for spontaneous activation at 37°C of ∼400 yr, corresponding to an
energy barrier of ∼22 kcal/mol for thermal isomerization of the 11-cis retinal
chromophore (Baylor et al. 1980). In mammalian rods, which have 4 × 107
rhodopsins per cell, this works out to only approximately one spontaneous
activation event per minute per rod. This very low level of receptor noise
represents a critical performance feature that sets the absolute sensitivity of
dim light vision (Hecht et al. 1942; Baylor et al. 1980). In contrast,
mammalian cones operate at light levels that are several orders of magnitude
higher than the rod operating range, and cones are correspondingly several
orders of magnitude noisier than rods (Schnapf et al. 1990; Schneeweis and
Schnapf 1999).
Visual pigment spectral sensitivity is one of the most intensively studied
systems in which sensory receptor evolution has been explored at the
molecular, organismal, and ecological levels. In the ocean, chlorophyll and
other biomolecules in photosynthetic microorganisms selectively deplete
longer-wavelength sunlight, and, as a result, there is a corresponding
blueshift in the visual pigment spectral sensitivities of fish that live at greater
ocean depths (Lythgoe 1979). Similarly, dolphin rhodopsin and the dolphin
long-wavelength cone pigment are blueshifted relative to their terrestrial
counterparts as an adaptation to the aquatic environment (Fasick et al. 1998;
Fasick and Robinson 2000). At extreme depths, where little sunlight
penetrates, visual pigment spectral sensitivities are under entirely different
selective pressures: Bioluminescence permits communication among
organisms of the same species, and visual pigments are tuned to the emitting
wavelengths, in some cases at the far-red end of the spectrum (Douglas et al.
1998).
Visual pigment spectral tuning has also been studied in relation to
discrimination among natural objects that are behaviorally relevant (Osorio
and Vorobyev 2008). Comparing the color space defined by honeybee
rhabdomeric photoreceptors, which have sensitivity maxima at ∼350, ∼425,
and ∼550 nm, with that defined by human cone photoreceptors, which have
sensitivity maxima of ∼440, ∼530, and ∼560 nm (representative of Old World
primate cone sensitivities) (Fig. 4), reveals a wider dispersion of floral hues
in honeybee color space compared with primate color space (Fig. 5). This
pattern supports the general idea that color vision in pollinator species such
as honeybees, butterflies, and hummingbirds coevolved with floral pigments
to enhance discrimination among floral species, an arrangement that enhances
both feeding and pollination. Similarly, the surface hues of fruits that are
consumed by primates occupy a wider swath of primate color space
compared with honeybee color space (Fig. 5), a pattern that suggests that
primate trichromacy may have coevolved with fruit coloration to enhance
both fruit consumption and seed dispersal (Regan et al. 2001). In particular,
yellow, orange, or red fruit is readily detected against a background of
dappled green foliage if an animal compares the extents of excitation of a pair
of visual pigments in the 500–600-nm region of the spectrum, as nearly all
Old World and some New World primates can (Mollon 1989; Regan et al.
2001). Such discrimination is difficult with the single longer-wavelength-
sensitive pigment typical of non-primate mammals. A chromatic
discrimination task similar to this one is the basis of the Ishihara test for color
vision deficiency in humans.

Figure 5. Chromaticity diagrams for humans and honeybees. The triangles represent a plane within a
3D receptor space, with each vertex corresponding to the point at which the plane intersects an axis
representing the degree of excitation of one receptor type. (S) Short-wavelength receptor axis; (M)
medium-wavelength receptor axis; (L) long-wavelength receptor axis. The small black circles within
the triangles represent the chromaticities of a set of fruits that are consumed by primates (upper panels)
or a set of flower petals (lower panels). The line within each chromaticity diagrams represents the locus
of spectrally pure lights, with black circles and the adjacent numbers marking steps of 50 nm. (Adapted
from Osorio and Vorobyev 2008; reprinted, with permission, from Elsevier © 2008.)

Fruit flies and mosquitoes provide an analogous instance of species-

specific differences in the patterning of salient odorants in olfactory receptor
space. Drosophila melanogaster feeds on ripe or rotting fruit and appears to
be especially good at discriminating among esters, the dominant volatiles
emitted by fruit (Su et al. 2009; Carey et al. 2010). In contrast, Anopheles
gambiae feeds on human blood and is better at discriminating aromatics,
including several that are characteristic of humans (Carey et al. 2010). High
salience is also seen in the heterodimeric carbon dioxide receptors of
Drosophila and Anopheles. In Drosophila, this odorant represents a stress
signal, whereas in Anopheles, it is one of the chemotactic signals used to
identify a human host (Jones et al. 2007; Lu et al. 2007). Finally, receptors
specific for within-species olfactory communication, most especially in the
context of pheromones, have been identified in both vertebrates and
invertebrates (Touhara and Vosshall 2009). In Drosophila, cis-vaccenyl
acetate (cVA) is produced by males, increases female receptivity, and then—
upon transfer to the female partner during mating—decreases female
attractiveness to other males. Interestingly, the responses to cVA are
mediated through at least two odorant receptors (Or67d and Or65a), a two-
transmembrane-domain coreceptor (SNMP), and a soluble odorant-binding
protein (LUSH) (Vosshall 2008).
As noted in the discussion above on primate color vision and fruit
consumption, an immobile plant has much to gain if it can encourage animal
foragers to help spread its seeds. Avian foragers present the best opportunity
for widespread seed dispersal, and, therefore, one might expect that plants
and the birds that disperse their seeds would have coevolved a strategy that
discourages competition from less desirable foragers such as mammals. A
striking example of this phenomenon is seen in the insensitivity of the avian
vanilloid thermosensory channel (TRPV1) to activation by capsaicin, which
is the source of the painfully “hot” sensation elicited by chili peppers (Jordt
and Julius 2002). Capsaicin thus appears to selectively repel mammals by
activating their temperature/pain receptors, thereby preserving the pepper
seeds for consumption and transport by birds. The avoidance or non-
avoidance of food constitutes a major point of intersection between animal
and plant ecologies. In this respect, it is interesting that the vertebrate bitter
receptor (T2R) gene repertoire—which represents a sum of all undesirable
edible compounds, many of which are plant-derived—is evolving rapidly
(Dong et al. 2009).
The most extreme change in sensory signaling is a complete loss of
receptor function. In the genomes of the bushbaby (Otolemur crassicaudatus)
and owl monkey (Aotus trivirgatus)—primates with predominantly nocturnal
lifestyles—the shortwave-sensitive cone pigment gene has decayed into a
pseudogene, leaving only a single class of longer-wavelength cones to
mediate daytime vision, with no possibility of color vision (Jacobs et al.
1996; Kawamura and Kubotera 2004). Similarly, in humans, all of the
sequences coding for vomeronasal receptors of the V2R class, which mediate
pheromone sensing in rodents, are pseudogenes (Touhara and Vosshall
2009). Among mammalian genes that encode the principal receptors of the
main olfactory epithelium, the fraction that are pseudogenes ranges from
∼15% to ∼80%, depending on the species (Niimura and Nei 2007; Niimura
2009; Touhara and Vosshall 2009). In dolphins, all genes encoding class II
receptors of the main olfactory epithelium, which are mostly specialized for
volatile hydrophobic ligands, appear to be inactivated by mutation (Freitag et
al. 1998; Niimura 2009).
Any discussion of sensory signaling would be incomplete without
considering the evolution of specialized anatomic structures that facilitate
sensory function. The frequency-dependent conversion of sound pressure
waves to localized basilar membrane distortions in the cochlea represents a
dramatic example of these auxiliary structures in action. This micro-
mechanical system is remarkably sensitive: the threshold for detecting basilar
membrane displacement is <1 nm (Johnstone et al. 1986). A larger-scale
anatomic specialization is the increase in head width in hammerhead sharks.
The great distance between left and right nasal openings, together with the
evolution of olfactory receptor neurons that have sub-nanomolar affinities for
amino acids, allows the hammerhead to sense shallow concentration
gradients of dilute amino acids by comparing the excitation of left and right
olfactory sensory epithelia (Tricas et al. 2009; Gardiner and Atema 2010).
This spatial differencing strategy is the olfactory analog of binaural sound
localization in mammals.
A particularly striking instance of anatomic specialization is seen in the
pit organs of vipers such as rattlesnakes. Stretched across the pit is an
extremely thin tissue that is densely innervated by thermosensory fibers. The
fibers express a TRP channel that senses the minute increase in temperature
evoked by infrared radiation from a nearby warm-blooded animal (Gracheva
et al. 2010). The low body temperature of the viper relative to its prey and the
small heat capacity of the sensory tissue are important for optimal pit organ
function. Recent calculations suggest that the sensory fibers of the pit organ
can respond to temperature changes as small as 0.001°C (Bakken and
Krochmal 2007).

6 CONCLUDING REMARKS
The sensory receptors and signaling systems described here represent only a
small sampling of the many that have been investigated. Because of space
limitations, we have not discussed plant sensory systems, and we have only
briefly touched on microbial systems. Even with this limited sampling, the
diversity of sensory systems is striking, and it is evident that each lifestyle
and ecological niche is accompanied by a distinctive set of adaptations in
sensory system structure and function.
Over the past 30 years, the identities and primary structures of most of the
major classes of vertebrate sensory receptor proteins have been defined. The
one exception is the mechanosensory channels in the auditory and vestibular
system, which remain enigmatic. The signaling cascades downstream from
GPCR-type sensory receptors have also been largely defined. In vertebrate
photoreception, the best studied of all GPCR signaling cascades, it is likely
that all of the components involved in signaling and adaptation are now
known. In less experimentally accessible systems, such as the taste and
vomeronasal systems, additional signaling components remain to be
identified, and the interactions between signal activation pathways and
feedback loops are still incompletely understood.
A full molecular understanding of sensory receptor function requires the
3D structures of receptors and signaling components in their various active
and inactive conformations, and, in some cases, in complex with each other.
This has been achieved for rhodopsin, transducin, and the bacterial MscL
mechanosensory channel, and it is an area of active investigation for other
classes of sensory receptors and their downstream effectors. Structural
studies can be expected to play a critical role in elucidating the molecular
basis of receptor–ligand specificity in chemosensory systems.
A further challenge in the field of sensory biology comes from the need to
diagnose and treat diseases that affect sensory signaling, including those
associated with chronic pain or the loss of vision or hearing. Understanding
sensory signaling at the molecular and cellular levels will inform these
clinical investigations and will continue to be one of nature’s grand scientific
challenges for biologists, chemists, physicists, and engineers.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005991
CHAPTER 12

Synaptic Signaling in Learning and

Memory

Mary B. Kennedy
Division of Biology and Biological Engineering, California Institute of
Technology, Pasadena, California 91125
Correspondence: kennedym@its.caltech.edu

SUMMARY

Learning and memory require the formation of new neural networks in

the brain. A key mechanism underlying this process is synaptic plasticity
at excitatory synapses, which connect neurons into networks. Excitatory
synaptic transmission happens when glutamate, the excitatory
neurotransmitter, activates receptors on the postsynaptic neuron.
Synaptic plasticity is a higher-level process in which the strength of
excitatory synapses is altered in response to the pattern of activity at the
synapse. It is initiated in the postsynaptic compartment, where the
precise pattern of influx of calcium through activated glutamate
receptors leads either to the addition of new receptors and enlargement
of the synapse (long-term potentiation) or the removal of receptors and
shrinkage of the synapse (long-term depression). Calcium/calmodulin-
regulated enzymes and small GTPases collaborate to control this highly
tuned mechanism.
Outline
1 Introduction
2 Spine synapses
3 Signaling molecules in spine synapses
4 Regulation of the number of AMPARs in a synapse
5 The role of small GTPases in synaptic plasticity
6 Concluding remarks
References

1 INTRODUCTION
The function of the brain is to process and store information about the
environment and direct behavior in response to that information. Three major
cell types in the brain—excitatory neurons that use glutamate as their
transmitter, inhibitory neurons that use γ-aminobutyric acid (GABA) as their
transmitter, and glial cells—work together to respond to the environment
while maintaining the overall connectivity among neurons within an
acceptable homeostatic range. Excitatory neurons, the most numerous in the
brain, each receive thousands of synaptic inputs and, in turn, make thousands
of synaptic connections onto other neurons. A human brain contains, on
average, 86 billion neurons (Herculano-Houzel 2009) that in toto make
trillions of synaptic connections.
A typical cortical excitatory neuron (Fig. 1) comprises a neuronal soma
(cell body), several branched dendrites, and a single axon that can extend for
many millimeters and often branches to make thousands of individual
synaptic connections. The soma is the site of the nucleus and most of the
neuron’s protein synthetic machinery. Most inhibitory synaptic contacts
occur on the somal plasma membrane. In contrast, the highly branched
dendrites receive most of the excitatory synaptic contacts, which are made
onto small membrane protuberances called dendritic spines (Fig. 1). When
the neuronal membrane becomes depolarized to a threshold level, an action
potential is initiated at the base of the axon near the soma; this wave of
depolarization travels unabated to each of the thousands of presynaptic
endings along the axon. Depolarization causes membranous synaptic vesicles
within the presynaptic terminals to fuse with the plasma membrane at the
“active zone” opposite the postsynaptic site and flood the synaptic cleft with
neurotransmitters. Some of the transmitter molecules bind to specific
receptors, which are ligand-gated channels in the postsynaptic membrane.
Sodium and potassium ions flow through the channels of the activated
receptors, decreasing the gradient in their concentration across the membrane
and thus producing a localized depolarization called an excitatory
postsynaptic potential (EPSP). If the synapse fires repeatedly, or if several
different synapses on a neuron fire at the same time, the EPSPs can sum to
produce a depolarization that extends to the soma and initiates an action
potential.

Figure 1. Features of excitatory neurons in the brain. The cell body (soma) of a typical excitatory
pyramidal neuron is ∼10 μm in diameter and is located in one of several sheets of tightly packed somas
that define the layers of the neocortex and hippocampus. Apical and basal dendrites extend from the
soma, reaching into adjacent areas that are referred to as neuropil. Postsynaptic structures are located in
tiny membrane protuberances called spines that can be seen along the dendrites. Each soma gives rise
to one axon, which has a smaller diameter than the numerous dendrites. The axon can extend for
millimeters from the soma and branches to form thousands of presynaptic terminals where transmitter
is released onto the postsynaptic sites of other neurons. The axon hillock is located at the base of the
axon. Action potentials are usually initiated at this site; they travel along the axon (arrows) to
presynaptic terminals keeping a uniform amplitude of depolarization. Back-propagating action
potentials travel in the opposite direction through the soma and into the dendrites. The size of their
depolarization decreases as they travel and is regulated by the composition of dendritic ion channels.
(From Peters and Kaiserman-Abramof 1970; modified, with permission, © Wiley.)

Information is stored when individual synapses that connect a particular

group of neurons become more able (or less able) to generate an action
potential in the postsynaptic neuron in response to environmental signals.
Memories are stored initially in the hippocampus, where synapses among
excitatory neurons begin to form new circuits within seconds of the events to
be remembered. An increase in the strength of a relatively small number of
synapses can bind connected neurons into a circuit that stores a new memory.
A deceptively simple principle guides the direction and amplitude of this
synaptic plasticity: neurons that fire together, wire together. When release of
transmitter at a synapse is repeatedly correlated with firing of action
potentials in the postsynaptic neuron, they become stronger. In contrast, when
release of transmitter at a synapse repeatedly fails to correlate with
postsynaptic firing, because the resulting EPSPs do not sum to produce the
required threshold depolarization, they become gradually weaker and may
disappear altogether. Essentially all of the glutamatergic synapses between
excitatory neurons in the hippocampus and cortex of the mammalian brain
display this behavior. Such synapses are referred to as Hebbian synapses,
after Donald Hebb, who first suggested a similar principle in 1949.
Nonglutamatergic synapses do not display this Hebbian behavior. The unique
plasticity of excitatory glutamatergic synapses is an essential mechanism of
memory formation. Glutamatergic synaptic plasticity, and thus memory
formation, can be modulated by release of the inhibitory transmitter GABA
and by the influence of acetylcholine, biogenic amines, small peptides, and
larger protein hormones released from neurons and glial cells; but synapses
that release these other transmitters do not display Hebbian behavior.
The ongoing pattern of electrical activity through Hebbian synapses
influences cellular processes in the postsynaptic neuron at many time scales.
In a few seconds, changes can be triggered in the structure of the postsynapse
itself. Over minutes, the summation of synaptic activity can result in
increased levels of the classical second messengers cAMP and calcium and
activation of the mTOR and MAPK pathways (Kennedy et al. 2005), leading
to up-regulation of translation of mRNAs stored in the dendritic shaft near
active synapses (Ho et al. 2011). Local translation is believed to provide
proteins needed for remodeling of synapses and dendrites in response to high
synaptic activity. Over a few hours, activity-dependent nuclear transcription
factors stored near the synapse can become activated and travel from the
synapse into the nucleus (Flavell and Greenberg 2008; Ch’ng and Martin
2011). These changes in dendritic protein synthesis and in nuclear
transcription can influence the structure of the neuron and its role in neuronal
networks for hours, days, or a lifetime. For example, new ion channels may
be transcribed and inserted into the membrane to change the intrinsic
electrical firing pattern of the neuron, or the overall production of excitatory
receptors may be dampened to maintain homeostatic balance.
Here I focus on the specialized machinery that underlies Hebbian
behavior of synapses between excitatory neurons. At this time, signal
transduction involved in memory formation is best understood, although still
incompletely, for the early phases of plasticity.

2 SPINE SYNAPSES
In the brain, most synapses between excitatory neurons are located on spines,
tiny compartments that protrude from the neuron’s highly branched dendrites
(Fig. 2). A typical excitatory pyramidal neuron in the hippocampus or cortex
has ∼10,000 such synapses, most spines hosting just a single synapse. Spines
vary in size from ∼0.5–2 µm in length, from ∼0.25–1 µm in width, and from
∼10–100 attoliters in volume. The synaptic contact itself usually occurs at the
tip of the spine. It comprises the presynaptic active zone, the synaptic cleft,
and the postsynaptic receptor cluster and varies in diameter from ∼0.1 to 0.8
µm. Spines also vary in shape from stubby to thin to “mushroom-shaped.” In
general, the larger, mushroom-shaped spines contain stronger synapses.
Functionally, a stronger synapse is defined as one that contributes more
depolarization to the neuronal membrane upon activation than a weaker one;
thus, its activation is more likely to generate an action potential in the
postsynaptic neuron.

Figure 2. Synaptic plasticity. At the cellular level, one of the most essential elements of memory
formation is the adjustment in synaptic strength of excitatory synapses between neurons. AMPA-type
glutamate receptors (yellow) allow passage of sodium and potassium through their channel. Their
principal function is to depolarize the membrane, producing an excitatory postsynaptic potential
(EPSP). NMDA-type glutamate receptors (blue) also depolarize the membrane, but in addition to
sodium and potassium, calcium flows through their channel and can initiate synaptic plasticity. A long-
lasting increase in synaptic strength is referred to as long-term potentiation (LTP). LTP involves the
addition of new synaptic AMPA-type glutamate receptors (AMPARs) and an increase in the size of the
head of the postsynaptic spine, supported by an increase in the size and branching of the actin
cytoskeleton. Long-term depression (LTD) is a long-lasting decrease in synaptic strength that involves
a decrease in the number of synaptic AMPARs and shrinkage of the spine head. LTP is induced when
repeated firing of an action potential in the presynaptic terminal and the resulting release of glutamate
cause firing of action potentials in the postsynaptic neuron. LTD is induced when repeated firing of an
action potential in the presynaptic terminal does not cause firing of action potentials in the postsynaptic
neuron.

The postsynaptic membranes of spine synapses contain two distinct types

of ligand-gated channels that are receptors for the neurotransmitter glutamate
but are distinguishable by their ability to respond to pharmacological agents.
For 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA)-
type glutamate receptors (AMPARs), binding of glutamate triggers a small,
relatively rapid EPSP, resulting from an influx of potassium and sodium ions.
AMPA-type receptors produce an EPSP each time glutamate is released at a
synapse. The other class, N-methyl-D-aspartate (NMDA)-type glutamate
receptors (NMDARs), are evolutionarily related to AMPARs but are more
complex; they are responsible for the Hebbian behavior of spine synapses
(Mayer et al. 1984; Nowak et al. 1984). As I discuss in detail below, the
channel in NMDARs allows passage of calcium ions, as well as potassium
and sodium, and opens only if two conditions are met: glutamate is bound
and the synaptic membrane is strongly depolarized, as occurs when release of
transmitter is correlated with firing of action potentials in the postsynaptic
neuron.
Synaptic plasticity is most often studied by recording electrical responses
from synapses of the Schaffer-collateral pathway, which connects two
different sets of excitatory neurons in the hippocampus (Lüscher and
Malenka 2012). Repeated activation of this pathway, and thus its spine
synapses, at a frequency between ∼10 and 100 Hz for a few seconds usually
initiates a process referred to as long-term potentiation (LTP), in which the
activated synapses increase in size and more effectively depolarize the
postsynaptic membrane. In contrast, activation of these synapses at a lower
frequency, between ∼1 and 5 Hz, for several minutes usually initiates a
process called long-term depression (LTD), in which the activated synapses
decrease in size and less effectively depolarize the postsynaptic membrane.
The change in strength of the synapses can last for hours to a lifetime,
depending on how often the stimulation is repeated.
Intriguingly, although these processes have opposite effects on the
strength of the synapse, both are controlled by the influx of calcium caused
by activation of NMDARs. Differences in the timing and amount of calcium
entry into the spine through NMDARs account for the opposite outcomes
after stimulation of synapses in the two different frequency ranges (Franks
and Sejnowski 2002; Sjostrom and Nelson 2002). The higher frequency,
larger amplitude influx of calcium leads to two important molecular changes
in the spine synapse over the next 15–60 sec (Fig. 2). More AMPARs are
inserted into the postsynaptic membrane, increasing the size of the EPSP in
the spine upon subsequent synaptic activation. At the same time, the actin
cytoskeleton, which gives the spine its shape, is remodeled, producing a
larger and more branched cytoskeleton that supports a larger spine head. In
contrast, lower frequency, lower amplitude, but more prolonged influx of
calcium produces the opposite effect on the spine. The number of AMPARs
in the postsynaptic membrane is reduced, resulting in a smaller EPSP upon
subsequent activation, and the actin cytoskeleton shrinks, leaving a smaller
spine head. We do not yet know precisely how this delicate differential
regulation by calcium influx is achieved. However, much has been learned
about the molecular machinery that is responsible.
Although NMDARs initiate synaptic plasticity, their numbers are not
altered by the processes that alter AMPA receptor numbers during LTP or
LTD. A variety of modulatory agents in the brain can adjust the flux of
calcium through NMDARs (Salter and Kalia 2004) or the frequency range at
which the switch between LTP and LTD occurs, a process called
metaplasticity (Abraham and Bear 1996; Yang et al. 2012). Homeostatic
regulation can lead to slow changes in the steady-state levels of NMDARs
and AMPARs; however, these changes are not directly related to storage of
memories.

3 SIGNALING MOLECULES IN SPINE SYNAPSES

3.1 The NMDA-Type Glutamate Receptor and the Hebbian
Response
Both AMPARs and NMDARs are homologous tetramers arranged such that a
channel is formed by the intersection of their intramembrane domains (Mayer
2006). Binding of glutamate to two sites on the extracellular portion of the
receptor opens the channel. However, the similarity between AMPARs and
NMDARs ends there. Opening of the AMPAR channel produces the
predictable rapid EPSP (Seeburg 1993). In contrast, opening of the NMDAR
channel alone is not sufficient to produce depolarization because the mouth
of the channel is blocked by a bound magnesium ion, which acts like a cork
in a bottle. Ions can flow through the open channel of the NMDAR only if
the membrane is sufficiently depolarized to loosen the binding of the
magnesium ion and relieve the “magnesium block” (Ascher and Nowak
1988).
One event that can depolarize the spine membrane sufficiently is a “back-
propagating action potential,” a type of dendritic depolarization that was
discovered relatively recently (Spruston et al. 1995; Magee and Johnston
1997; Stuart et al. 1997; Magee et al. 1998). Axonal action potentials are
initiated near the neuronal cell body at the base of the axon, where the
threshold for triggering an action potential is lowest. They then propagate to
synapses at the end of the axon in a nondecremental fashion; that is, the size
of the depolarizing wave does not decrease as it moves along the axon.
Dendritic action potentials are also believed to begin at the base of the axon,
but they are decremental, decreasing in size as they back-propagate from the
base of the axon through the cell body and into the dendrites. The size of the
back-propagating potential and the length that it travels depend on the
configuration of potassium and sodium ion channels in each dendrite.
However, the depolarization produced can be sufficient to relieve the
magnesium block at spines along the dendrite. Therefore, the coincidence of
glutamate binding to receptors at a spine and the arrival of a back-
propagating action potential will allow the NMDAR channels to open.
Electrophysiologists still debate whether there are additional circumstances
associated with firing of the postsynaptic neuron that result in strong local
depolarization of dendrites during synaptic activity. Nonetheless, it is clear
that when a synapse repeatedly contributes to the triggering of postsynaptic
action potentials, NMDARs in that synapse will be powerfully activated.
 A second important difference between NMDARs and AMPARs is the
mixture of ions that flow through their channels. Most AMPARs only allow
passage of sodium and potassium ions, which produces depolarization. In
contrast, calcium passes through NMDAR channels along with sodium and
potassium (MacDermott et al. 1986) and acts as a second messenger in the
spine. NMDARs stay activated for several tens of milliseconds, during which
the channel flickers open in short bursts, partly because magnesium bounces
in and out of the mouth of the channel and partly because opening and
closing of any protein channel is stochastic in nature. As a result, calcium
flows into the spine in irregular bursts, for tens of milliseconds, and is rapidly
pumped out by calcium-ATPases and sodium/calcium exchangers (see p. 95
[Bootman 2012]). Proteins in the spine cytosol that are sensitive to calcium
are thus subjected to rapidly fluctuating levels of the ion that may never reach
a stable equilibrium as long as the channel is open. In contrast, AMPARs are
active for just a few milliseconds and produce only a brief, transient
depolarization.
Finally, NMDARs contain very long carboxy-terminal “tails” (∼600
residues in length) that extend into the cytosol and help to organize the
postsynaptic signaling machinery. In adults, the tetrameric NMDAR is
assembled from mixtures of five receptor subunit isoforms: GluN1, GluN2A,
GluN2B, GluN2C, and GluN2D (Kutsuwada et al. 1992; Monyer et al. 1992).
Each individual receptor contains two GluN1 subunits, which are necessary
for formation of the channel, and a pair of GluN2 subunits (Furukawa et al.
2005; Mayer 2006), which contribute the long cytosolic tails. The carboxy-
terminal tail of GluN1 is shorter (∼100–120 residues) compared with those of
the GluN2 subunits. In the hippocampus and cortex, the predominant GluN2
subunits are GluN2A and GluN2B (Monyer et al. 1994). Their 600-residue
tails associate with distinct but overlapping sets of signaling enzymes and
scaffold proteins (Foster et al. 2010). Both of them can associate with the
primary postsynaptic density scaffold protein PSD95 and with calcium-
/calmodulin-dependent protein kinase II (CaMKII); however, the affinity of
CaMKII for GluN2B is considerably higher than for GluN2A (Gardoni et al.
2001).
3.2 Calcium-Regulated Signaling Enzymes in the
Postsynaptic Density
The postsynaptic density (PSD) is the name that was given by electron
microscopists to a densely staining plaque of proteinaceous material attached
to the cytosolic face of the postsynaptic membrane opposite presynaptic
vesicle release sites at excitatory synapses. Subcellular fractionation and
biochemical analyses (see Kennedy 1997) revealed that it contains
specialized scaffold proteins that physically link NMDARs, AMPARs, and
signaling enzymes responsible for synaptic plasticity (Kennedy 2000; Sheng
and Kim 2011).
About eight calcium-sensitive enzymes reside in significant numbers in or
near the PSD, although some appear to have a more central role in synaptic
plasticity than others (Table 1). CaMKII and the phosphoprotein phosphatase
calcineurin (also known as PP2B) are required for NMDAR-dependent
induction of LTP and LTD, respectively. The others participate in signaling
pathways that can regulate synaptic function in response to modulatory
agents, such as acetylcholine, biogenic amines, or neuropeptides.

Table 1. Calcium-sensitive enzymes in or near the PSD

Calcium/CaM regulated
Calcium-/calmodulin-dependent protein kinase II (CaMKII)
Calcineurin (also known as protein phosphatase 2B)
Calcium/CaM-stimulated adenylyl cyclase (AC1)
Neuronal nitric oxide synthase (nNOS)
Calcium/CaM-dependent phosphodiesterase (PDE1)
Ras-GRF1/2
Calcium regulated
Protein kinase C (PKC)
Calpain protease

3.2.1 CaMKII
CaMKII makes up ∼1% of total protein in the forebrain and ∼2% in the
hippocampus (Bennett et al. 1983; Erondu and Kennedy 1985). These high
levels of expression, at least 10 times higher than those of other signaling
enzymes, are a specialization of excitatory neurons, which represent most of
the mass of the forebrain (Sik et al. 1998). CaMKII is present throughout the
cytosol of somas, axons, and dendrites, including spines in which it is present
both in the cytosol and the PSD (Kennedy et al. 1983; Chen et al. 2005; Khan
et al. 2012). Activation of synaptic NMDARs increases association of
CaMKII with spines and the PSD; however, the role and mechanism of this
translocation are still incompletely understood (Khan et al. 2012).
CaMKII is a complex holoenzyme, the structure of which has interesting
consequences for the dynamics of its activation by calcium/CaM (Fig. 3).
Each holoenzyme comprises 12 catalytic subunits held together by their
carboxy-terminal association domains (Bennett et al. 1983; Kolb et al. 1998;
Rosenberg et al. 2005, 2006). Mammalian genomes encode four highly
similar CaMKII subunits: α, β, γ, and δ (Gaertner et al. 2004). They can form
stable homo-oligomers or hetero-oligomers that contain differing numbers of
each isoform; the numbers depend on their relative rates of synthesis. Their
major sequence differences occur in the linker region between the catalytic
and association domains. Only the α and β subunits are highly expressed in
brain; and the α subunit is only expressed in neurons. In forebrain, the α:β
ratio is ∼3:1 (Bennett et al. 1983). Thus, the unusually high levels of CaMKII
in forebrain are primarily a result of the level of expression of the α subunit.

Figure 3. CaMKII. CaMKII is a ring of six dimers of calcium-/calmodulin (CaM)-activated catalytic

subunits. The subunits are bound together by a central “hub” structure (light orange) formed from the
carboxy-terminal association domains of each subunit. The inactive dimers (light and dark blue) are
docked against the central hub by interactions among helices in the association domains (red) and
residues in their inhibitory domains (light yellow). Binding of activated calmodulin to the subunit
dimers is cooperative because binding to one subunit dissociates the dimer and makes the other subunit
more available for calmodulin binding. The activated subunits are mobile and, in addition to
phosphorylating other synaptic proteins, they can autophosphorylate each other at a critical threonine
residue that locks the subunit in an active state until it is dephosphorylated by phosphatase 1 or
phosphatase 2A. (From Rosenberg et al. 2005; modified, with permission, © Elsevier.)

Atomic structures of holoenzymes from Caenorhabditis elegans

(Rosenberg et al. 2005) and human (Rellos et al. 2010; Chao et al. 2011)
revealed that the 12 subunits are arranged in two closely apposed rings (Fig.
3). The association domains are located at the center of the ring and the
catalytic domains are around the outside. When the enzyme is inactive, each
catalytic domain in the upper ring forms a dimer with a corresponding
subunit in the lower ring. Binding of calcium-CaM to one of the subunits in a
dimer activates that subunit and frees its partner, increasing its availability to
bind calcium-CaM. The magnitude of the resulting cooperativity for binding
of calcium-CaM depends on the nature of the subunit dimer interface and the
length of the linker region between the catalytic and association domains
(Chao et al. 2010, 2011). Thus, the sensitivity of CaMKII to activation by
calcium is highly tuned, differing among different species and among
individual isoforms in the same species.
When CaMKII is inactive, an inhibitory domain blocks the substrate-
binding site. Upon activation by calcium-CaM, CaMKII subunits become
autophosphorylated. The first autophosphorylation occurs on a threonine
residue (T286) in the inhibitory domain, preventing its binding to the
substrate-binding site. The phosphothreonine keeps the active site open even
after the calcium concentration returns to baseline and calcium-CaM
dissociates (Miller and Kennedy 1986; Miller et al. 1988; Schworer et al.
1988). This first autophosphorylation event is intersubunit; occurring when
one activated subunit in the holoenzyme binds to and phosphorylates the
inhibitory domain of a neighboring subunit (Hanson et al. 1994). The target
subunit must also have bound calcium-CaM so that its inhibitory domain is
exposed (Fig. 3) (Hanson et al. 1994; Rellos et al. 2010). Thus, conversion of
CaMKII to a calcium-independent state depends on the square of the active
CaM concentration. This combined cooperativity of CaM binding and CaM-
induced autophosphorylation contributes to the dependence of CaMKII
activity on the frequency of calcium influx into the dendritic spine (Chao et
al. 2010).
A second autophosphorylation occurs on threonine residues located in the
CaM-binding domain (T305 or T306) and blocks binding of calcium-CaM to
the kinase (Patton et al. 1990). This event desensitizes CaMKII to subsequent
activation by calcium/CaM. Reversal of calcium-independent activity and
alleviation of desensitization requires dephosphorylation of the respective
sites by protein phosphatase PP1 or PP2A (Shields et al. 1985). Thus, the
duration of activation of CaMKII at the postsynaptic site by synaptic activity
is regulated reversibly by the magnitude of the transient formation of
calcium-CaM during synaptic activity and by local regulation of protein
phosphatase activity, which is incompletely understood.
The earliest evidence for a role of CaMKII in synaptic plasticity came
from pharmacological experiments in which inhibitors of protein kinases
injected postsynaptically blocked induction of LTP (e.g., Malinow et al.
1989). However, definitive evidence for its involvement was obtained when
disruptions in learning behavior and synaptic plasticity were observed in a
series of mouse mutants. Deletion of the α subunit of CaMKII, for example,
results in a deficiency in LTP and impaired spatial learning (Silva et al.
1992a,b). Remarkably, mutation of T286 to alanine in the α subunit abolishes
LTP and spatial learning altogether, establishing that autophosphorylation of
T286 in CaMKII plays a central role in induction of LTP (Giese et al. 1998).
The concentration of CaMKII in spines and the PSD is highly variable,
and regulated by synaptic activity. Estimates of the number of holoenzymes
in an average spine (64 attoliters in volume) vary from ∼200 to ∼1000
(Bennett et al. 1983; Erondu and Kennedy 1985; Lee et al. 2009). Estimates
of the number of CaMKII holoenzymes in an average PSD are more variable,
ranging from ∼10 to ∼100 (Chen et al. 2005; Ding et al. 2013). Synaptic
activity has been observed to recruit CaMKII to postsynaptic sites, although
the mechanism of this movement is not clear (Shen and Meyer 1999). Once
in the spine, CaMKII can attach to several docking sites: F-actin filaments,
which bind a site on the β subunit (Shen et al. 1998); the cytosolic tails of
NR2 subunits of NMDARs (Leonard et al. 2002), which bind a site on both
the α and β subunits; and a scaffold protein termed densin (Walikonis et al.
2001; Carlisle et al. 2011), which binds specifically to α subunits. The
significance and precise dynamics of these regulated movements of the
CaMKII holoenzyme remain to be determined.
The spine and PSD contain many potential targets for phosphorylation by
CaMKII, including AMPAR subunit GluA1, the neuronal GTPase-activating
protein (GAP) synGAP, and AMPAR-associated transmembrane AMPAR
regulatory proteins (TARPs) (see below), all of which are important for
orchestrating the early stages of synaptic plasticity. However, the precise
timing and coordination of phosphorylation events following influx of
calcium are unknown.

3.2.2 Calcineurin
Calcineurin is a calcium/CaM-activated protein phosphatase found in many
cell types. Injection of inhibitors of calcineurin into postsynaptic neurons in
hippocampal slices first suggested that its activity is required for induction of
LTD (Mulkey et al. 1994). Both brain isoforms of its catalytic subunit
(CNAα and CNAβ) bind calcium-CaM and form a heterodimer with the
regulatory subunit CNB1, which also binds calcium (Kuno et al. 1992). The
requirement of calcineurin for induction of LTD was confirmed in
hippocampal slices from mice with a forebrain-specific deletion of CNB1.
The magnitude of LTD was reduced in these slices and the frequency
threshold for the transition from induction of LTD to induction of LTP was
shifted to a lower value (Zeng et al. 2001). These findings led to the
hypothesis that the relative activation of CaMKII and calcineurin determines
whether LTP or LTD will be induced—a hypothesis that remains to be
proven. An implication of this hypothesis is that the steady-state number of
AMPARs at a synapse and the steady-state size of the actin cytoskeleton are
maintained by a balance of CaMKII and calcineurin activities. More recent
work reveals that the mechanisms controlling the size and strength of the
postsynapse involve the action of several signaling enzymes. For example,
the broader-specificity protein phosphatases PP1 and PP2A also appear to be
necessary for induction of LTP (Mulkey et al. 1993). It is not yet clear how
synaptic activity regulates these two phosphatases. Furthermore, protein
kinases PKA and PKC, which mediate the action of other major second
messenger pathways, can regulate synaptic plasticity when activated by any
of several modulatory neurotransmitters, including acetylcholine, biogenic
amines, and neuropeptides (Abeliovich et al. 1993; Blitzer et al. 1995;
Kennedy et al. 2005).
A possible link between PKA, calcineurin, and PP1 involves a cycle
similar to that found in glycogen metabolism in which a small protein
inhibitor of PP1 (inhibitor 1) is activated by phosphorylation by PKA and
inactivated by dephosphorylation by calcineurin (Huang and Glinsmann
1976; Lisman 1989). However, deletion of inhibitor 1 in the mouse has no
effect on LTP in the Schaffer-collateral pathway or in the medial perforant
pathway that arises in the entorhinal cortex, but reduces LTP in synapses
from the lateral perforant path (Allen et al. 2000). Importantly, the mutation
has no effect on performance of spatial learning tasks. Paralogs of inhibitor 1
that regulate PP1 have been identified in the basal ganglia and cerebellum,
but none has been found in the hippocampus or cortex. The divergent effects
of inhibitor 1 deletion on different synaptic pathways in the hippocampus
show that distinct, heterogeneous molecular mechanisms underlie synaptic
plasticity both in different dendritic subregions and in different neuronal
subtypes. Two other PP1 regulatory proteins, spinophilin and neurabin, can
regulate PP1 activity and its association with the actin cytoskeleton. Deletion
of spinophilin eliminates LTD induced by low-frequency stimulation of the
Schaffer-collateral pathway (Feng et al. 2000). This is consistent with a
requirement for PP1 activity but does not shed light on the relationship of
calcium influx to PP1 activity during induction of LTD.
Calcineurin is a more selective phosphatase than PP1 or PP2A, requiring
upstream motifs, such as LxVP, in its substrates (Grigoriu et al. 2013).
Although there is a small amount of overlap in the target sites for CaMKII
and calcineurin, many of their target sites do not overlap. Therefore, the shift
in phosphorylation status of individual proteins in the spine during and after
an influx of calcium is difficult to predict and is still the subject of study.
3.2.3 Modulatory Calcium-Sensitive Enzymes
The calcium/CaM-stimulated adenylyl cyclase isoform AC1 (Wang and
Storm 2003), the calcium/CaM-activated cyclic nucleotide phosphodiesterase
PDE1 (Sharma et al. 2006), the calcium/CaM-regulated neuronal nitric oxide
synthase isoform (nNOS) (Salerno et al. 2013), members of a family of
calcium-sensitive guanine nucleotide exchange factors (GEFs) called
RasGRF1 and RasGRF2 (Feig 2011), calcium-sensitive PKC isoforms (Lipp
and Reither 2011), and the calcium-dependent protease calpain (Croall and
DeMartino 1991) are all present at low and varying levels in spines and can
modulate the sensitivity of a synapse to induction of synaptic plasticity or the
magnitude and duration of plastic changes. These modulatory mechanisms
are outside our scope here, however.

3.3 Scaffold Proteins in the PSD

Signaling enzymes are organized within the PSD by three major classes of
scaffold proteins: the PSD95 family (also called MAGUKS [for membrane-
associated guanylate kinases]); the SHANK family (for SH3 domain and
ankyrin repeat domain proteins, also called ProSAPs [for proline-rich-
synapse-associated proteins]); and the Homer family. Additional scaffold
proteins, including A-kinase-anchoring protein (AKAP) 79 (see Ch. 3
[Newton et al. 2014]), neurabin, and spinophilin, serve to position enzymes
such as PKA, PKC, calcineurin, and PP1 in the PSD. TARPs, also called
stargazins, associate tightly with AMPARs and help to control their
trafficking into the synapse (Fig. 4) (Hastie et al. 2013).
Figure 4. Schematic diagram of the postsynaptic density (PSD) scaffold. NMDARs are immobilized in
the postsynaptic membrane by association with PSD95. AMPARs associate with TARPs that bind
PSD95. Signaling enzymes are positioned near the receptors by association with PSD95 or other
scaffolds. For example, PKA, PKC, and calcineurin bind to AKAP79, which binds to PSD95 and actin
in the spine. AMPARs are recruited to the PSD during induction of LTP by diffusional trapping at new
docking sites. This diffusional trapping may involve phosphorylation of TARPs by CaMKII, which
increases their binding affinity for PSD95. AMPARs are added to or removed from the extrasynaptic
plasma membrane pool by exocytosis or endocytosis, respectively. (Modified from Sheng and Kim
2011.)

PSD95 and its three relatives SAP97, SAP102, and PSD93 are centered
∼12–20 nm from the postsynaptic membrane (Valtschanoff and Weinberg
2001; Chen et al. 2008) and link glutamate receptors to the PSD structure and
to proximal signaling enzymes. Each contains three PDZ domains, an SH3
domain, and a carboxy-terminal degenerate guanylate kinase (GuK) domain,
all of which act as protein-docking sites (Kornau et al. 1997; Sheng and Kim
2011). The first two PDZ domains of the PSD95 family can bind to several
synaptic membrane proteins via PDZ-binding motifs in their carboxyl
termini, including the GluN subunits of NMDARs, TARPs, and neuroligin, a
transmembrane adhesion protein. In adult mammals, PSD95 is the most
abundant of the family members and associates preferentially with GluN2A,
whereas SAP102 predominates in synapses during the first few weeks of
development and associates preferentially with GluN2B (Sans et al. 2000).
These two scaffold proteins facilitate the developmental shift from NMDARs
that contain predominantly GluN2B to the adult form that contains a mixture
of GluN2A and GluN2B (Sans et al. 2000; Elias et al. 2006). The transition is
important for synaptic signaling because complexes formed by PSD95 and
SAP102 contain different sets of signaling enzymes.
The PDZ domains can also bind to cytosolic signaling enzymes, including
nNOS and synGAP. The carboxy-terminal SH3 domains of the PSD95 family
have an unusual split structure created by the insertion of the GuK domain
between two α helices (McGee et al. 2001; Tavares et al. 2001). This
structure may help regulate oligomerization at the postsynaptic site. The SH3
domain can also bind directly to an AKAP79/150 scaffold protein that
localizes PKA, PKC, and calcineurin to the PSD (Gold et al. 2011). Finally,
the terminal GuK domain forms a docking site for linker proteins of the
GKAP family (for guanylate kinase-associating protein), providing a critical
bridge between the PSD95 family and the next layer of postsynaptic scaffold
proteins, the SHANK family (Kim et al. 1997; Takeuchi et al. 1997; Naisbitt
et al. 1999).
SHANK proteins act as a “scaffold of scaffolds” within the PSD (Fig. 4)
(see Sheng and Kim 2011). They form an interacting network centered ∼25
nm from the postsynaptic membrane and contain multiple, differentially
spliced protein-interaction domains (Sheng and Kim 2000; Boeckers et al.
2002). GKAP binds to a PDZ domain in SHANK, linking it to the PSD95
scaffold (Fig. 4). A proline-rich domain in SHANK links it to the actin
cytoskeleton via the SH3 domain of cortactin. Cortactin is an F-actin-binding
protein that also enhances binding of actin to the ARP2/3 complex,
facilitating branching of actin filaments.
A second proline-rich domain in SHANK interacts with the Homer family
of scaffold proteins (Fig. 4). Homer proteins form tetramers linked by their
carboxy-terminal coiled-coil domains. Each tetramer contains four identical
amino-terminal EVH domains that bind to proline-rich sites on three classes
of synaptic proteins: SHANK, the cytosolic tails of metabotropic glutamate
receptors (mGluRs), and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs).
Thus, the Homer proteins can form a bridge between mGluRs and internal
calcium stores located in vesicles of smooth endoplasmic reticulum that
contain IP3Rs. Not all spines contain such stores, but when they are present
calcium is released into the cytoplasm when IP3 binds to the IP3Rs (p. 95
[Bootman 2012]). Finally, the EVH domains link all of the Homer complexes
directly to SHANK.
The dense protein network formed by these three families of scaffold
proteins is highly dynamic. For example, PSD95 exchanges between
neighboring spines with a median retention time of ∼30–100 min (Gray et al.
2006). Retention time decreases during sensory deprivation (Gray et al. 2006)
and also after synaptic activity (Steiner et al. 2008). The composition and size
of PSDs thus appear to be regulated in an ongoing way by neural activity.
This dynamic scaffold provides the underlying spatial organization for the
signaling events that regulate the strength of the synapse (Fig. 4).
Genome-wide association (GWAS) studies have identified mutations or
copy-number variants in PSD scaffolds as risk factors for autism spectrum
disorders. In humans and in mice, deletion of SHANK scaffold proteins has
been repeatedly linked to autistic behaviors (Durand et al. 2007; Herbert
2011; Peca et al. 2011). Similarly, deletion of PSD95 in mice results in
behaviors associated with autism, and two human single-nucleotide
polymorphisms in the gene encoding PSD95 are associated with
characteristics of William’s syndrome, a genetic disorder that includes a
highly social personality and cognitive difficulties (Feyder et al. 2010).

4 REGULATION OF THE NUMBER OF AMPARs IN A

SYNAPSE
One of the two central processes associated with LTP and LTD is regulation
of the number of synaptic AMPARs. What is the link between activation of
calcium-dependent enzymes and control of the number of AMPARs at a
synapse? AMPAR number appears to be regulated at several levels, and we
are beginning to unravel some of the signaling pathways involved.
AMPARs are tetramers formed from mixtures of four subunits: GluA1,
GluA2, GluA3, and GluA4. In adult forebrain neurons, the most prominent
subunit combinations are GluA1-GluA2 (so-called GluA1-2 receptors) and
GluA2-GluA3 (so-called GluA2-3 receptors) (Lu et al. 2009). Each subunit
can be alternatively spliced to form additional isomers. The tetramer
combinations are formed during synthesis in the soma and dendrites, and
vesicles containing the receptors are then carried to synaptic sites by
constitutive membrane trafficking. One important functional distinction
among the isomers is the sequence of the cytoplasmic tail (Malinow and
Malenka 2002). GluA1, GluA2L, and GluA4 each have a cytoplasmic tail of
∼100 residues, whereas GluA2 and GluA3 have shorter tails (∼50–70
residues). The two types of tails each contain distinct phosphorylation sites
and distinct PDZ-domain ligands that govern trafficking of the receptor and
movement into the synaptic membrane. GluA1-2 receptors are less abundant
in PSDs, but they are the receptors that are added to synapses during
induction of LTP (Shi et al. 2001). They are also the receptors that are
removed when recently potentiated synapses undergo activity-dependent
LTD. In contrast, GluA2-3 receptors cycle constitutively into and out of the
PSD, independently of synaptic activity. The GluA2-3 tetramers gradually
replace GluA1-2 tetramers during periods of low activity, while maintaining
the total steady-state number of AMPA receptors (Shi et al. 2001).
Experiments in which individually tagged surface AMPARs are tracked in
real time have provided evidence for a three-step mechanism of activity-
induced movement of GluA1-2 tetramers to the synapse. The steps include:
(1) exocytosis of AMPARs at extrasynaptic and perisynaptic sites, (2) lateral
diffusion into synapses, and (3) rate-limiting diffusional trapping in the PSD
(Opazo and Choquet 2011). In this model, activation of NMDARs initiates
two parallel signaling cascades: one that facilitates diffusional trapping of
AMPARs in the PSD, and a second that increases AMPAR exocytosis
perisynaptically (Fig. 5) (Makino and Malinow 2009; Petrini et al. 2009).
Thus, a relatively rapid increase in the number of AMPARs at the synapse
(less than a minute) is accomplished by diffusional trapping (Opazo et al.
2010); concurrently, the mobile population of AMPARs in the dendritic and
perisynaptic region is replenished by exocytotic delivery of new AMPARs
over a period of minutes (Makino and Malinow 2009).
Figure 5. Signaling pathways in the spine. (A) Schematic diagram of major signaling enzymes that
mediate changes in synaptic plasticity driven by calcium influx through activated NMDARs. Critical
early targets of CaMKII include AMPARs, TARPs, and SynGAP. RasGRF can activate both Ras and
Rac. The critical targets of calcineurin are not known. One possibility is the cofilin phosphatase
Slingshot, which dephosphorylates and activates the F-actin regulator cofilin. Calcium/CaM-dependent
AC and cAMP-phosphodiesterase (PDE) are both present in spines; their responses to calcium could
generate a transient spike in cAMP, activating Epac. Ras and Rap regulate trafficking of AMPARs, but
their downstream targets, beyond the MAPKs, are not yet known. RasGRF and Ras, acting through the
GTPase exchange factor TIAM1 can activate Rac, which regulates pathways that control
polymerization of actin. TrkB receptors that respond to the central nervous system (CNS) hormone
brain-derived neural factor (BDNF) can provide tonic activation of Ras and Rap. (B) Synaptic
regulation of AMPAR trafficking. A critical step in the induction of LTP is the trapping of additional
AMPARs in the PSD scaffold through association of TARP and PSD95, which is increased by
phosphorylation of residues on TARP by CaMKII. Dephosphorylation of these residues by PP1 can
produce loss of AMPARs and depotentiation. Calcineurin, a calcium/CaM-dependent phosphatase,
regulates PP1 and endocytosis. Addition of AMPARs to the dendritic plasma membrane by exocytosis,
and their removal by endocytosis, occurs at perisynaptic sites in the spine and along the dendritic shaft.
Changes in the activity of Ras and Rap are regulated by downstream targets of calcium/CaM, including
RasGRF, SynGAP, and adenylyl cyclase (AC). Active Ras and Rap, in turn, activate the MAPKs
ERK1/2 and p38. Pathways downstream from ERK facilitate exocytosis, whereas those downstream
from p38 facilitate endocytosis. Many of the intermediate steps in the processes downstream from
MAPKs are unknown.

Recent work has clarified the mechanism of diffusional trapping. Opazo

et al. (2010) found that, in the absence of recent NMDAR activation,
AMPARs on the dendritic surface are highly mobile and exchange rapidly
between extrasynaptic and synaptic sites. Under these basal conditions,
CaMKII is not highly enriched in the PSD (Ding et al. 2013). Activation of
NMDARs causes influx of calcium into the spine, which activates CaMKII
and causes it to translocate into the PSD, where it phosphorylates several
sites on the cytoplasmic carboxyl terminus of the AMPAR-associated TARPs
(Tomita et al. 2005). This phosphorylation facilitates binding of the TARPs
to PDZ domains on PSD95, and thus restricts diffusion of AMPARs, trapping
them in the PSD (Opazo et al. 2010). This diffusional trapping mechanism
(see Fig. 5A) may result in the addition of new placeholders (slots) for
AMPARs, as suggested by Shi et al. (2001).
New AMPARs are added by exocytosis to the plasma membrane of spines
and adjoining dendrites during a period lasting several seconds to a few
minutes following induction of LTP (Patterson et al. 2010). The biochemical
mechanisms by which activation of NMDARs and subsequent activation of
CaMKII lead to increased exocytosis of AMPARs are unknown. One clue is
that inhibition of the Ras-ERK1/2 pathway partially blocks the activity-
induced increase in exocytosis rate (Patterson et al. 2010).

5 THE ROLE OF SMALL GTPases IN SYNAPTIC

PLASTICITY
The Ras and Rho families of small GTPases play important roles in synaptic
plasticity, regulating both insertion and removal of AMPARs at the
membrane, and growth and shrinkage of the spine actin cytoskeleton (Fig. 5).
Their activity influences both homeostatic maintenance of synaptic structure
and alterations in structure that occur during changes in synaptic strength.
However, most of what we know about their roles is still phenomenological;
many mechanistic questions remain about how they are activated in the spine
by synaptic activity and which downstream targets drive changes in synaptic
structure.
Ras and the downstream MAPKK (MEK), which activates the MAPK
ERK1/2, appear to be important for activity-driven increases in the number of
synaptic AMPARs during induction of LTP (Fig. 5A). Inhibition of MEK
before electrophysiological induction of LTP blocks the normal increase in
the number of synaptic AMPARs (Zhu et al. 2002). Furthermore,
transfections of primary cultures of hippocampal neurons with wild-type,
constitutively active, or dominant-negative forms of Ras for 15 h cause
changes in AMPAR-mediated synaptic currents, which is consistent with the
notion that endogenous Ras activity contributes to an increase in the number
of synaptic AMPARs. Similar experiments with the GTPase Rap suggest that
endogenous Rap contributes to a decrease in synaptic AMPAR levels and that
inhibition of the downstream MAPK p38 blocks the normal decrease in the
number of synaptic AMPARs produced after LTD induction. Because of the
relatively long timescale of these experimental manipulations of the Ras and
Rap pathways, it has been difficult to separate their effects on homeostatic
maintenance of synaptic structure from their roles in acute changes in
synaptic strength.
Ras and Rap are activated by several mechanisms in spines (Kennedy et
al. 2005). Ras can be activated directly by the calcium/CaM-dependent
RasGRF (Feig 2011). A variety of hormones in the brain stimulate receptor
tyrosine kinases to activate Ras and Rap. A prominent example in spines is
TrkB, which responds to the peptide hormone brain-derived neurotrophic
factor (BDNF) and regulates synaptic function in a variety of ways during
development and in the adult (Minichiello 2009; Yoshii and Constantine-
Paton 2010). Active, phosphorylated TrkB can activate Ras or Rap1,
depending on which adapter proteins it binds (York et al. 1998; Stork 2003).
In addition, Rap is activated by Epac2, a cAMP-activated Rap GEF
(RapGEF), which responds to cAMP formed in the spine by either
calcium/CaM-dependent or G-protein-activated adenylyl cyclases (Grewal et
al. 1999; Penzes et al. 2011; p. 99 [Sassone-Corsi 2012]).
Downstream Ras effectors in spines include Raf1, which initiates the
phosphorylation cascade leading to activation of ERK1/2; phosphoinositide
3-kinase (PI3K), an enzyme that forms 3′ phosphorylated
phosphatidylinositol lipids such as PIP3, an activator of several membrane-
bound signaling proteins (p. 87 [Hemmings and Restuccia 2012]); and
TIAM1, a membrane-associated RacGEF (Tolias et al. 2005) that links
activation of Ras to activation of Rac. The principal Rap effector in spines is
B-Raf, a form of Raf that, in neurons, can activate the p38 MAPK cascade as
well as the ERK1/2 cascade (Shi et al. 2005).
A second line of evidence for critical roles of Ras and Rap in regulation
of synaptic plasticity comes from studies of synGAP (Chen et al. 1998; Kim
et al. 1998). SynGAP is abundant in the PSD and its GAP activity against
both Ras and Rap is increased by phosphorylation by CaMKII (Oh et al.
2002, 2004; Krapivinsky et al. 2004). Thus, activation of CaMKII by
NMDARs produces a decrease in levels of active Ras. However, this occurs
with a delay, and because of the presence of calcium/CaM-sensitive RasGRF
in the spine, there is first a transient spike in the level of active Ras (Carlisle
et al. 2008). Mice lacking synGAP entirely die shortly after birth (Kim et al.
2003; Vazquez et al. 2004). However, mice heterozygous for synGAP
survive and exhibit defects in dendritic spine development, a reduced
amplitude of hippocampal LTP, and defective learning behavior (Komiyama
et al. 2002; Kim et al. 2003; Vazquez et al. 2004; Carlisle et al. 2008). Thus,
synGAP is unusual among PSD proteins in that it exhibits a gene-dosage
effect; simply reducing the amount of synGAP by half produces a striking
phenotype. The importance of synGAP for cognition is underscored by
reports that copy-number variants or single point mutations in the gene
encoding it are found in ∼5% of human patients with nonsyndromic
intellectual disability (NSID) and none are found in controls (Hamdan et al.
2009, 2011).
Phenotypes of cultured synGAP−/− neurons and synGAP+/− heterozygous
mice highlight the role of Ras and Rap in modification of the spine actin
cytoskeleton. Cultured synGAP−/− hippocampal neurons show accelerated
spine development, and at maturity their spines are significantly larger than
those of wild type (Vazquez et al. 2004). Neurons in intact brains of
synGAP+/− heterozygotes also have larger spines, display markedly increased
activation of Ras and Rac, and increased phosphorylation of cofilin, a
regulator of actin polymerization (Carlisle et al. 2008). In neurons cultured
from wild-type mice, activation of NMDARs causes transient
dephosphorylation of cofilin, most likely by the specific phosphatase
Slingshot, which is activated by calcineurin (Carlisle et al. 2008). The
dephosphorylation activates cofilin, leading to depolymerization of actin.
Subsequent activation of the kinase PAK leads to phosphorylation and
activation of LIM kinase, which then phosphorylates and inactivates cofilin,
so that the actin cytoskeleton can repolymerize in a new form. In synGAP−/−
neurons, the basal level of active PAK is elevated, and the initial transient
dephosphorylation of cofilin is blunted (Carlisle et al. 2008), apparently
tipping the balance toward increasing actin polymerization and larger spines.
SynGAP−/− neurons also have increased levels of synaptic AMPARs
(Vazquez et al. 2004; Rumbaugh et al. 2006) and alterations in the regulation
of ERK1/2 and p38 (Krapivinsky et al. 2004; Rumbaugh et al. 2006; Carlisle
et al. 2008). Thus, it appears that synGAP exerts a carefully balanced
restrictive effect both on spine size and on synaptic strength through its
regulation of Ras and Rap activity.
The precise timing of activation of Ras and Rap by their various effectors
during induction of LTP or LTD has not been studied with precision; thus,
we do not yet know the exact mechanisms by which their activation is
coordinated to control transient changes during synaptic plasticity versus
homeostatic regulation of neuronal excitability.

6 CONCLUDING REMARKS
Synapses in the brain release a number of different neurotransmitters
including GABA, acetylcholine, the biogenic amines serotonin, dopamine,
and norepinephrine, and a wide variety of peptide neurohormones. However,
as far as we now know, it is only excitatory glutamatergic synapses that
display the Hebbian form of regulation discussed here. The sculpting of
excitatory connections in response to input from the environment is the
principal mechanism of memory formation in the brain. As excitatory
connections are altered by the Hebbian mechanism, new neural networks are
formed, and others are weakened or strengthened. All of the other types of
synapses contribute to regulation of Hebbian plasticity and help to determine
the conditions under which specific memories are formed, as well as how
long the memories will last. We know much less about regulation of the size
of the signal and response in these other synaptic types, which are fewer in
number and are dispersed among the more abundant glutamatergic synapses,
making them less accessible to molecular manipulation or measurement.
Another obstacle to our full understanding of synaptic regulation is the
subtle variation in mechanisms of synaptic plasticity in spines of different
excitatory neuronal types and among neurons in different brain regions.
These differences effectively obscure our vision because most experimental
methods either sample blindly from the mixture of synapses in a preparation
or record average changes from a poorly understood mixture of synaptic
types. As we learn which receptors and enzymes play critical roles in
modulating synaptic plasticity, new anatomical techniques such as array
tomography (Micheva et al. 2010) and superresolution confocal microscopy
(Dani et al. 2010) will help to sort out distinct synaptic types.
A final experimental frontier concerns the delicate timing of synaptic
regulation required for healthy brain function. To paraphrase Marc Kirschner
describing regulation of embryonic development, “In the regulation of the
brain, as in the theater, timing is everything. Imagine if, one night, the actors
in a play were to miss every single cue, delivering each line perfectly, but
always too early or too late. The evening would be a disaster. The same is
true in brain function. Starting at the moment when the environment
stimulates sensory endings, neurons in the brain send signals to each other to
coordinate sensory perception, emotional and motor responses, and the
laying down of memories. Not only do the signals have to be correct, they
also must be perfectly timed. Otherwise, disasters like mental illness can
result” (paraphrased words in italics) (see kirschner.med.harvard.edu).
A challenge arises from the fact that the biochemical reactions that initiate
and sculpt changes in spine structure underlying activity-dependent synaptic
plasticity occur in a tiny compartment that contains tens to several hundred
copies of the requisite enzymes and effectors. Some of these are immobilized
by scaffold proteins that hold them in close proximity to the most important
downstream targets. Additional complexity arises from the fact that the
initiating calcium signal is always fluctuating, driven by the stuttering
kinetics of the NMDAR channel and active calcium pumps in the spine
membrane. Thus, time-resolved, high-resolution mass spectroscopy and
engineered biochemical real-time sensors, in concert with modeling methods
such as those afforded by the spatially accurate, stochastic modeling program
MCell (e.g., see Kennedy et al. 2005), will be needed to help resolve rapid,
transient molecular events involved in memory formation from those
underlying homeostatic mechanisms.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a016824
CHAPTER 13

Signaling in Muscle Contraction

Ivana Y. Kuo1 and Barbara E. Ehrlich1,2

1Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520
2Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven,
Connecticut 06520
Correspondence: barbara.ehrlich@yale.edu

SUMMARY

Signaling pathways regulate contraction of striated (skeletal and cardiac)

and smooth muscle. Although these are similar, there are striking
differences in the pathways that can be attributed to the distinct
functional roles of the different muscle types. Muscles contract in
response to depolarization, activation of G-protein-coupled receptors,
and other stimuli. The actomyosin fibers responsible for contraction
require an increase in the cytosolic levels of calcium, which signaling
pathways induce by promoting influx from extracellular sources or
release from intracellular stores. Increases in cytosolic calcium stimulate
numerous downstream calcium-dependent signaling pathways, which
can also regulate contraction. Alterations to the signaling pathways that
initiate and sustain contraction and relaxation occur as a consequence of
exercise and pathophysiological conditions.

Outline
 1 Introduction
2 Skeletal muscle contraction
3 Skeletal muscle fiber types and exercise
4 Malignant hyperthermia in skeletal muscle
5 Cardiac muscle contraction
6 Exercise hypertrophy in cardiac muscle
7 Pathophysiological cardiac hypertrophy
8 Heart failure
9 Smooth muscle types
10 The contractile process in smooth muscle
11 Calcium sensitization
12 Vascular smooth muscle in disease
13 Concluding remarks
References

1 INTRODUCTION
Muscle can be subdivided into two general categories: striated muscle, which
includes skeletal and cardiac muscles; and nonstriated muscle, which
includes smooth muscle such as vascular, respiratory, uterine, and
gastrointestinal muscles. In all muscle types, the contractile apparatus
consists of two main proteins: actin and myosin. Striated muscle is so called
because the regular arrangement of alternating actomyosin fibers gives it a
striped appearance. This arrangement allows coordinated contraction of the
whole muscle in response to neuronal stimulation through a voltage- and
calcium-dependent process known as excitation–contraction coupling. The
coupling enables the rapid and coordinated contraction required of skeletal
muscles and the heart. Smooth muscle does not contain regular striations or
undergo the same type of excitation–contraction coupling. Instead, it
typically uses second messenger signaling to open intracellular channels that
release the calcium ions that control the contractile apparatus. These
processes, in contrast to excitation–contraction coupling, are slow and thus
suitable for the slower and more sustained contractions required of smooth
muscle. The actomyosin contractile apparatus is both calcium- and
phosphorylation-dependent, and restoration of basal calcium levels or its
phosphorylation status returns an actively contracting muscle to a
noncontractile state. Muscle-specific signals modulate these processes,
depending on the type of muscle, its function, and the amount of force
required.
In all muscle cells, contraction thus depends on an increase in cytosolic
calcium concentration (Fig. 1). Calcium has an extracellular concentration of
2–4 mM and a resting cytosolic concentration of ∼100 nM. It is also stored
inside cells within the sarcoplasmic (SR, referring to skeletal and cardiac
muscle) and endoplasmic reticulum (ER, referring to smooth muscle) at a
concentration of ∼0.4 mM (p. 95 [Bootman 2012]). In striated muscle, the
increase in calcium levels is due to its release from the SR stores via
ryanodine receptor (RyRs). Neurotransmitters such as acetylcholine bind to
receptors on the muscle surface and elicit a depolarization by causing
sodium/calcium ions to enter through associated channels. This shifts the
resting membrane potential to a more positive value, which in turn activates
voltage-gated channels, resulting in an action potential (the “excitation” part).
The action potential stimulates L-type calcium channels (also known as
dihydropyridine receptors). In skeletal muscle, these are mechanically
coupled to the SR RyRs and open them directly. In cardiac muscle, calcium
influx through the L-type channels opens RyRs via calcium-induced calcium
release (CICR) (p. 95 [Bootman 2012]). The RyR is a large tetrameric six-
transmembrane-span calcium-release channel. Of the three RyR subtypes,
RyR1 is predominantly found in skeletal muscle (see review by Klein et al.
1996), and RyR2 is predominantly found in cardiac muscle (Cheng et al.
1993).
 Figure 1. Overview of muscle contraction signals in striated (A) and smooth (B) muscle.

Smooth muscle also contains voltage-gated calcium channels and RyRs

responsible for increases in intracellular calcium concentration (see below).
Depolarization causes L-type calcium channels to open, enabling calcium to
enter down its concentration gradient into the cell (Fig. 1B). Opening of
RyRs is usually associated with CICR. As the intracellular calcium
concentration rises, calcium binds to RyRs, whose consequent opening
further enhances the increase in cytoplasmic calcium concentration. Another
major mechanism controlling contraction in these cells, however, involves a
different tetrameric six-transmembrane-span calcium channel: the inositol
1,4,5-trisphosphate (IP3) receptor (IP3R). Circulating hormones (e.g.,
vasopressin and bradykinin) and neurotransmitters released by sympathetic
nerves (e.g., endothelin and norepinephrine) act through G-protein-coupled
receptors (GPCRs) to generate the second messenger IP3 via activation of
phospholipase C (PLC). IP3 binds to and opens IP3Rs on the ER/SR, causing
the calcium release that drives contraction. IP3Rs are present in both skeletal
and cardiac muscle; however, they do not contribute significantly to the
excitation–contraction coupling in striated muscle. Note that both RyRs and
IP3Rs are stimulated by low concentrations of cytoplasmic calcium but close
when the concentration gets higher, showing bell-shaped response curves
(Bezprozvanny et al. 1991; Finch et al. 1991).
Once intracellular calcium levels are raised, calcium binds to either
troponin C on actin filaments (in striated muscle) or calmodulin (CaM),
which regulates myosin filaments (in smooth muscle). In striated muscle,
calcium causes a shift in the position of the troponin complex on actin
filaments, which exposes myosin-binding sites (Fig. 2A). Myosin bound by
ADP and inorganic phosphate (Pi) can then form cross-bridges with actin,
and the release of ADP and Pi produces the power stroke that drives
contraction. This force causes the thin actin filament to slide past the thick
myosin filament and shortens the muscle. Binding of ATP to myosin then
releases myosin from actin, and myosin hydrolyzes ATP to repeat the process
(Fig. 2B).
Figure 2. Calcium triggers contraction in striated muscle. (A) Actomyosin in striated muscle. (1)
Striated muscle in the relaxed state has tropomyosin covering myosin-binding sites on actin. (2)
Calcium binds to troponin C, which induces a conformational change in the troponin complex. This
causes tropomyosin to move deeper into the actin groove, revealing the myosin-binding sites. (B)
Cross-bridge cycle in striated muscle. (1) Calcium binds to troponin C, causing the conformational shift
in tropomyosin that reveals myosin-binding sites on actin. (2) ATP then binds to myosin. (3) ATP is
then hydrolyzed. (4) A cross-bridge forms and myosin binds to a new position on actin. (5) Pi is
released and myosin changes conformation, resulting in the power stroke that causes the filaments to
slide past each other. (6) ADP is then released. (C) Contraction in smooth muscle. In smooth muscle,
calcium binds to calmodulin and causes the activation of myosin light chain (MLC) kinase (MLCK).
This phosphorylates MLC, which then binds to actin to form phosphorylated actomyosin, enabling the
cross-bridge cycle to start.
 In smooth muscle, by contrast, calcium binds to CaM, which then
interacts with myosin light-chain kinase (MLCK), causing it to phosphorylate
the myosin light-chain (MLC) at S19 or Y18. The phosphorylated MLC then
forms cross-bridges with actin, producing phosphorylated actomyosin, which
leads to contraction (Fig. 2C). Note that striated muscle contraction can also
be regulated by calcium-bound CaM and MLCK; however, this is not the
dominant mechanism. Finally, calcium and calcium-CaM also bind to various
other proteins in muscle cells, including the phosphatase calcineurin and
protein kinases such as CaMKIV, respectively. These regulate other cellular
targets, including transcription factors such as NFAT and CREB, which
control gene expression programs that can have longer-term effects on
muscle physiology.
These different calcium-release mechanisms all also stimulate the
pumping of calcium from the cytoplasm back into intracellular stores via the
SR/ER calcium ATPase (SERCA) pump. The plasma membrane calcium
ATPase (PMCA) pump and the sodium/calcium exchanger (NCX), both of
which reside on the plasma membrane, can also remove calcium from the
cytosol. Calcium dissociates from troponin C or calmodulin as the cytosolic
calcium concentration decreases as a consequence, which terminates the
contraction process.
The main pathways promoting muscle relaxation involve the second
messengers cAMP and cyclic guanosine monophosphate (cGMP). cAMP is
generated by adenylyl cyclases, downstream from the β-adrenergic GS-
coupled receptor, which is activated by noradrenaline. Note that the cAMP
pathway generally promotes contraction in cardiac muscle; however, in
smooth muscle, activation of cAMP causes relaxation. The cGMP pathway
can be activated either by nitric oxide (NO) or natriuretic peptides (NPs). In
the case of blood vessels and other smooth muscles, NO produced by
endothelial NO synthase (eNOS) diffuses across the muscle cell membrane to
activate soluble guanylyl cyclase (sGC), which in turn increases levels of
cGMP. NPs, such as atrial (ANP, released by the heart atria under high blood
pressure), brain (BNP, primarily released by the heart ventricle), and c-type
(CNP, mainly involved in pathological conditions, and released by the
vascular and central nervous system), instead bind to transmembrane
guanylyl cyclase, whose intracellular domain possesses the enzymatic
activity (Nishikimi et al. 2011). The cAMP and cGMP generated act via the
protein kinase PKA and PKG on the contractile process in multiple ways: (1)
their phosphorylation of calcium pumps leads to increased activity; (2)
activation of MLC phosphatase (MLCP) by PKG antagonizes MLCK; and
(3) both PKA and PKG cause a reduction in the sensitivity of the contractile
machinery by inhibiting the GTPase RhoA (this increases MLCP activity and
causes MLC dephosphorylation and muscle relaxation). The levels of cAMP
and cGMP are in turn regulated through their degradation by
phosphodiesterases to yield the inactive metabolites 5′-AMP and 5′-GMP.
Below, we examine the key differences between the signaling
mechanisms controlling contraction of skeletal, cardiac, and smooth muscle,
and how these relate to their differing functions. In addition, we discuss the
changes to the signaling pathways that occur as a consequence of exercise
and pathological situations.

2 SKELETAL MUSCLE CONTRACTION

Skeletal muscles comprise multiple individual muscle fibers that are
stimulated by motor neurons stemming from the spinal cord. They are
grouped together to form “motor units” and more than one type of muscle
fiber can be present within each motor unit. Muscle fibers can be divided into
fast- and slow-twitch muscles. Fast-twitch muscles use glycolytic metabolism
and are recruited for phasic activity (an active contraction). Slow-twitch
muscles (also known as red muscles) are rich in myoglobin, mitochondria,
and oxidative enzymes and specialized for sustained or tonic activity. See
Schiaffino and Reggiani (2011) for a more complete discussion of skeletal
muscle types and the types of myosin isoforms that make up fast- and slow-
twitch muscles.
The neuromuscular junction (NMJ) that connects skeletal muscle with the
nerves that innervate them consists of three distinct parts: the distal motor
nerve ending, the synaptic cleft, and the postsynaptic region, located on the
muscle membrane. Motor neurons branch into multiple termini, which are
juxtaposed to motor endplates, specialized regions of muscle where
neurotransmitter receptors are concentrated (Fig. 3A). The transfer of
information between the nerve and muscle is mediated by the release of
acetylcholine from the motor neuron, which diffuses across the synaptic cleft,
and binds to and activates the ligand-gated, nicotinic acetylcholine receptors
(nAChRs) on the endplate. Activation of the nAChR leads to an influx of
cations (sodium and calcium) that causes depolarization of the muscle cell
membrane. This depolarization in turn activates a high density of voltage-
gated sodium channels on the muscle membrane, eliciting an action potential.

Figure 3. Skeletal muscle contraction and changes with exercise. (A) Neurotransmitter (acetylcholine,
ACh) released from nerve endings binds to receptors (AChRs) on the muscle surface. The ensuing
depolarization causes sodium channels to open, which elicits an action potential that propagates along
the cell. The action potential invades T-tubules and causes the L-type calcium channels to open, which
in turn causes ryanodine receptors (RyRs) in the SR to open and release calcium, which stimulates
contraction. Calcium is pumped back into the SR by (SR/ER calcium ATPase SERCA) pumps. The
decreasing cytosolic calcium levels cause calcium to disassociate from troponin C and, consequently,
tropomyosin reverts to a conformation that covers the myosin-binding sites. (B) Signaling in exercised
skeletal muscle. Both calcium and calcium-independent signals stimulate the transcriptional coactivator
PGC1α. This activates a number of transcription factors that regulate genes associated with
mitochondrial biogenesis, glucose, and lipid homeostasis.
 The action potential runs along the top of the muscle and invades the T-
tubules (specialized invaginations of the membrane containing numerous ion
channels). The opening of voltage-gated sodium channels activates L-type
voltage-gated calcium channels lining the T-tubule. A conformational change
in these enables release of calcium on the closely apposed SR via activation
of RyR1. Calcium then binds to troponin as described above, initiating the
contraction process. Calcium-bound CaM also activates MLCK, whose
phosphorylation of the MLC changes cross-bridge properties. This modulates
the troponin-dependent contraction, although there is no effect on the ATPase
activity of MLC. MLC phosphorylation instead enhances force development
at submaximal saturating calcium concentrations (see below). The phosphate
group is subsequently removed by protein phosphatase 1 (PP1).

3 SKELETAL MUSCLE FIBER TYPES AND EXERCISE

Skeletal muscle is plastic. Exercise can lead to pronounced changes in its
metabolic properties and, sometimes, a change in the fiber type. Physical
differences between fast- and slow-twitch muscles underlie the functional
roles of these fibers, including the type of myosin used and differing resting
calcium levels. The free calcium level is twofold higher in slow-twitch
muscle, even though the SR volume is greater. The level of MLC
phosphorylation is higher in fast-twitch muscle, however, because of higher
levels of expression of MLCK (Bozzo et al. 2005). The force enhancement
produced by MLC phosphorylation, under submaximal saturating calcium
concentrations, counteracts the reduction in force caused by fatigue in fast-
twitch muscle fibers (Schiaffino and Reggiani 2011).
Fast- and slow-twitch fibers also have different calcium-sequestering and
-buffering systems. Different SERCA isoforms are present: SERCA2A is the
main isoform in slow-twitch muscle fibers, whereas SERCA1A is expressed
in fast-twitch muscle fibers. Similarly, different cytosolic calcium buffers are
expressed. Calsequestrin (CSQ) is the main SR-luminal calcium-buffering
protein. It is a high-capacity, low-affinity calcium-binding protein that binds
calcium cooperatively (Campbell et al. 1983). When the muscle is at rest, the
SR is primed to release large amounts of calcium, because CSQ is
polymerized, which reduces its ability to bind calcium. In cardiac muscle,
only CSQ2 is expressed. In skeletal muscle, CSQ1 and CSQ2 are found in
slow-twitch muscle fibers, but only CSQ1 is found in fast-twitch muscle
fibers. The two isoforms differ in their carboxy-terminal tail; functionally,
CSQ1 reduces the activity of RyR1, whereas CSQ2 increases the open
probability of RyR1 and RyR2 (Wei et al. 2009).
Other differences between the muscle fiber types include posttranslational
modifications such as phosphorylation of RyR by PKA, and interactions
between RyR and other proteins, such as CaM and FK506-binding protein
(FKBP) 12 and FKBP12.6. Phosphorylation of RyR by either PKA or
CaMKII fully activates the channel. PKA and CaMKII can also
phosphorylate phospholamban, a protein that inhibits SERCA;
phosphorylation causes phospholamban to dissociate from SERCA. The
FKBPs are immunophilins that bind to immunosuppressants such as
rapamycin and FK506. FKBP12 and FKBP12.6 have differing expression
levels in muscle tissue, but both bind all three forms of the RyR and stabilize
its closed state. Collectively, these calcium-dependent differences between
fast- and slow-twitch muscle fibers, in addition to differences in the myosin
isoform used and the number of mitochondria, account for the different
functional outputs of the two muscle fiber types.
Long-term exercise causes a general shift in muscle fiber type from slow
twitch to fast twitch. It induces a number of changes, including altered
expression and activity of membrane transporters and mitochondrial
metabolic enzymes, together with increased blood supply to skeletal muscle
(Ch. 14 [Hardie 2012]). These, in turn, enhance the oxidative capacity and
increase expression of enzymes preventing damage by reactive oxygen
species (ROS). One major signaling pathway is through the peroxisome-
proliferator-activated receptor (PPAR) γ coactivator (PGC) 1α (Fig. 3B).
PGC1α coactivates a number of transcription factors that regulate genes
important for muscle function. These include PPARs (which regulate glucose
and lipid homeostasis, proliferation, and differentiation), nuclear respiratory
factors (NRFs, which regulate metabolism and mitochondrial biosynthesis),
myocyte enhancer factor 2 (MEF2, which is involved in development and
hematopoesis), and Forkhead box O (FoxO) family transcription factors
(which counter oxidative stress and promote cell-cycle arrest and apoptosis)
(Handschin and Spiegelman 2006; Ronnebaum and Patterson 2010). In
addition to PGC1α, calcium-dependent processes are also involved. Rises in
cytosolic calcium result in the activation of calcineurin, which then
dephosphorylates NFAT. Translocation of NFAT to the nucleus results in
activation of slow-fiber gene expression. Rises in nuclear calcium levels also
cause calcium-dependent signaling molecules to become active. These
include the phosphorylation of histone deacetylases (HDACs) by nuclear
calmodulin-dependent protein kinase. HDACs repress transcription by
causing DNA to be tightly wrapped around histones. Removal of HDACs
enables transcription factors such as MEF2 to bind and enable induction of
genes encoding proteins found in slow fibers (Liu et al. 2005).
As mentioned above, exercise induces an increase in the levels of
mitochondrial metabolic enzymes to compensate for the increased metabolic
demand on skeletal muscle. Unsurprisingly, PGC1α is a potent stimulator of
mitochondrial biogenesis (see review by Olesen et al. 2010). This was shown
elegantly by experiments in which overexpression of PGC1α in white,
glycolytic skeletal muscle could turn it into red, oxidative muscle by
increasing the levels and activity of a number of mitochondrial proteins (Lin
et al. 2002; Wenz et al. 2009). These proteins include most components of
the mitochondrial respiratory chain and ATP synthase, as well as several
enzymes in the Krebs cycle and enzymes involved in fatty acid oxidation.

4 MALIGNANT HYPERTHERMIA IN SKELETAL

MUSCLE
Mutations in RyR and CSQ isoforms cause malignant hyperthermia,
demonstrating the importance of proteins involved in calcium signaling in
skeletal muscle. The mutations in RyR1 appear to increase its open
probability when levels of luminal calcium are low and account for the
majority of malignant hyperthermia cases (80%); the remainder are caused by
mutations to CSQ1.
In the case of RyR1 mutations, volatile anesthetics (inhaled anesthetics
such as isoflurane or halothane) lead to a rapid opening of RyR1 and an
uncontrolled release of calcium from the SR, which in turn leads to sustained
skeletal muscle contraction (Robinson et al. 2006). In response to the
elevated calcium levels, there is activation of SERCA to pump calcium, using
ATP, back into the SR. However, the continual activation of SERCA
consumes excessive ATP, leading to hypermetabolism. This then leads to a
drop in ATP levels, acidosis, tachycardia, and an abnormal increase in body
temperature. These symptoms can be treated with dantrolene, an inhibitor of
the RyR signaling pathway. The mutations in RyR1 associated with
malignant hyperthermia are clustered in three hot spots on the 500 kDa
protein (Lanner et al. 2010). The first cluster is near the amino terminus and
the second cluster is in the middle of the protein. The third cluster lies in the
carboxy-terminal region surrounding the channel-forming domains. How
mutations in all three regions exert similar effects is yet to be determined.
Mutations in CSQ can also result in malignant hyperthermia. A lack of
buffering causes uncontrolled calcium transients that lead to lethal malignant
hyperthermia in response to heat stress and volatile anesthetics (Dainese et al.
2009).

5 CARDIAC MUSCLE CONTRACTION

In cardiac muscle, depolarization starts in the pacemaker cells (modified
cardiac myocytes that set the heart rate and are rich in signaling molecules) in
the sinoatrial node, which is innervated by both parasympathetic and
sympathetic nerves. The external stimuli modulate the activity of the
pacemaker cells—they undergo spontaneous self-depolarization to produce
action potentials. This is achieved by a slow leak of potassium ions and a
concurrent influx of sodium and calcium ions. The action potential then
traverses to the cardiac myocytes, where it invades the T-tubule. However,
unlike skeletal muscle, where L-type calcium channels are directly coupled to
RyRs, in cardiomyocytes the influx of calcium across the plasma membrane
elicits calcium release from the SR via RyRs by CICR (Fig. 4B). The
predominant isoform in the heart is RyR2. As in skeletal muscle, contraction
is controlled by phosphorylation of troponin but can also be modulated by
calcium-CaM and MLCK. Mice with a nonphosphorylatable MLC in
ventricular myocytes display depressed contractile function and develop atrial
hypertrophy and dilatation (Sanbe et al. 1999).

Figure 4. Cardiac muscle contraction and changes with exercise. (A) Cardiac muscle contraction can
occur as a consequence of calcium entry through L-type calcium channels, which activate ryanodine
receptor (RyR) channels in the SR. Alternatively, β-adrenergic receptors on the cell membrane lead to
activation of adenylyl cyclase (AC), which stimulates PKA. This can promote contraction by
phosphorylating RyR and L-type calcium channels or relaxation by phosphorylating the SERCA pump
inhibitor phospholamban. (B) Changes with exercise lead to an activation of the PI3K/Akt pathway,
and a down-regulation of NFAT and calcinurin.

Catecholamines, such as adrenaline and noradrenaline, act on β-

adrenergic receptors (metabotropic GPCRs) to release cAMP that in turn
activates PKA. PKA can be viewed as a primary regulator of the contractile
pathway, as it phosphorylates a number of targets, including L-type calcium
channels and RyRs. In most cases, phosphorylation of these proteins
increases calcium release (for example, phosphorylation of RyR increases its
open probability), and thus the outcome is to stimulate contraction (Ibrahim
et al. 2011). Another target of PKA is phospholamban (an inhibitor of
SERCA), which, when phosphorylated, loses its inhibitory effect on SERCA.

6 EXERCISE HYPERTROPHY IN CARDIAC MUSCLE

Cardiac hypertrophy is an abnormal enlargement of the heart that occurs
because of increases in cell size and proliferation of nonmuscle cells. These
changes can either be beneficial (e.g., exercised hearts), in which changes are
correlated with increased contractility, or detrimental, in which changes lead
to decreased contractility and subsequent heart failure.
Exercised hearts develop a form of mild cardiac hypertrophy that does not
lead to cardiac failure. The main structural changes include a thickening of
the ventricle wall, which leads to increased contractility and thus a greater
ability to pump blood. Within myocytes, expression of the α myosin heavy
chain increases, which leads to high ATPase activity and increased
contractility (Fig. 4B). Various signals are involved, including growth factors
such as insulin-like growth factor (IGF1), vascular endothelial growth factor
(VEGF), and hepatocyte growth factor (HGF) (Fig. 4B) (p. 87 [Hemmings
and Restuccia 2012]). There is increased signaling through the
phosphoinositide 3 kinase (PI3K)/Akt pathway, which leads to proliferation
and growth of cardiomyocytes (Matsui et al. 2003). Transcription factors up-
regulated in exercised hearts include GATA4, which regulates genes
involved in myocardial differentiation. Other pathways, such as the
calcineurin/NFAT pathway are down-regulated (Oliveira et al. 2009). Cardiac
muscle, like skeletal muscle, consumes tremendous amounts of ATP. Thus,
PGC1α is also up-regulated in exercised hearts, facilitating transcription of
metabolic and oxidative genes (Ventura-Clapier et al. 2007; Watson et al.
2007).

7 PATHOPHYSIOLOGICAL CARDIAC HYPERTROPHY

The main pathways driving pathological cardiac hypertrophy are
overstimulation of the sympathetic nervous system, increased oxidative
stress, and inflammatory signaling (Balakumar and Jagadeesh 2010). These
collectively lead to induction of fetal isoforms of heart proteins and a
corresponding decrease in adult forms (Chien 1999), including the myosin
heavy chain (see below). The signals responsible include the GPCR agonist
endothelin 1, peptide growth factors such as platelet-derived growth factor
(PDGF), epidermal growth factor (EGF), and cytokines such as cardiotrophin
and leukemia inhibitory factor (LIF). Mechanical stress can also induce
hypertrophy. In each case, activation of the ERK mitogen-activated protein
kinase (MAPK) pathway (p. 81 [Morrison 2012]) is often observed in
hypertrophy and leads to regulation of transcription factors that alter
expression of the myosin heavy chain, IP3R2, and other proteins (see below).
In hypertrophy, paracrine and autocrine neurohormonal factors that
activate the heterotrimeric G protein Gq, and consequently PLCβ, are
released. This results in an increase in cytosolic calcium levels and activation
of PKC by diacylglycerol (DAG) as well as activation of CaMKII (Mishra et
al. 2010). The importance of the Gq pathway in hypertrophy has been shown
in studies of transgenic mice: mice overexpressing Gq have heart failure
(D’Angelo et al. 1997), whereas mice with reduced Gq levels are protected
against hypertrophy (Wettschureck et al. 2001). There is also a switch from
the α form of the myosin heavy chain to the fetal β isoform (Miyata et al.
2000). This has a lower ATPase activity and a lower rate of contraction.
Other changes include increased SERCA2A activity (Hasenfuss et al. 1994;
Meyer et al. 1995), up-regulation of IP3R2 (Harzheim et al. 2010), and
changes to a neuronal calcium sensor (NCS1). NCS1 is a calcium-binding
protein that also interacts with IP3R (Schlecker et al. 2006).
8 HEART FAILURE
The structural organization of the T-tubules breaks down in heart failure. This
breakdown, caused by myocardial insults (such as myocardial infarction
causing ischemia) among other factors, leads to impaired contractility owing
to reduced, asynchronous, and chaotic calcium release. Several signaling
pathways are compromised in heart failure. Initially, there can be
reorganization of the β-adrenergic system. Activation of the β2-adrenergic
receptor is normally limited to the T-tubule, whereas in heart failure, there is
a redistribution of the receptor across the entire plasma membrane (Nikolaev
et al. 2010). With chronic adrenergic activation, the hyperphosphorylation of
RyRs results in leaky RyR channels, leading to a reduction of SR calcium
and, thus, weaker contractions.
Other alterations to RyR2 that are observed include increased
nitrosylation and loss of the regulatory protein FKBP12.6 (Andersson and
Marks 2010). Both of these changes result in increased RyR activity.
Moreover, mutations in RyR2 that result in leaky channels have been linked
to catecholaminergic polymorphic ventricular tachycardia (CPVT) and
arrhythmogenic right ventricular dysplasia type 2.
Mutations in CSQ2 cause CPVT (Postma et al. 2002) by lowering
buffering capacity within the SR, which results in premature calcium release
and thus arrhythmias. Another important modulator of RyRs is junctin. Its
levels are reduced in heart failure, which may be a compensatory mechanism
to increase contractility (Pritchard and Kranias 2009).
Heart failure also leads to up-regulation of molecules that may have a
protective function. One pathway is through cGMP, which promotes
relaxation. The cGMP pathway is regulated by cGMP-targeted
phosphodiesterases, of which one, PDE5A, looks to be a promising target for
protective therapy against hypertrophy.

9 SMOOTH MUSCLE TYPES

Smooth muscle is found lining the walls of various organs and tubular
structures in the body, including the intestine, bladder, airway, uterus, blood
vessels, and stomach. It receives neural innervation from the autonomic
nervous system, and its contractile state is also controlled by hormonal and
autocrine/paracrine stimuli. Smooth muscle can be divided into two types:
unitary and multiunit smooth muscles. In unitary smooth muscle, individual
smooth muscle cells are coupled to neighboring cells by gap junctions. These
gap junctions permit cell-to-cell passage of small molecules such as ATP and
ions. These include those mobilized in response to electrical signals causing
depolarization, which enable the whole area (known as a syncytium) to
coordinate activity. In contrast, in multiunit smooth muscle, cells are not
coupled to each other and are intermingled with connective tissue. Smooth
muscle can undergo tonic (sustained) or phasic contractions. In the case of
vascular smooth muscle, a sustained contraction is required to provide vessel
tone. This enables the regulation of blood flow. Blood vessels are divided
into the larger diameter conduit vessels (e.g., the thoracic aorta) and the
smaller diameter resistance vessels. The resistance blood vessels display a
myogenic response, in which increasing pressures over the physiological
range (∼70–100 mmHg) result in a sustained contractile state. However,
overconstriction of the vessels leads to hypertension (see below). Other
smooth muscle, such as that found in the gut, including the stomach, small
intestine, or gall bladder, shows variable tone and rhythmic contractions
known as slow waves.

10 THE CONTRACTILE PROCESS IN

SMOOTH MUSCLE
The important distinction between striated muscle and smooth muscle is that
calcium mediates contraction by regulating the availability of actin filaments
in striated muscle, whereas in smooth muscle MLC is the target (Fig. 5). The
source of the increase in cytosolic calcium levels can be extracellular or
intracellular, or a combination of the two. In the case of tonic constriction of
blood vessels, a constant supply of calcium comes from influx via the L-type
calcium channels. The resting membrane potential of smooth muscle
(between −50 and −40 mV) is such that it lies in an overlap (the window
current) between the activation and inactivation curves of the L-type channel.
Thus a small population of the L-type channels is always open. An
alternatively spliced high-voltage-gated form of T-type channels may also
contribute to calcium influx (Kuo et al. 2011), along with stretch-activated
channels residing on the plasma membrane, such as TRPC6.

Figure 5. Smooth muscle contraction. Calcium released by L-type calcium channels or IP3Rs
downstream from Gq-coupled cell-surface receptors causes smooth muscle contraction. It binds to
calmodulin (CaM) and the resulting complex stimulates myosin light-chain (MLC) kinase (MLCK).
This phosphorylates MLC to promote contraction. A RhoA/ROCK pathway and a diacylglycerol
(DAG) pathway contribute to calcium sensitization by altering the phosphorylation status of myosin
light-chain phosphatase (MLCP). Relaxation is mediated through the cGMP/PKG pathway downstream
from nitric oxide (NO) and agonists such as atrial natriuretic peptide (ANP).

In stomach muscle, the rhythmic contractions are due to the activity of

pacemaker cells, but activation of voltage-gated calcium channels can trigger
calcium entry and contraction. Sympathetic nerves run along the vascular
smooth muscle and can release stimuli such as acetylcholine, norepinephrine,
angiotensin, and endothelin. Moreover, circulating blood factors such as
cytokines and diffusible factors such as nitric oxide can also act on receptors
in the plasma membrane or cross the plasma membrane, respectively, to
regulate pathways controlling intracellular calcium levels. The activation of
receptor-operated channels (ROCs) also causes calcium influx, which enables
additional calcium release from intracellular stores. GPCRs activate PLCβ to
generate IP3, which releases calcium via IP3Rs. In vascular smooth muscle
and the circular smooth muscle of the gut, the main isoform is IP3R1. Note,
however, that there is some heterogeneity. In longitudinal smooth muscle of
the gut, RyRs, rather than IP3Rs, are expressed. Agonists such as
cholecystokinin bind to the GPCR cholecystokinin A receptor (CCK-AR),
which activates phospholipase A2, which in turn produces arachidonic acid.
Arachidonic acid (AA) can also be generated through the cleavage of DAG.
AA activates chloride channels, which depolarize the cell membrane,
enabling the opening of voltage-gated calcium channels and an initial influx
of calcium. This calcium can either act directly on the RyR causing CICR or
enable the release of cyclic ADP ribose, which interacts with RyRs to
enhance CICR.
In all smooth muscle, calcium-bound CaM then binds to MLCK,
stimulating phosphorylation of MLC, which leads to muscle contraction. The
necessity for MLCK has been shown in MLCK-knockout mice, in which
smooth muscle MLC cannot be phosphorylated by other kinases (He et al.
2008; Zhang et al. 2010). The dephosphorylation of MLC is catalyzed by
MLCP and a complex of the myosin-targeting protein MYPT1 and the
phosphatase PP1 and results in relaxation.

11 CALCIUM SENSITIZATION
Calcium sensitization is an essentially calcium-independent process that
enables the amount of constriction in smooth muscle to be tuned by an
alteration in the sensitivity of MLC to calcium (Fig. 5). This process enables
the muscle to sustain a contraction once the initial calcium transient has
dissipated. There are two mechanisms for calcium sensitization: a DAG-PLC-
PKC pathway and a RhoA pathway (Lincoln 2007).
Diacylglycerol (DAG) is produced by PLCβ downstream from certain
GPCRs and activates the conventional and novel protein kinase C (cPKC and
nPKC), but not atypical PKC (aPKC) (Steinberg 2008). PKC has a variety of
downstream targets, such as MLCK and C-kinase potentiated protein
phosphatase 1 inhibitor, molecular mass 17 kDa (CPI-17), both of which
enhance constriction. CPI-17 is a smooth-muscle-specific inhibitor of MLCP
that binds to its catalytic subunit and inhibits phosphatase activity, allowing
contraction to persist.
Several agonists, including angiotensin II, norepinephrine, and endothelin,
activate the small G protein RhoA. RhoA in turn activates Rho kinase
(ROCK), which can mediate calcium sensitization through two main
pathways. First, ROCK stimulates phosphorylation of MYPT1 (Feng et al.
1999). This can be direct, at T695 or T853, with a preference for T853.
Alternatively, it can phosphorylate another kinase, zipper-interacting protein
kinase (ZIPK, also known as DAPK3), which phosphorylates MYPT1
primarily at T695 (Kiss et al. 2002). ZIPK also phosphorylates MLC at
T18/S19. Phosphorylation of MYPT1 interferes with binding of MLCP to
MLC, and thus is believed to decrease phosphatase activity. ROCK can also
phosphorylate CPI-17 (MacDonald et al. 2001).
The preference for the MYPT1 or CPI-17 pathway depends on the type of
smooth muscle. Whereas MYPT1 is ubiquitously expressed in smooth
muscle, CPI-17 is differentially expressed. Moreover, RhoA and associated
proteins are expressed at lower levels in phasic smooth muscle compared
with tonic smooth muscle (Patel and Rattan 2006). Note that PKC can also
phosphorylate CPI-17 to prevent MLCP activity. Within resistance arteries,
an increase in vascular pressure also activates the RhoA pathway; however,
the signaling intermediates linking the change in vascular pressure and the
activation of RhoA remain unknown (Cole and Welsh 2011).
12 VASCULAR SMOOTH MUSCLE IN
DISEASE
Smooth muscle cells are remarkably plastic, altering their phenotype in
response to conditions such as vascular injury, altered blood flow conditions,
or disease states. The changes in phenotype that can occur include cell
proliferation, apoptosis, and cell migration and are induced by many factors,
including cytokines and growth factors, mechanical forces, neuronal stimuli,
and genetic factors. Here we limit our discussion to hypertension.
In hypertension, there is often a change in the sympathetic nervous system
and the renin–angiotensin system that leads to increased blood pressure.
Angiotensinogen is converted to angiotensin I by renin, which in turn is
converted to angiotensin II by angiotensin-converting enzyme (ACE).
Increased circulating angiotensin II acts on the angiotensin receptors (AT1
and AT2), which, when activated, cause increased peripheral resistance. The
consequence for smooth muscle cells is they become hypercontractile.
Treatments include ACE inhibitors (which inhibit the conversion of
angiotensin I to angiotensin II), α1-adrenergic antagonists (which block the
AT1 and AT2 GPCRs), and calcium channel blockers (such as
dihydropyridines, which inhibit the voltage-gated calcium channels). All of
these treatments aim to reduce the contractility of smooth muscle. Interfering
with downstream targets such as RhoA signaling in hypertensive animals has
also been shown to be effective (Uehata et al. 1997; Seko et al. 2003; Moriki
et al. 2004).
The sustained contractile state of vascular smooth muscle is associated
with the activation of calcium-dependent transcription factors. These include
SRF, FOS, NFAT, and CREB. SRF, which is activated by the RhoA
pathway, promotes the expression of genes encoding components of the
contractile apparatus. Calcium-stimulated CaMKII activates and causes the
translocation of CaMKIV to the nucleus, where it can activate CREB, which
promotes transcription of components of the contractile apparatus and other
targets. However, CaMKII can also activate a phosphatase that
dephosphorylates and thus inactivates CREB (Matchkov et al. 2012). NFAT
is activated on dephosphorylation by calcium-activated calcineurin, which
induces genes associated with proliferation and migration.
NO produced by eNOS in endothelial cells protects against the changes
observed in hypertension: the cGMP pathway inhibits DNA synthesis,
mitogenesis, and cell proliferation (Forstermann and Sessa 2012). However,
endothelial dysfunction is a hallmark of vascular disease, including
hypertension. In many types of vascular diseases, eNOS is up-regulated but
owing to reduced oxygen availability it is converted to a dysfunctional
enzyme that produces superoxides, which contribute to vascular oxidative
stress (Forstermann and Sessa 2012).
In some disease states, smooth muscle cells adopt a noncontractile
phenotype. Although these cells still have signaling machinery that increases
intracellular calcium levels, they have significantly reduced calcium influx
through voltage-gated calcium channels. Thus, there is a shift to intracellular-
store-operated calcium release, similar to the changes observed in cardiac
hypertrophy. Concomitant with decreases in the levels of SERCA, RyR2,
PMCA1, and the sodium/calcium exchanger, the levels of STIM, ORAI
(proteins associated with refilling of intracellular calcium stores; see p. 95
[Bootman 2012]), SERCA2B, and IP3R increase and there is a change in RyR
receptor subtypes from RyR2 to RyR3 (Lipskaia and Lompre 2004; Berra-
Romani et al. 2008; Baryshnikov et al. 2009; Matchkov et al. 2012). These
changes collectively reflect a less contractile phenotype.

13 CONCLUDING REMARKS
Signal transduction is essential for the function of contractile cells. The
stimulatory signal results in an increase in cytosolic calcium levels, which
activates muscle contraction. We now know the main contributors to the
various types of muscle contraction, and have a better appreciation of the
changes that occur to the contractile apparatus under exercise and
pathophysiological conditions. For example, the identification of PGC1α as a
master regulator of transcription factors up-regulated in both exercised and
pathological striated muscle provides new avenues to modulate muscles in a
therapeutic setting. It is also apparent that many signaling proteins in both
smooth and striated muscles are activated by changes in cytosolic calcium
levels, and these signaling pathways often lead to alterations in gene
expression. Because we now have a better appreciation of the changes that
occur to the contractile apparatus under pathophysiological conditions, this
knowledge can be harnessed to allow us to treat disease strategically.

ACKNOWLEDGMENTS
Research in the Ehrlich Laboratory is supported by National Institutes of
Health funds. I.Y.K. is an American Heart Association postdoctoral fellow.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006023
CHAPTER 14

Organismal Carbohydrate and Lipid

Homeostasis

D. Grahame Hardie
College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland,
United Kingdom
Correspondence: d.g.hardie@dundee.ac.uk

SUMMARY

All living organisms maintain a high ATP:ADP ratio to drive energy-

requiring processes. They therefore need mechanisms to maintain
energy balance at the cellular level. In addition, multicellular eukaryotes
have assigned the task of storing energy to specialized cells such as
adipocytes, and therefore also need a means of intercellular
communication to signal the needs of individual tissues and to maintain
overall energy balance at the whole body level. Such signaling allows
animals to survive periods of fasting or starvation when food is not
available and is mainly achieved by hormonal and nervous
communication. Insulin, adipokines, epinephrine, and other agonists
thus stimulate pathways that regulate the activities of key enzymes
involved in control of metabolism to integrate organismal carbohydrate
and lipid metabolism. Overnutrition can dysregulate these pathways and
have damaging consequences, causing insulin resistance and type 2
diabetes.
Outline
1 Introduction
2 Maintaining energy homeostasis—Hormones and adipokines
3 Muscle—Acute activation of glycogen breakdown
4 Muscle—Acute regulation of glucose uptake and glycogen synthesis,
5 Muscle—Acute regulation of fatty acid oxidation,
6 Muscle—Long-term adaptation to exercise
7 Liver—Acute regulation of carbohydrate metabolism
8 Liver—Long-term regulation of gluconeogenesis via effects on gene
expression
9 Liver—Regulation of fatty acid, triglyceride, and cholesterol
metabolism
10 Adipocytes—Regulation of fatty acid metabolism
11 Brown adipocytes—Regulation of fatty acid oxidation and heat
production
12 Insulin resistance and type 2 diabetes—A response to overnutrition?
13 Concluding remarks
References

1 INTRODUCTION
Heterotrophic organisms, including mammals, gain energy from the ingestion
and breakdown (catabolism) of reduced carbon compounds, mainly
carbohydrates, fats, and proteins. A large proportion of the energy released,
rather than appearing simply as heat, is used to convert ADP and inorganic
phosphate (Pi) into ATP. The high intracellular ratio of ATP to ADP thus
created is analogous to the fully charged state of a rechargeable battery,
representing a store of energy that can be used to drive energy-requiring
processes, including the anabolic pathways required for cell maintenance and
growth. Individual cells must constantly adjust their rates of nutrient uptake
and catabolism to balance their rate of ATP consumption, so that they can
maintain a constant high ratio of ATP to ADP. The main control mechanism
used to achieve this energy homeostasis is AMP-activated protein kinase
(AMPK) (see Box 1) (Hardie 2011).

BOX 1. THE ENERGY CHARGE HYPOTHESIS AND ENERGY

SENSING BY AMP-ACTIVATED PROTEIN KINASE

Catabolism generates ATP from ADP, whereas anabolism and most

other cellular processes, such as the action of motor proteins or
membrane pumps, require energy and is usually driven by hydrolysis of
ATP to ADP. There is no reason a priori why these opposing processes
should automatically remain in balance, and the fact that cellular
ATP:ADP ratios are usually rather constant indicates that there are
systems inside cells that maintain energy balance. Daniel Atkinson
proposed in his energy charge hypothesis that the major signals that
regulate cellular energy homeostasis would be ATP, ADP, and AMP
(Ramaiah et al. 1964). AMP, like ADP, is a good indicator of energy
stress, because its concentration increases as the ADP:ATP ratio
increases, owing to displacement of the near-equilibrium reaction
catalyzed by adenylate kinase (2ADP ↔ ATP + AMP). Atkinson’s
hypothesis was based on findings that two enzymes, glycogen
phosphorylase and phosphofructokinase, which catalyze key control
points in glycogen breakdown and glycolysis (see main text), are
activated by AMP and inhibited by ATP. The discovery of the AMP-
activated protein kinase (AMPK) revitalized this concept. Regulation of
AMPK by adenine nucleotides is surprisingly complex (Hardie et al.
2011), but provides great flexibility.
 In the diagram above, the numerals below each form refer to its
activity relative to the basal state (top left). AMPK is activated >100-
fold by phosphorylation of a threonine residue (172) within the
activation loop of the kinase domain. This is mainly catalyzed by the
upstream kinase LKB1, which has a high basal activity. One of the
regulatory subunits of AMPK contains two sites that competitively bind
the adenine nucleotides AMP, ADP, or ATP. Binding of ADP or AMP
(but not ATP) to the first site (top center) causes a conformational
change that promotes net phosphorylation of 172 (Oakhill et al. 2011;
Xiao et al. 2011), thus causing a switch to the active, phosphorylated
form (bottom center; during a mild metabolic stress, the concentration of
AMP is much lower than that of ADP, so binding of ADP may be the
key event responsible for this change). As stress becomes more severe,
binding of AMP (but not ADP or ATP) at the second site causes a
further 10-fold allosteric activation (bottom right), the combination of
the two effects yielding >1000-fold activation overall. As metabolic
stress subsides, AMP and ADP concentrations will decrease and they
will be replaced in the two regulatory sites by ATP (moving from right
to left on the bottom row). This initially causes a loss of the allosteric
activation, then a conformational change that promotes
dephosphorylation, so that the kinase returns to the basal state (top left).
This complex mechanism allows AMPK to be activated in a sensitive
but dynamic manner over a wide range of ADP:ATP and AMP:ATP
ratios, phosphorylating more and more downstream targets as stress
becomes more severe.
 One potential problem for heterotrophic organisms is that there may be
prolonged periods when food is not available. They must therefore store
molecules like glucose and fatty acids during “times of plenty” to act as
reserves for use during periods of fasting or starvation. In multicellular
organisms, much of this energy storage function has been devolved to
specialized cells. For example, although all mammalian cells (with the
possible exception of neurons) can store some glucose in the form of
glycogen, large quantities are stored only in muscle and the liver. Similarly,
most mammalian cells can store fatty acids in the form of triglyceride
droplets in the cytoplasm, but storing large amounts appears to be harmful to
many cells (see Sec. 12). Adipocytes in white adipose tissue have therefore
become specialized for triglyceride storage, releasing fatty acids when other
tissues need them. In this chapter, we examine the signaling pathways that
coordinate carbohydrate and lipid metabolism between energy-utilizing
tissues such as muscle, energy-storing tissues such as adipose tissue, and the
liver (an organ that coordinates whole body metabolism).

2 MAINTAINING ENERGY HOMEOSTASIS—

HORMONES AND ADIPOKINES
The development of multicellular organisms during eukaryotic evolution
required the acquisition of systems of hormonal and neuronal signaling that
allowed tissues to communicate their needs to each other. In this section, the
critical endocrine glands and nerve centers involved in regulation of whole
body energy homeostasis, and the hormones and cytokines they produce, are
briefly discussed; they are also summarized in Fig. 1. Subsequently, our main
focus will be the signaling pathways by which target cells respond to these
agents.
Figure 1. Summary of endocrine systems that regulate energy balance by modulating carbohydrate and
lipid metabolism. Neurons in the hypothalamus promote feeding, and the successive release of
corticotrophin-releasing hormone (CRH), adenocorticotrophin-releasing hormone (ACTH), and cortisol
from the hypothalamus, pituitary, and adrenal cortex, respectively, and release of epinephrine from the
adrenal medulla. Hypothalamic neurones are activated by low glucose, the thyroid hormone T3, and
adiponectin, while being inhibited by leptin and insulin. The α and β cells in the pancreas monitor
blood glucose independently, releasing glucagon and insulin, respectively. Insulin and leptin are
hormones that represent nutrient surplus, whereas cortisol, epinephrine, and adiponectin are hormones
that represent either deprivation of nutrients (e.g., starvation) or demand for energy (e.g., during
exercise).

2.1 The Hypothalamus and Pituitary Gland

The hypothalamus is a small region at the base of the brain that controls
critical functions such as body temperature, thirst, hunger, and circadian
rhythms. It modulates feeding behavior by producing neuropeptides that
promote or repress appetite. The hypothalamus also produces “releasing
hormones” (e.g., corticotrophin-releasing hormone [CRH] and thyrotropin-
releasing hormone [TRH]) that travel the short distance to the neighboring
pituitary gland. Here they trigger release of peptide hormones (e.g.,
adrenocorticotrophic hormone [ACTH] and thyroid-stimulating hormone
[TSH], released in response to CRH and TRH, respectively).

2.2 The Adrenal Gland

The adrenal gland contains two distinct regions: a central medulla and an
outer cortex. The medulla contains modified sympathetic neurons that release
epinephrine (a catecholamine also known as adrenaline, formed by oxidation
and deamination of the amino acid tyrosine) into the bloodstream. The
hypothalamus has projections that connect with sympathetic nerves and can
thus trigger epinephrine release. For example, this occurs when blood glucose
levels drop, a response that appears to involve activation of AMPK in
glucose-sensitive neurons within the hypothalamus (McCrimmon et al.
2008). Release of epinephrine also occurs during exercise and other stressful
situations and triggers the “fight or flight response.” As well as altering blood
flow via effects on the heart and vasculature, epinephrine has many metabolic
effects, acting via G-protein-coupled receptors (GPCRs) to increase
intracellular cyclic AMP or calcium (Ch. 1 [Heldin et al. 2014]). In muscle, it
stimulates glycogen and triglyceride breakdown, providing fuels for
accelerated ATP production. In the liver, it promotes release of glucose from
glycogen into the bloodstream. In adipose tissue, it mobilizes triglyceride
stores, the fatty acids derived either being used as catabolic fuels or, in brown
adipose tissue, to generate heat.
The adrenal cortex releases the glucocorticoid cortisol, which (like other
steroid hormones) is synthesized from cholesterol. This also occurs in
response to starvation or stressful conditions, but in this case is triggered by
release of ACTH from the pituitary gland. Acting via nuclear receptors that
directly regulate transcription, glucocorticoids promote gluconeogenesis in
the liver, protein breakdown in muscle, and triglyceride breakdown in
adipose tissue, while reducing insulin-stimulated glucose uptake by muscle.
2.3 The Thyroid Gland
The thyroid gland synthesizes the thyroid hormone thyroxine (also known as
T4 because it contains four iodine atoms), via iodination and subsequent
combination of two molecules of the amino acid tyrosine present within a
large precursor protein called thyroglobulin. Proteolytic breakdown of
thyroglobulin releases T4, but the more potent hormone tri-iodothyronine
(T3) is produced by removal of one iodine atom from T4 in other tissues,
including the liver. T3 acts, like glucocorticoids, by binding to nuclear
receptors that regulate transcription, and has effects on almost all cells.
However, a major effect of T3 on whole body energy homeostasis involves
inhibition of AMPK in the hypothalamus (Lopez et al. 2010). This promotes
firing of sympathetic nerves that trigger release of epinephrine from the
adrenal medulla. This in turn increases energy expenditure by stimulating
white adipose tissue to release fatty acids (which are oxidized in other
tissues) and by promoting fatty acid oxidation and heat production in brown
adipose tissue.

2.4 Pancreatic Islets

The pancreas contains small islands of endocrine tissue called the islets of
Langerhans, which are responsible for the secretion of the hormones insulin
and glucagon. The β cells synthesize insulin, a peptide hormone with two
disulfide-linked chains formed by cleavage of a single precursor polypeptide,
proinsulin. They release insulin in response to high concentrations of glucose
and amino acids that are derived from the gut after feeding, i.e., during “times
of plenty.” Almost all cells in the body express the insulin receptor, which
(like the IGF1 receptor) has two disulfide-linked polypeptide chains, one with
a cytoplasmic tyrosine kinase domain. Binding of insulin to this receptor
activates phosphoinositide (PI) 3-kinase, which synthesizes
phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol
4,5-bisphosphate (PIP2). The membrane lipid PIP3 is a second messenger that
activates the Akt signaling pathway (p. 87 [Hemmings and Restuccia 2012]),
promoting the cellular uptake of glucose and amino acids, as well as
synthesis of fatty acids, and the conversion of these components to forms in
which they are stored, i.e., glycogen, proteins, and triglycerides.
The α cells within the islets release another peptide hormone, glucagon, in
response to low blood glucose during fasting or starvation. Glucagon binds to
Gα-linked receptors that increase cyclic AMP levels in the liver (see below),
causing a switch from glycolysis to glucose production by gluconeogenesis.
It also mobilizes triglyceride stores in adipose tissue, releasing fatty acids into
the bloodstream by the process of lipolysis.

2.5 Adipose Tissue

The main function of adipose tissue is to store triglycerides, but it also acts as
an endocrine organ, releasing peptide hormones termed adipokines that play
key roles in regulating whole body energy balance. One is leptin, a small
globular protein whose concentration is elevated in the blood of obese
individuals, which suggests that it represents a signal that fat reserves are
adequate (Friedman and Halaas 1998). The leptin receptor is a member of the
cytokine receptor family, a single-pass membrane protein that lacks intrinsic
kinase activity but is coupled via Janus kinases (JAKs) to transcription
factors of the STAT family (p. 177 [Harrison 2012]). Leptin receptors are
expressed in the hypothalamus, and their activation represses synthesis of
neuropeptides that promote feelings of hunger, while enhancing synthesis of
those that promote satiety, thus reducing appetite. Unfortunately, many obese
people appear to have become resistant to the appetite-suppressing effects of
leptin.
Another adipokine, adiponectin, is an unusual peptide hormone that has a
globular domain linked to a collagen-like sequence that causes it to form
disulphide-linked trimers as well as higher-order oligomers (Shetty et al.
2009). Although secreted by adipocytes, adiponectin paradoxically displays
high plasma concentrations in lean individuals and low levels in obese
individuals, which suggests that it is a signal indicating that fat stores are low.
Adiponectin binds to two receptors (AdipoRI and AdipoRII) that are
predicted to have seven transmembrane helices, although they differ from
GPCRs in that their amino termini are intracellular (Kadowaki and Yamauchi
2005). In general, adiponectin has catabolic and anti-anabolic effects: binding
to AdipoRI activates AMPK (via a mechanism that remains unclear),
promoting fat oxidation in the liver and muscle, and inhibiting glucose
production by the liver. Adiponectin also increases appetite by activating
AMPK in the hypothalamus, thus opposing the effects of leptin (Kubota et al.
2007).

3 MUSCLE—ACUTE ACTIVATION OF GLYCOGEN

BREAKDOWN
The mechanisms by which target cells respond to the hormones and cytokines
described in the previous section are discussed below. Skeletal muscle
represents the major site of glycogen storage within the body, although
because it lacks glucose-6-phosphatase it cannot release glucose back into the
bloodstream. Muscle glycogen breakdown is therefore used entirely to meet
the energy demands of the muscle itself, and is especially important during
periods of intense exercise. The enzyme phosphorylase uses phosphate to
split the terminal glycosidic linkages of the outer chains of glycogen,
releasing glucose 1-phosphate, which immediately enters glycolysis to
generate ATP. 5′-AMP allosterically activates phosphorylase, with ATP
antagonizing this effect. Thus, phosphorylase should be activated by an
increase in the cellular AMP:ATP ratio, a signal that the energy status of the
cell is compromised (see Box 1). One of the key glycolytic enzymes in
muscle, phosphofructokinase (PFK1), is also allosterically activated by AMP
and inhibited by ATP, so glycolysis should be activated at the same time
(Fig. 2).
Figure 2. Regulation of glycogen breakdown in skeletal muscle. Muscle contraction increases ADP
and AMP and decreases ATP, activating both phosphorylase b (phos b) and phosphofructokinase (PFK)
through allosteric regulation, thus promoting glycogen breakdown and glycolysis to generate ATP.
However, contraction (initiated by firing of motor nerves that release acetyl choline) is triggered by
release of calcium from channels in the sarcoplasmic reticulum membrane, also activating
phosphorylase kinase. The latter phosphorylates phosphorylase and converts it to the a form (phos a),
which no longer requires AMP for activity. Increases in cyclic AMP levels, triggered by binding of
epinephrine to receptors on the plasma membrane, activate cyclic-AMP-dependent protein kinase
(PKA). PKA phosphorylates phosphorylase kinase and amplifies its activation by the calcium-
dependent mechanism.

Phosphorylase occurs not only as the form activated by AMP (called

phosphorylase b), but also as a second form (phosphorylase a) that is
phosphorylated at a serine residue near the amino terminus and active even in
the absence of AMP. The enzyme that catalyzes the b-to-a transition,
phosphorylase kinase, is activated by calcium. When a muscle is stimulated
to contract, the neurotransmitter acetylcholine is released at the specialized
synapse between the motor nerve and the muscle (the neuromuscular
junction). Activation of nicotinic acetylcholine receptors on the muscle cell
then causes firing of action potentials that pass down the transverse tubules,
triggering opening of voltage-gated calcium channels. This in turn causes
opening of calcium-activated calcium channels (ryanodine receptors) on the
sarcoplasmic reticulum, leading to a sudden release of calcium from there
into the cytoplasm (Ch. 13 [Kuo and Ehrlich 2014]). This calcium influx
triggers muscle contraction (creating a massive demand for ATP), while at
the same time activating phosphorylase kinase. Thus, contraction is
synchronized with glycogen breakdown, which helps to satisfy the demand
for ATP. Phosphorylase kinase was the first of >500 mammalian protein
kinases to be identified (Fischer and Krebs 1989). It is a large multisubunit
complex (α4β4 γ4δ4), with the γ subunit carrying the kinase activity and the δ
subunit being a tightly bound molecule of the calcium-binding protein
calmodulin, responsible for activation by calcium. Cyclic-AMP-dependent
protein kinase (PKA), which is activated by increases in cyclic AMP when
epinephrine acts on muscle, phosphorylates both the α and β subunits. This
greatly increases the kinase activity of the γ subunit, while also making the
complex more sensitive to calcium. Note that the PKA → phosphorylase
kinase pathway was the first protein kinase cascade to be described.
Why does phosphorylase need three tiers of regulation, mediated by
AMP, calcium, and cyclic AMP (Fig. 2)? Imagine a mouse suddenly
encountering a cat: if the mouse had no phosphorylase kinase (and therefore
only had the AMP-activated b form of phosphorylase), it could start to run,
but glycogen breakdown would not occur until some ATP had been used up
and AMP had increased. The delay involved in this feedback mechanism
might be fatal. Indeed, humans with muscle phosphorylase kinase deficiency
have been described, and they experience muscle weakness or pain during
exercise. If, however, the mouse had phosphorylase kinase, calcium-
dependent phosphorylation of phosphorylase would now occur, overriding
the allosteric mechanism, and the onset of glycogen breakdown would be
synchronized with the onset of muscle contraction. This feed-forward effect
of calcium would anticipate the demand for ATP and remove the delay
implicit in the allosteric mechanism, thus increasing the chances of escape.
Finally, if the mouse is foraging in a place where it might expect trouble, it
will be nervous and have high levels of circulating epinephrine. This causes
phosphorylation of phosphorylase kinase by PKA, so that when calcium goes
up in response to contraction, the conversion of phosphorylase to the more
active a form is even more rapid, maximizing the chances of escape.

4 MUSCLE—ACUTE REGULATION OF GLUCOSE

UPTAKE AND GLYCOGEN SYNTHESIS
Muscle stores of glycogen are finite and cannot maintain rapid ATP
production for long periods. Prolonged exercise therefore requires increased
uptake of glucose from the bloodstream. In addition, glycogen must be
replenished when exercise terminates, and this also requires increased
glucose uptake. Muscle expresses the “insulin-sensitive” glucose transporter
GLUT4. In the fasted state, it is mainly present in intracellular GLUT4
storage vesicles (GSVs) but insulin released after a meal causes these to fuse
with the plasma membrane, increasing the number of transporters at the
membrane and hence the rate of glucose uptake (Fig. 3). Fusion of GSVs
with the membrane is promoted by small proteins of the Rab family. Under
basal conditions, these are maintained in their inactive GDP-bound form by
proteins with Rab-GTPase activator protein (Rab-GAP) domains that bind to
GSVs. One of these is TBC1D4 (also known as AS160). Binding of insulin to
its receptor causes activation of PI 3-kinase (p. 87 [Hemmings and Restuccia
2012]), causing the formation of PIP3 and thus activation of Akt. Akt
phosphorylates TBC1D4 at multiple sites, causing it to interact with 14-3-3
proteins and dissociate from GSVs. No longer restrained by the Rab-GAP
activity of TBC1D4, Rab proteins are converted to their active, GTP-bound
forms, stimulating fusion of GSVs with the plasma membrane and increasing
glucose uptake (Chen et al. 2011).
Figure 3. Regulation of muscle glucose uptake and glycogen synthesis by insulin and contraction. In
resting muscle in the fed state, insulin binding to its receptor activates the PI-3-kinase → Akt pathway.
Akt phosphorylates the Rab-GAP protein TBC1D4 (AS160) attached to GLUT4 storage vesicles
(GSVs), causing its dissociation and promoting Rab:GTP-mediated fusion of GSVs with the plasma
membrane and increased glucose uptake. This causes accumulation of glucose 6-phosphate (G6P),
which activates glycogen synthase; the latter is also dephosphorylated and activated following
inactivation of GSK3 by Akt. Muscle contraction, by contrast, causes increases in ADP and AMP
levels that activate AMPK. AMPK phosphorylates TBC1D1, causing GSVs to fuse with the membrane.
In this case, G6P does not accumulate because of the demand for ATP, and AMPK also inactivates
glycogen synthase. This drives flux from increased glucose uptake into ATP production rather than
glycogen synthesis.

Glucose uptake triggered by muscle contraction uses a similar

mechanism, but is switched on by different signaling pathways. In this case,
members of the AMPK family (Box 1) are the key mediators. Exercise
consumes ATP and thus increases muscle ADP:ATP and AMP:ATP ratios,
activating AMPK. The use of pharmacological activators of AMPK in
perfused muscle shows that activating AMPK is sufficient for increased
glucose uptake (Merrill et al. 1997), whereas the use of muscle-specific-
AMPK-knockout mice shows that AMPK is necessary for a full response,
although a small response does remain in its absence (O’Neill et al. 2011).
Because a muscle-specific knockout of the upstream kinase for AMPK,
LKB1, does appear to abolish the response (Sakamoto et al. 2005), it is
possible that another kinase downstream from LKB1 compensates when
AMPK is absent. One candidate is the SNF1- and AMPK-related kinase
(SNARK, also known as NUAK2) (Koh et al. 2010), although how it is
activated during muscle contraction remains unclear.
AMPK stimulates glucose uptake, at least in part, by phosphorylating
TBC1D1 (Sakamoto and Holman 2008). As with phosphorylation of its close
relative TBC1D4 (also known as AS160) by Akt, this causes association with
14-3-3 proteins and dissociation from GSVs, promoting their fusion with the
plasma membrane. Because the kinase domains of AMPK and
SNARK/NUAK2 have closely related sequences, SNARK/NUAK2 might
also phosphorylate TBC1D1. Thus, insulin and contraction increase glucose
uptake via parallel signaling pathways that converge on the activation of Rab
proteins.
The effects of insulin on muscle are anabolic, and the increased flux
through GLUT4 is mainly directed into glycogen synthesis. By contrast, the
effects of AMPK are catabolic and the increased flux through GLUT4 is
directed into glycolysis and glucose oxidation instead. How are these
different metabolic fates determined? Insulin stimulates glucose uptake in
resting muscle, when there would not be a large demand for ATP. Glucose 6-
phosphate (G6P) would therefore not be metabolized rapidly and would
accumulate and activate glycogen synthase, for which G6P is a critical
allosteric activator (Bouskila et al. 2010). In addition, glycogen synthase is
inactivated by phosphorylation at carboxy-terminal sites by glycogen
synthase kinase 3 (GSK3). However, in the presence of insulin, Akt
phosphorylates and inactivates GSK3, thus causing a net dephosphorylation
and activation of glycogen synthase (McManus et al. 2005)—this represents a
second mechanism by which insulin stimulates glycogen synthesis. By
contrast, AMPK is activated in contracting muscle, when glycolysis would be
activated and G6P would not accumulate. In addition, AMPK itself
inactivates glycogen synthase by phosphorylating sites distinct from those
phosphorylated by GSK3 (Jorgensen et al. 2004). Thus, whereas insulin
activates glycogen synthase, driving flux from increased glucose uptake into
glycogen synthesis, AMPK inactivates glycogen synthase, driving flux from
increased glucose uptake into glycolysis and glucose oxidation instead (Fig.
3).

5 MUSCLE—ACUTE REGULATION OF FATTY ACID

OXIDATION
During prolonged, low-intensity exercise, ATP is partly generated by the
mitochondrial oxidation of fatty acids. Muscle uptake of plasma fatty acids is
catalyzed by transporters such as CD36 that translocate from intracellular
vesicles to the membrane. Like GLUT4 translocation, this process is
stimulated by AMPK (Bonen et al. 2007), although the mechanism remains
unclear in this case. Fatty acids then enter the mitochondrion for oxidation in
the form of acyl-carnitine esters, which requires a carnitine: palmitoyl
transferase, CPT1, on the outer mitochondrial membrane. CPT1 is inhibited
by malonyl-CoA, a metabolic intermediate produced in muscle by the ACC2
isoform of acetyl-CoA carboxylase. ACC2 is phosphorylated and inactivated
by AMPK, which lowers malonyl-CoA levels and thus stimulates fatty acid
oxidation during exercise (Merrill et al. 1997).

6 MUSCLE—LONG-TERM ADAPTATION TO EXERCISE

Athletes who train for endurance events have elevated mitochondrial content,
allowing them to produce ATP more rapidly by glucose and fatty acid
oxidation. Regular endurance exercise increases mitochondrial biogenesis in
part through effects of AMPK on the transcriptional coactivator PPAR-γ
coactivator-1α (PGC-1α). PGC-1α is recruited to DNA by transcription
factors that bind to promoters of nuclear genes encoding mitochondrial
proteins (Lin et al. 2005). These include the nuclear respiratory factors NRF1
and NRF2, which switch on expression of mitochondrial transcription factor
A (TFAM), a mitochondrial matrix protein required for the replication of
mitochondrial DNA. PGC-1α is also recruited to promoters by PPAR-α,
PPAR-δ, and estrogen-related receptor α (ERR-α), which switch on genes
involved in mitochondrial fatty acid oxidation. One of these encodes pyruvate
dehydrogenase kinase 4 (PDK4) (Wende et al. 2005). By phosphorylating
and inactivating pyruvate dehydrogenase, PDK4 reduces entry of pyruvate
into the TCA cycle and thus favors oxidation of fatty acids rather than
carbohydrates.
AMPK may stimulate PGC-1α in part via direct phosphorylation, which is
proposed to promote its ability to activate its own transcription, in a positive-
feedback loop (Jager et al. 2007). However, AMPK activation also causes
deacetylation of PGC-1α (Canto et al. 2010). Up to 13 lysine residues on
PGC-1α are modified by acetylation, a reaction catalyzed by
acetyltransferases of the GCN5 and SRC families. This causes PGC-1α to
relocalize within the nucleus, inhibiting its transcriptional activity. The acetyl
groups on PGC-1α are removed by the NAD+-dependent deacetylase SIRT1,
which reactivates PGC-1α. AMPK may increase the activity of SIRT1 by
increasing the concentration of cytoplasmic NAD+, although the exact
mechanism remains uncertain. The “nutraceutical” resveratrol, which is
produced by plants in response to fungal infection and is present in small
amounts in red wine, has garnered much interest because it extends lifespan
in nematode worms and in mice fed a high-fat diet (Baur et al. 2006). It was
originally thought to be a direct activator of SIRT1, but it now appears that it
may activate SIRT1 indirectly by inhibiting mitochondrial ATP synthesis and
thus activating AMPK (Hawley et al. 2010; Um et al. 2010).

7 LIVER—ACUTE REGULATION OF CARBOHYDRATE

METABOLISM
During starvation, blood glucose levels must be maintained to provide fuel
for catabolism, particularly in neurons, which cannot use fatty acids. During
short-term fasting, liver glycogen breakdown is the major source of glucose.
Epinephrine (acting via Gq-linked, α1 adrenergic receptors coupled to release
of inositol 1,4,5-trisphosphate [IP3] by phospholipase C) increases
intracellular calcium levels, whereas glucagon (acting via a Gα-linked
receptor) increases the levels of cyclic AMP. Calcium and cyclic AMP then
trigger glycogen breakdown in the liver via essentially the same mechanisms
as those described above for muscle (Fig. 4). One important difference is that
liver cells express glucose-6-phosphatase (and an associated transporter that
carries G6P into the ER lumen), which are required for the release of glucose
derived from glycogen breakdown into the bloodstream (van Schaftingen and
Gerin 2002). The liver also expresses a different isoform of phosphorylase
that is not sensitive to AMP. This is consistent with the view that liver
glycogen is a store of glucose for use by other tissues, rather than for internal
use during periods of energy deficit, as in muscle.

Figure 4. Acute regulation of glycolysis and gluconeogenesis in the liver. Reaction steps unique to
glucose release via gluconeogenesis or glycogenolysis are shown in blue. The steps opposing liver
pyruvate kinase (L-PK) in gluconeogenesis are not shown in detail. During fasting or starvation,
epinephrine and glucagon increase calcium and cyclic AMP (cAMP) levels, activating phosphorylase
kinase and cAMP-dependent protein kinase (PKA), which act together to promote glycogen
breakdown. During starvation, PKA also phosphorylates L-PK and 6-phosphofructo-2-kinase/fructose
2,6-bisphosphatase (PFK2/F2BPase), inactivating the former, and inhibiting the kinase and activating
the phosphatase activity of the latter. This causes a drop in fructose 2,6-bisphosphate levels, which
triggers a net switch from glycolysis to gluconeogenesis.

In the liver, glycolysis is most active in the fed state and is an anabolic
pathway, because it provides precursors for lipid biosynthesis. The liver is
also the major site of gluconeogenesis, the synthesis of glucose from
noncarbohydrate precursors, which is essentially a reversal of glycolysis
except for three irreversible steps in which different reactions are used (blue
arrows in Fig. 4). Gluconeogenesis becomes particularly important as a
source of glucose during starvation, particularly for the brain, which cannot
use fatty acids. The liver therefore must have mechanisms to trigger a switch
from glycolysis to gluconeogenesis during the transition from the fed to the
starved state. A key mediator of this switch is a metabolite that has a purely
regulatory role, fructose 2-6-bisphosphate, which is synthesized and broken
down to fructose 6-phosphate by distinct domains of a single bienzyme
polypeptide termed 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
(PFK2/FBPase) (Fig. 4).
A key step in glycolysis is the conversion of fructose 6-phosphate to
fructose 1,6-bisphosphate, catalyzed by 6-phosphofructo-1-kinase (PFK1).
PFK1 is allosterically activated by fructose 2, 6-bisphosphate, which also
inhibits the opposing reaction in gluconeogenesis, catalyzed by fructose-1,6-
bisphosphatase. On the transition from the fed to the starved state, glucagon
is released, increasing cyclic AMP levels in the liver and activating PKA.
PKA phosphorylates the liver isoform of PFK2/FBPase, inhibiting its kinase
and activating its phosphatase activity (Rider et al. 2004). The consequent
drop in fructose 2,6-bisphosphate levels both reduces PFK1 activation and
relieves inhibition of fructose-1,6-bisphosphatase, causing a net switch from
glycolysis to gluconeogenesis. In addition, PKA phosphorylates and
inactivates the liver isoform of pyruvate kinase (L-PK) (Riou et al. 1978),
causing additional inhibition of glycolysis at a later step (Fig. 4).
On returning to the fed state again, blood glucose increases, glucagon
levels decrease, and the effects just described are reversed. Some of the
increased flux of glucose into the liver caused by the high blood glucose
levels also enters the pentose phosphate pathway, generating the intermediate
xylulose 5-phosphate. Xylulose 5-phosphate has been found to activate a
protein phosphatase that dephosphorylates PFK2/FBPase, thus switching it
back to the state that favors fructose 2,6-bisphosphate synthesis (Nishimura et
al. 1994).
In muscle, where gluconeogenesis is absent and glycolysis has a purely
catabolic role, it would not make sense for hormones that increase cyclic
AMP (such as epinephrine) to inhibit glycolysis. Indeed, muscle expresses
different isoforms of pyruvate kinase and PFK2/FBPase, which lack the PKA
sites.

8 LIVER—LONG-TERM REGULATION OF
GLUCONEOGENESIS VIA EFFECTS ON GENE
EXPRESSION
Another important tier of regulation of glycolysis and gluconeogenesis occurs
at the level of transcription. Although expression of most genes involved in
these pathways is regulated, research has particularly focused on the genes
encoding the catalytic subunit of glucose-6-phosphatase (G6Pc), and
phosphoenolpyruvate carboxykinase (PEPCK) (Yabaluri and Bashyam
2010). Although often referred to as “gluconeogenic genes,” in fact neither is
involved exclusively with that pathway. Thus, glucose-6-phosphatase
releases into the bloodstream glucose derived from glycogen breakdown as
well as gluconeogenesis (Fig. 4), whereas phosphoenolpyruvate produced by
PEPCK is used as a precursor for biosynthesis of products other than glucose,
including glycerol 3-phosphate used in triglyceride synthesis.
Three important hormonal regulators of transcription of these genes are
glucocorticoids and glucagon (which are released during fasting or starvation
and increase transcription) and insulin (which is released after carbohydrate
feeding and represses transcription). The promoters for these genes contain
hormone response units that bind the critical transcription factors and are
most well defined in the case of the G6Pc promoter (Fig. 5).
Figure 5. Regulation of the G6Pc promoter, showing the approximate location of elements binding the
key transcription factors. Glucocorticoids such as cortisol, in complex with the glucocorticoid receptor
(GR), bind to three sites within the glucocorticoid response unit, enhancing transcription. Cyclic AMP-
dependent protein kinase (PKA) phosphorylates cyclic AMP response element binding protein (CREB),
recruiting CREB-binding protein (CBP), and activating transcription. Finally, Akt phosphorylates
FoxO at multiple sites, triggering the binding of 14-3-3 proteins and their nuclear exclusion, thus
inhibiting transcription. Not shown are the roles of coactivators other than CBP described in the text,
i.e., PGC-1α and CRTC2.

The Glucocorticoid Response Unit. The promoter contains three

glucocorticoid response elements (GREs) that bind the glucocorticoid-
receptor complex, which activates transcription. However, accessory
elements that bind additional transcription factors, including hepatocyte
nuclear factors (HNF1/3β/4α/6) and FOXO1, are necessary for a full
response.
The Cyclic AMP Response Unit. The promoter contains two cyclic-AMP-
response elements (CREs), sequence elements that bind the transcription
factor CRE-binding protein (CREB). Glucagon activates PKA, which
phosphorylates CREB at 133, promoting binding of the CBP transactivator to
activate transcription.
The Insulin Response Unit. The promoter contains two insulin-response
elements that bind FoxO1, a transcription factor whose loss leads to
decreased expression of both PEPCK and G6Pc on fasting. FoxO1, along
with other members of the forkhead box family, is phosphorylated at multiple
conserved sites by Akt, causing its relocalization from the nucleus to the
cytoplasm owing to binding of 14-3-3 proteins (Brunet et al. 1999). Insulin
also induces expression of COP1, an E3 ubiquitin ligase that promotes
FoxO1 degradation by the proteasome (Kato et al. 2008).
Transcription of these genes is also regulated by modulation of various
coactivators not shown in Fig. 5, including PGC-1α. The PGC-1α promoter
contains CREs, and in the liver PGC-1α is a cyclic-AMP-induced gene
activated by glucagon. PGC-1α promotes PEPCK expression in part by
interacting with the glucocorticoid receptor. The NAD+-dependent
deacetylase SIRT1 is also induced in the liver on fasting and, as described
above, it deacetylates PGC-1α, increasing transcription of target genes.
SIRT1 also deacetylates FOXO1, thus enhancing transcription of G6Pc.
Interestingly, some of these signaling events appear to have arisen during
early eukaryotic evolution. In the nematode worm Caenorhabditis elegans,
restricting the diet in early life switches development to the long-lived Dauer
larval form, and this can be mimicked by mutations in genes encoding
orthologues of the insulin/IGF1 receptor (daf-2) or PI-3-kinase (age-1), or
overexpression of orthologues of SIRT1 (Sir-2.1) and FoxO1 (Daf-16)
(Tissenbaum and Guarente 2001). Thus, dietary restriction extends life span
in part by preventing activation of the insulin-like receptor → PI-3-kinase →
Akt pathway, which in turn inhibits the transcription factor FoxO1 (Daf-16)
by triggering its phosphorylation and acetylation. Dietary restriction also
activates the C. elegans orthologue of AMPK, which activates FoxO1 by
phosphorylation at sites different from those targeted by Akt (Greer et al.
2007), and perhaps also by deacetylation. All of these pathways are
conserved in mammals, and there is currently intense interest as to whether
they regulate mammalian life span in the same manner.
Another transcriptional coactivator that regulates PEPCK expression is
CREB-regulated transcription coactivator 2 (CRTC2, formerly called
TORC2), which is recruited to the PEPCK promoter by CREB. CRTC2 is
phosphorylated by the protein kinase salt-inducible kinase 1 (SIK1), which
triggers binding of 14-3-3 proteins and its relocation from the nucleus to the
cytoplasm. However, PKA phosphorylates SIK1, causing its relocalization
from the nucleus to the cytoplasm, thus preventing CRTC2 phosphorylation
and promoting PEPCK expression. SIK1 is a member of the AMPK-related
kinase family and AMPK also phosphorylates CRTC2 at the same site,
explaining how AMPK activation switches off PEPCK expression.
Interestingly, phosphorylation of the CRTC2 orthologue by AMPK is
conserved in C. elegans, and is required for extension of life span by AMPK
(Mair et al. 2011).
When would AMPK switch off gluconeogenesis? Adiponectin activates
AMPK in the liver via the AdipoRI receptor (see above). This explains how it
inhibits hepatic glucose production, and why low adiponectin levels in obese
humans correlate with elevated liver glucose production. In addition, AMPK
is activated by the antidiabetic drug metformin (see below), which lowers
blood glucose levels mainly by repressing gluconeogenesis.

9 LIVER—REGULATION OF FATTY ACID,

TRIGLYCERIDE, AND CHOLESTEROL METABOLISM
Liver cells carry out both the synthesis and oxidation of fatty acids, and
express both isoforms of acetyl-CoA carboxylase (ACC1 and ACC2). By
phosphorylating ACC1 and ACC2 at conserved sites to cause their
inactivation, AMPK switches off fatty acid synthesis to conserve energy,
while switching on fatty acid oxidation to generate more ATP (Hardie 2007).
This occurs, for example, when AMPK is activated in the liver by
adiponectin. AMPK activation also causes inactivation of glycerol phosphate
acyl transferase (GPAT), the first enzyme in the pathway of triglyceride and
phospholipid synthesis, although it has not yet been shown to be a direct
target for AMPK. Finally, AMPK inhibits cholesterol synthesis by
phosphorylation of HMG-CoA reductase (ACC1 and HMG-CoA reductase
were, in fact, the first AMPK targets to be identified (Munday et al. 1988;
Clarke and Hardie 1990).
In addition to these acute effects, fatty acid and triglyceride synthesis are
regulated in the longer term at the level of gene expression. The genes
targeted include those whose products are involved directly in these pathways
(ACC1, fatty acid synthase, stearoyl-CoA desaturase and GPAT), in the
glycolytic pathway that provides the precursors for lipid synthesis
(glucokinase, PFK1, aldolase, and L-PK), and in pathways that provide
NADPH for the reductive steps in lipid synthesis (glucose-6-phosphate
dehydrogenase and malic enzyme). These are referred to collectively as
lipogenic enzymes, and their expression is up-regulated by carbohydrate
feeding, so that excess dietary carbohydrates are converted to triglycerides.
The latter are then exported from the liver as very-low-density lipoproteins
(VLDL) and carried to adipose tissue for long-term storage.
Carbohydrate feeding causes an increase in blood glucose that also
triggers insulin release. Studies with cultured liver cells suggest that increases
in both glucose and insulin are necessary for the increased transcription of
most lipogenic genes. A transcription factor involved in the effects of insulin
is sterol response element-binding protein 1c (SREBP1c). SREBP1a and
SREBP1c are derived from the same gene by use of alternate transcription
start sites and are closely related to the product of another gene, SREBP2
(Raghow et al. 2008). All three have amino-terminal transcription factor
domains (TFDs) linked to carboxy-terminal regulatory domains by two
transmembrane α helices that anchor them within the endoplasmic reticulum
membrane (Fig. 6). The TFDs are released from the membrane by regulated
proteolytic processing; the TFD from SREBP1a/1c targets mainly lipogenic
genes, whereas that from SREBP2 targets genes involved in cholesterol
biosynthesis and uptake (including HMG-CoA reductase).

Figure 6. Regulation of processing of SREBPs. The precursor forms of SREBPs bind to the membrane
protein SCAP through interactions between their carboxy-terminal regulatory domain (RD) and the
WD repeat domain (WDD) of SCAP. The SREBP-SCAP complex is retained in the endoplasmic
reticulum by interaction with Insigs. Reduced binding of sterols to the sterol-binding domain (SBD) of
Insig1 and the sterol sensor domain (SSD) of SCAP causes their dissociation, and the SCAP-SREBP2
complex then translocates to the Golgi, where the site 1 and site 2 proteases (S1P and S2P) cleave
SREBP2, releasing the transcription factor domain (TFD) that translocates to the nucleus. Regulation of
SREB1c is similar, except that there appears to be multiple mechanisms that trigger its release from the
ER, including insulin-induced degradation of Insig2.

The carboxy-terminal domains of SREBPs bind to ER membrane proteins

called SREB cleavage activator protein (SCAP) and Insigs (Insig1/Insig2).
When membrane sterol levels are high, they bind to SCAP and Insig1,
causing SREBP2 to be retained within the ER. Conversely, when membrane
sterol levels are low, SCAP dissociates from Insig2 and the SCAP–SREBP2
complex moves to the Golgi apparatus, where proteinases release the TFD
(Yang et al. 2002). Whereas SREBP2 appears to be regulated by sterols
mainly at the proteolytic processing step (i.e., sterols inhibit processing),
insulin appears to regulate SREBP1c at additional levels, enhancing its
transcription and reducing its degradation, and enhancing degradation of
Insig2.
Mice lacking SREBP1c show reduced expression of most lipogenic
genes, but their response to carbohydrate feeding is not entirely eliminated.
This suggests that other transcription factors also play a role. It has been
proposed that the response of lipogenic genes to glucagon and fatty acids
(which down-regulate their expression), and high glucose (which induces
expression), involves the carbohydrate response element-binding protein
(ChREBP) (Uyeda and Repa 2006), a transcription factor with a DNA-
binding domain related to that of SREBP-1. ChREBP is phosphorylated at
two sites by PKA in response to glucagon, which prevents its nuclear import
and inhibits promoter binding. Phosphorylation of a third site by AMPK also
inhibits promoter binding and may account for down-regulation of lipogenic
genes by adiponectin. It has also been proposed that generation of AMP
during conversion of fatty acids to their CoA esters could activate AMPK and
explain the effects of fatty acids on lipogenic gene expression. One
hypothesis to explain the effect of high glucose is that it is metabolized by the
pentose phosphate pathway to xylulose 5-phosphate, which activates the
phosphatase that dephosphorylates PFK2/FBPase (see above). This
phosphatase is also thought to dephosphorylate the PKA/AMPK sites on
ChREBP, promoting its nuclear localization and promoter binding
(Kabashima et al. 2003).

10 ADIPOCYTES—REGULATION OF FATTY
ACID METABOLISM
Although subcutaneous fat provides thermal insulation, the main metabolic
function of adipocytes is to store fatty acids as triglycerides, neutral lipids
that are very insoluble in water and are deposited in lipid droplets. White
adipocytes, unlike other cells, have a single central triglyceride droplet that
occupies almost the entire volume of the cell. The phospholipid monolayer
that forms its cytoplasmic face is lined with a protein called peripilin1
(Brasaemle 2007). To release fatty acids back into the circulation, the
triglycerides in the lipid droplet must be hydrolyzed back to free fatty acids
(lipolysis). Three lipases are involved, which remove the first, second, and
third fatty acids: (1) adipose tissue triglyceride lipase (ATGL), (2) hormone-
sensitive lipase (HSL), and (3) monacylglycerol lipase. Lipolysis is greatly
enhanced during fasting by glucagon and/or epinephrine, acting via increases
in cyclic AMP; insulin opposes this because Akt phosphorylates and activates
the cyclic AMP phosphodiesterase PDE3B, thus lowering cyclic AMP levels
(Berggreen et al. 2009). HSL is directly phosphorylated and activated by
PKA, although the effect on activity is modest (about twofold) compared
with the effects on lipolysis (at least 100-fold). Phosphorylation of HSL also
triggers its translocation from the cytoplasm to the lipid droplet, thus
increasing its accessibility to substrate (Clifford et al. 2000). However, some
hormone-stimulated release of fatty acids still occurs even in adipocytes from
HSL-deficient mice (Haemmerle et al. 2002). This may be because perilipin1,
which seems to regulate access of lipolytic enzymes to the surface of the lipid
droplet, is also phosphorylated by PKA (Clifford et al. 2000). Adipocytes
from perilipin1 knockout mice have a high basal lipolytic rate that is only
marginally stimulated by cyclic-AMP-elevating agents (Tansey et al. 2001).
The crucial effect of PKA may therefore be to phosphorylate perilipin1,
which alters the accessibility of triglycerides within the lipid droplet to both
ATGL and HSL.
Activation of AMPK opposes the effects of epinephrine and glucagon on
lipolysis, in part because it phosphorylates HSL at sites close to the PKA
sites, antagonizing the activation and translocation induced by PKA (Daval et
al. 2005). Whether AMPK also antagonizes the effect of phosphorylation of
perilipin1 by PKA remains unclear. It might appear paradoxical that AMPK
inhibits lipolysis, because fatty acids are an excellent fuel for oxidative
catabolism. However, the fatty acids produced by lipolysis are not usually
oxidized within the adipocyte, but are released for use elsewhere. If the fatty
acids generated by lipolysis are not rapidly removed from adipocytes either
by export or by oxidative metabolism, they are recycled into triglycerides, an
energy-intensive process in which two molecules of ATP are consumed per
fatty acid. Thus, inhibition of lipolysis by AMPK may ensure that the rate of
lipolysis does not exceed the rate at which the fatty acids can be removed
from the system.

11 BROWN ADIPOCYTES—REGULATION OF
FATTY ACID OXIDATION AND HEAT
PRODUCTION
Brown adipocytes are so called because, unlike white fat cells, they have
abundant mitochondria containing cytochromes that produce their
characteristic color. Increases in cyclic AMP levels induced by epinephrine
trigger lipolysis as in white fat cells, but brown adipocytes differ in that the
fatty acids are not released but are oxidized within their own mitochondria.
Another unique feature of brown adipocytes is that they express uncoupling
protein1 (UCP1), which dissipates the electrochemical gradient produced by
pumping of protons across the inner mitochondrial membrane by the
respiratory chain. The energy expended in brown fat cells therefore mainly
appears in the form of heat, rather than ATP. This heat-generating system is
particularly important in neonatal animals (including humans), but also
occurs in adult rodents exposed to cold environments. Once thought to be
absent in adult humans, improved methodology has shown that brown fat
does indeed occur (van Marken Lichtenbelt et al. 2009). These findings have
rekindled interest in the idea that regulation of energy expenditure by brown
fat might be a way of controlling obesity.

12 INSULIN RESISTANCE AND TYPE 2

DIABETES—A RESPONSE TO
OVERNUTRITION?
Insulin resistance is a condition in which tissues become resistant to the
effects of the hormone, and individuals with type 2 diabetes have a high
fasting blood glucose level caused primarily by insulin resistance (rather than
lack of insulin as in type 1 diabetes). In insulin-resistant individuals, muscle
takes up less glucose in response to insulin, and the hormone is also less
effective at suppressing glucose production by the liver. Blood glucose
therefore remains elevated for longer periods after a carbohydrate meal, a
condition known as glucose intolerance. Many individuals who are insulin
resistant compensate by secreting more insulin so that, although they may
show glucose intolerance, in the fasting state their blood glucose is within the
normal range such that they are not classed as diabetic. However, this
compensation mechanism may eventually fail, and insulin-resistant
individuals often become diabetic.
As the world has become more developed and urbanized, there has been
an alarming increase in the prevalence of type 2 diabetes. By 2025 it is
predicted that the number with the disorder will increase to >300 million
(nearly 4% of the world population). The increase is particularly evident in
countries where economic development has been very rapid, like China,
where in 2010 >90 million adults were estimated to have diabetes (Yang et
al. 2010).
What is the reason for this dramatic increase? Type 2 diabetes is strongly
associated with obesity: In one large study, females who were obese (body
mass index [BMI] > 30 kg/m2) had a 20-fold higher risk of developing
diabetes compared with those who were lean (BMI < 23 kg/m2) (Hu et al.
2001). This is a serious problem, because over one-quarter of the U.S.
population now have a BMI > 30. Obesity is caused by excessive energy
intake (overnutrition) and/or reduced energy expenditure (less physical
activity). At the cellular level, insulin resistance can be regarded as a
response to excessive storage of nutrients, especially lipids, and it can be
reproduced in vitro by incubating cells with high concentrations of glucose or
fatty acids. Excessive storage of triglycerides appears to be a particular
culprit (Samuel et al. 2010). This is dramatically illustrated by lipodystrophy,
a lack of white adipose tissue that can be caused either by rare genetic
disorders, or as a side effect of antiviral drugs used to treat AIDS patients
(Garg 2004). In both cases, excessive amounts of triglyceride are stored in
the liver and muscle instead, and these tissues become profoundly insulin
resistant. It appears that large amounts of triglyceride can be stored with
relative safety in adipocytes, but that, if stored elsewhere, they become very
damaging. Indeed, the tendency to insulin resistance in obese people may be
because their adipose tissue stores are already full, leading to increased lipid
storage (steatosis) in the liver and muscle.
The current frontline drug used to treat type 2 diabetes is metformin,
prescribed to more than 100 million people in 2010. Derived from an ancient
herbal remedy, it activates AMPK, which it does either by inhibiting complex
I of the mitochondrial respiratory chain (Owen et al. 2000) or by inhibiting
AMP deaminase (Ouyang et al. 2011), both of which increase the cellular
AMP:ATP and ADP:ATP ratios (Hawley et al. 2010). By mechanisms
already discussed, AMPK activation inhibits synthesis and storage of fat and
promotes its oxidation instead, promotes glucose uptake and oxidation by
muscle, and inhibits gluconeogenesis in the liver. All of these effects would
be beneficial in individuals with insulin resistance and/or type 2 diabetes.
There remains some controversy as to whether AMPK activation explains all
therapeutic effects of metformin (Foretz et al. 2010). Nevertheless, it is
intriguing that a drug used to treat diabetes activates a signaling pathway
switched on by exercise. There is much evidence to show that regular
exercise protects against development of insulin resistance, and conversely
that physical inactivity is a factor contributing to its occurrence. Thus,
metformin may at least partly be acting as an exercise mimetic.
There is also much debate about the underlying causes of insulin
resistance at the molecular level. Disturbances in lipid metabolites,
inflammatory mediators, and adipokines have all been proposed. Whatever
the underlying mechanisms, insulin resistance is likely to be a feedback
mechanism that has evolved to limit nutrient uptake under conditions when
cellular stores of nutrients are already replete. The key to understanding
insulin resistance may lie in working out how cells detect when their nutrient
stores are full. Although leptin and adiponectin appear to be released from
adipocytes when their triglyceride stores are high and low, respectively, we
currently know very little at the molecular level about how the level of
intracellular stores of lipids (or other nutrients, like glycogen) are monitored.

13 CONCLUDING REMARKS
Energy balance in multicellular organisms involves a complex interplay
between energy-utilizing tissues such as muscle, energy-storing tissues such
as adipose tissue, and organs involved in metabolic coordination such as the
liver. These tissues signal to each other via hormones and cytokines that are
either secreted by specialized endocrine cells (e.g., the hypothalamus, islets
of Langerhans, pituitary, thyroid, and adrenal glands) or by the tissues
themselves (e.g., adipokines, released by adipocytes). These hormones either
act at receptors that switch on protein kinase signaling cascades triggered by
second messengers such as PIP3 (insulin), calcium (epinephrine acting at α1
receptors) or cyclic AMP (glucagon), or bind to nuclear receptors that are
transcription factors (cortisol and T3). These signaling cascades interact with
other signaling pathways involved in regulating energy balance at the cell-
autonomous level (e.g., AMPK). The net effect is modulation of carbohydrate
and lipid metabolism, both by direct phosphorylation of metabolic enzymes
and by effects of gene expression or protein turnover. One important
remaining challenge is to understand how cells monitor their levels of energy
reserves such as triglyceride, a process likely to be important in
understanding disorders such as obesity and type 2 diabetes.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006031
CHAPTER 15

Signaling in Innate Immunity and

Inflammation

Kim Newton and Vishva M. Dixit

Department of Physiological Chemistry, Genentech, Inc., South San
Francisco, California 94080
Correspondence: dixit@gene.com

SUMMARY

Inflammation is triggered when innate immune cells detect infection or

tissue injury. Surveillance mechanisms involve pattern recognition
receptors (PRRs) on the cell surface and in the cytoplasm. Most PRRs
respond to pathogen-associated molecular patterns (PAMPs) or host-
derived damage-associated molecular patterns (DAMPs) by triggering
activation of NF-κB, AP1, CREB, c/EBP, and IRF transcription factors.
Induction of genes encoding enzymes, chemokines, cytokines, adhesion
molecules, and regulators of the extracellular matrix promotes the
recruitment and activation of leukocytes, which are critical for
eliminating foreign particles and host debris. A subset of PRRs activates
the protease caspase 1, which causes maturation of the cytokines IL1β
and IL18. Cell adhesion molecules and chemokines facilitate leukocyte
extravasation from the circulation to the affected site, the chemokines
stimulating G-protein-coupled receptors (GPCRs). Binding initiates
signals that regulate leukocyte motility and effector functions. Other
 triggers of inflammation include allergens, which form antibody
complexes that stimulate Fc receptors on mast cells. Although the role
of inflammation is to resolve infection and injury, increasing evidence
indicates that chronic inflammation is a risk factor for cancer.

Outline
1 Introduction
2 DAMPs and PAMPs trigger the innate immune response
3 Toll-like receptors (TLRs)
4 RIG-I-like receptors (RLRs)
5 Nod-like receptors (NLRs)
6 The proinflammatory cytokine tumor necrosis factor (TNF)
7 Selectins and integrins
8 G-protein-coupled receptors (GPCRs)
9 Fc receptors
10 Inflammation as a risk factor for cancer
11 Concluding remarks
References

1 INTRODUCTION
The role of the inflammatory response is to combat infection and tissue
injury. Innate immune cells residing in tissues, such as macrophages,
fibroblasts, mast cells, and dendritic cells, as well as circulating leukocytes,
including monocytes and neutrophils, recognize pathogen invasion or cell
damage with intracellular or surface-expressed pattern recognition receptors
(PRRs). These receptors detect, either directly or indirectly, pathogen-
associated molecular patterns (PAMPs), such as microbial nucleic acids,
lipoproteins, and carbohydrates, or damage-associated molecular patterns
(DAMPs) released from injured cells. Activated PRRs then oligomerize and
assemble large multisubunit complexes that initiate signaling cascades that
trigger the release of factors that promote recruitment of leukocytes to the
region.
Vascular alterations play an important role in the inflammatory response
(Fig. 1). Histamine, prostaglandins, and nitric oxide act on vascular smooth
muscle to cause vasodilation, which increases blood flow and brings in
circulating leukocytes, whereas inflammatory mediators including histamine
and leukotrienes act on endothelial cells to increase vascular permeability and
allow plasma proteins and leukocytes to exit the circulation. Cytokines such
as tumor necrosis factor (TNF) and interleukin 1 (IL1) promote leukocyte
extravasation by increasing the levels of leukocyte adhesion molecules on
endothelial cells. Activated innate immune cells at the site of infection or
injury, including dendritic cells, macrophages, and neutrophils, remove
foreign particles and host debris by phagocytosis, plus they also secrete
cytokines that shape the slower, lymphocyte-mediated adaptive immune
response.
Figure 1. Cells and mediators of the inflammatory response. Molecules derived from plasma proteins
and cells in response to tissue damage or pathogens mediate inflammation by stimulating vascular
changes, plus leukocyte migration and activation. Granulocytes include neutrophils, basophils, and
eosinophils.

Below we examine how PRRs signal recognition of infection and injury.

We then describe how the ensuing inflammatory response is amplified by the
cytokines TNF and IL1β. Next, we discuss the mechanisms that get
leukocytes to where they are needed. Finally, we consider inflammatory
signaling pathways triggered during allergic or hypersensitivity reactions and
the possibility that chronic inflammation promotes tumor development.

2 DAMPs AND PAMPs TRIGGER THE INNATE IMMUNE

RESPONSE
DAMPs are endogenous molecules normally found in cells that get released
during necrosis and contribute to sterile inflammation. They include ATP, the
cytokine IL1α, uric acid, the calcium-binding, cytoplasmic proteins S100A8
and S100A9, and the DNA-binding nuclear protein HMGB1. Amyloid β
fibrils associated with Alzheimer’s disease have also been shown to be
proinflammatory. PAMPs, in contrast, are pathogen-derived, often essential
for microbe survival, and, like DAMPs, structurally diverse. PAMPs include
bacterial and viral nucleic acids, fungal β-glucan and α-mannan cell wall
components, the bacterial protein flagellin, components of the peptidoglycan
bacterial cell wall, and lipopolysaccharide (LPS) from Gram-negative
bacteria.

3 TOLL-LIKE RECEPTORS (TLRs)

Members of the TLR family are major PRRs in cells. They are type I
transmembrane proteins containing leucine-rich repeats (LRRs) that
recognize bacterial and viral PAMPs in the extracellular environment (TLR1,
TLR2, TLR4, TLR5, TLR6, and TLR11) or endolysosomes (TLR3, TLR7,
TLR8, TLR9, and TLR10). The ligands identified for the different TLRs are
listed in Table 1. Signal transduction by TLRs relies on a cytoplasmic
Toll/IL1 receptor (TIR) domain that serves as the docking site for TIR-
containing cytoplasmic adaptor proteins (Fig. 2). TIR domains in the receptor
for the proinflammatory cytokine IL1 function in a similar fashion.

Table 1. Agonists of mouse and human Toll-like receptors

TLR Ligand
1/2 Triacyl lipopeptides
2/6 Diacyl lipopeptides
3 dsRNA
4 Lipopolysaccharide
5 Flagellin
7 ssRNA
8 ssRNA in humans; unclear in mice
9 CpG DNA, malarial hemozoin
10a Unknown

11b Uropathogenic bacteria, Toxoplasma gondii profilin-like protein

12b Unknown

13b Unknown

aExpressed only in humans.

bExpressed only in mice.
Figure 2. Signaling by TLR4. (A) Domain structure of human TLR4. (B) Binding of LPS to TLR4 and
the coreceptor MD2 triggers interactions between the cytoplasmic TIR domain of TLR4 and TIR-
containing adaptor proteins (Mal, MyD88, and TRAM). MyD88 binds IRAK4, which requires its
kinase activity to bind the kinases IRAK1 and IRAK2 sequentially. The MyD88–IRAK complex also
engages the ubiquitin ligase TRAF6 to make polyubiquitin chains that activate the IKK complex for
NF-κB- and ERK-dependent gene transcription. Ubiquitin ligases cIAP1 and cIAP2 recruited to the
TLR4 signaling complex regulate translocation of a subset of signaling components to the cytoplasm,
where TAK1 activation initiates a MAPK cascade that stimulates gene expression. TLR4 activated at
the plasma membrane is endocytosed but can signal within the endosomal compartment via the
adaptors TRAM and TRIF. The kinase and ubiquitin ligase combination of RIP1 and Peli1 interacts
with TRIF to signal NF-κB activation, whereas TBK1 and TRAF3 stimulate IRF3-dependent
transcription. (C) Functional outputs of some of the genes up-regulated by TLR4 signaling.

3.1 MyD88-Dependent TAK1 and IKK Activation by TLRs

With the exception of TLR3, all known TLRs engage the adaptor MyD88
either directly (TLR5, TLR7, TLR8, TLR9, TLR10, and TLR11,
heterodimeric TLR1-TLR2 and TLR2-TLR6, and the IL1Rs) or in
combination with the adaptor TIRAP/Mal (TLR1-TLR2, TLR2-TLR6, and
TLR4). MyD88 contains a death domain (DD) in addition to a TIR domain,
and this mediates interactions with the DD of the serine/threonine kinase
IRAK4 (Lin et al. 2010). Clustering of IRAK4 within the receptor complex
probably results in its autophosphorylation. The kinase activity of IRAK4 is
required for its DD to bind the DD of the related kinases IRAK1 and IRAK2.
MyD88 and the IRAKs nucleate a larger complex, which in the case of TLR4
includes E3 ubiquitin ligases (TRAF6, cIAP1, and cIAP2) and the E2
ubiquitin-conjugating enzyme Ubc13 (Tseng et al. 2010). TRAF6 and Ubc13
catalyze the formation of polyubiquitin chains in which the carboxyl terminus
of one ubiquitin forms an isopeptide bond with the ε-amino group of K63 of
an adjacent ubiquitin. TRAF6 can build polyubiquitin chains on lysines
within itself and IRAK1, and it also promotes K63-linked polyubiquitylation
of cIAPs (Skaug et al. 2009; Tseng et al. 2010). These K63-linked chains are
thought to recruit the adaptor proteins TAB2 and TAB3, which exist in a
complex with the kinase TAK1. The regulatory subunit of the IκB kinase
(IKK) complex, known as IKKγ or NEMO, also binds K63-linked
polyubiquitin and is recruited to the TLR4 signaling complex (Laplantine et
al. 2009; Tseng et al. 2010).
Analyses of knockout mice indicate that both IKK and TAK1 are
important for activation of NF-κB transcription factors, whereas only TAK1
is required for activation of the mitogen-activated protein kinases (MAPKs)
p38α and JNK (Sato et al. 2005; Shim et al. 2005; Israel 2010). How TAK1
and IKK are activated, however, requires further clarification. Unanchored
K63-linked polyubiquitin chains synthesized by TRAF6 and Ubc13 were
proposed to activate TAK1 by inducing its autophosphorylation (Xia et al.
2009). Although TAK1 can phosphorylate the activation loops in the kinases
IKKβ and MKK6 in vitro, the latter a MAPK kinase upstream of p38α
(Skaug et al. 2009), it is not clear whether TAK1 phosphorylates IKKβ in
cells. A recent study suggested that TLR4-induced TAK1
autophosphorylation and activation, but not IKK activation, require
translocation of the MyD88–TRAF6–Ubc13–cIAP–TAK1–IKKγ signaling
complex from TLR4 into the cytosol (Tseng et al. 2010). This translocation
depends on TRAF6 and the cIAPs. Studies of gene-targeted mice expressing
a kinase-dead version of TAK1 would clarify whether TAK1 requires its
kinase activity to activate IKK.
IKK activation, like TAK1 activation, has been linked to polyubiquitin
binding, but the chain linkages and the key E2/E3 ubiquitin enzyme
combinations appear to differ. Both unanchored polyubiquitin formed by
TRAF6 with the E2 UbcH5c (Xia et al. 2009) and linear polyubiquitin
conjugated to IKKγ (Rahighi et al. 2009; Tokunaga et al. 2009) have been
shown to induce IKK activation. The linear ubiquitin chain assembly
complex (LUBAC) joins ubiquitins in a head-to-tail fashion with the
carboxy-terminal glycine residue of one ubiquitin linked to the amino
terminus of another ubiquitin. E2 enzymes that support LUBAC activity in
vitro include UbcH5c and UbcH7 (Gerlach et al. 2011; Ikeda et al. 2011;
Tokunaga et al. 2011). Binding of the carboxy-terminal UBAN domain in
IKKγ to linear ubiquitin chains may produce conformational changes
necessary for IKK activation. The importance of IKKγ binding to
polyubiquitin is supported by the identification of mutations in the UBAN
domain that impair NF-κB activation and cause immunodeficiency in humans
(Table 2) (Döffinger et al. 2001). These studies may explain why NF-κB
activation is largely normal in the absence of Ubc13 while MAPK activation
is compromised (Yamamoto et al. 2006).

Table 2. Genes regulating inflammation that are mutated in human disease

Gene Protein Disease
CIAS1 NLRP3 Familial cold autoinflammatory syndrome; Muckle–Wells syndrome; neonatal-
onset multisystem inflammatory disease
IKBKG IKKγ Anhidrotic ectodermal dysplasia with immunodeficiency
NOD2 NOD2 Crohn’s inflammatory bowel disease; Blau syndrome characterized by arthritis
and uveitis
TNFAIP3 A20 B-cell lymphomas

The MAPKs JNK and p38α, like TAK1, are activated downstream from
TLR2 and TLR4 in a cIAP-dependent manner. The TAK1-containing
signaling complex translocates into the cytosol and recruits the kinase MKK4
to phosphorylate and activate JNK (Tseng et al. 2010). It probably also
recruits MKK3 and MKK6 to activate p38α because both kinases associate
with TRAF6 in response to LPS (Wan et al. 2009). p38α is required for
activation of the transcription factors CREB and c/EBPβ, and it contributes to
the induction of several genes, including those encoding chemokines (Cxcl1,
Cxcl2), cytokines (IL10, IL12b, IL1a, and IL1b), and regulators of
extracellular matrix remodeling (Mmp13) and cell adhesion (Vcam1) (Kang
et al. 2008; Kim et al. 2008). JNK regulates the activity of the AP1
transcription factor and stimulates expression of proinflammatory mediators
such as TNF (Das et al. 2009).
Activation of the IKK complex is required for NF-κB-dependent
transcription as well as transcriptional responses downstream from the
MAPK ERK. IKKβ substrates include p105, the precursor of the p50 NF-κB1
transcription factor, as well as the IκB proteins that sequester NF-κB
transcription factors in the cytosol. Phosphorylation by IKKβ targets these
substrates for K48-linked polyubiquitylation by the E3 ubiquitin ligase SCFβ-
TrCP and subsequent proteasomal degradation (Kanarek et al. 2010).
Degradation of p105, which exists in a complex with the kinase Tpl2,
activates a Tpl2–MEK1–ERK kinase cascade that leads to the induction of
genes such as Ptgs2 by the CREB/ATF family of transcription factors
(Banerjee and Gerondakis 2007). The cyclooxygenase 2 (COX2) enzyme
encoded by Ptgs2 is involved in the synthesis of prostaglandins, which are
important mediators of pain, inflammation, and fever. IκB degradation allows
dimeric NF-κB transcription factors composed largely of RelA (p65) and NF-
κB1 (p50) subunits to accumulate in the nucleus and drive expression of a
large number of proinflammatory genes (Table 3 lists a subset of these
genes).

Table 3. Genes induced by the canonical IKKβ/NF-κB signaling pathway

Genea Protein Function
Ager RAGE PRR belonging to the immunoglobulin superfamily that recognizes
multiple ligands including HMGB1
Birc3 cIAP2 Ubiquitin ligase that regulates NF-κB activation
Casp4 Caspase 11 Aspartate-specific cysteine protease implicated in inflammation
Ccl2 MCP1 Chemokine for monocyte recruitment
Ccl3 MIP1α Chemokine for leukocyte recruitment
Ccl5 RANTES Chemokine for monocyte and T-cell recruitment
Cd200 CD200 Binds CD200R1 and inhibits macrophage activation
Cfb Complement Serine protease in the alternative complement activation pathway
factor B
Cflar c-FLIP Inhibitor of death receptor-induced apoptosis
Csf2 GM-CSF Growth factor that promotes differentiation and activation of DCs,
macrophages, and neutrophils
Cxcl1 KC Chemokine for neutrophil recruitment
Cxcl2 MIP2 Chemokine for neutrophil recruitment
F3 Tissue factor Coagulation factor
Icam1 ICAM1 Cell adhesion molecule that interacts with β2 integrins
Ifnb1 IFNβ Suppressor of virus replication
Il1b IL1β Cytokine that amplifies the inflammatory response
Il6 IL6 Pleiotropic cytokine that stimulates fever, production of hepatocyte acute
phase proteins, and lymphocyte differentiation
Il12b IL12 p40 Component of heterodimeric IL12 and IL23, which modulates NK cells
and lymphocyte effector functions
Mmp9 MMP9 Metalloproteinase that degrades extracellular matrix
Nfkbia IκBα Inhibitor of NF-κB signaling
Nfkbib IκBβ NF-κB transcriptional coactivator
Nos2 iNOS Enzyme that makes antimicrobial nitric oxide
Sele E-selectin Cell adhesion molecule
Selp P-selectin Cell adhesion molecule
Sod2 MnSOD Enzyme that converts superoxide to hydrogen peroxide
Tnf TNF Cytokine that amplifies the inflammatory response
Tnfaip3 A20 Inhibitor of NF-κB signaling by TLRs and TNFR1
Vcam1 VCAM1 Cell adhesion molecule that interacts with β1 integrin VLA-4

aMouse gene nomenclature used.

 NF-κB also induces genes that limit the duration and magnitude of the
inflammatory response, such as Tnfaip3 and Nfkbia (the latter encodes IκBα
and thus forms a negative-feedback loop). These genes prevent the
inflammatory response from causing more tissue damage than the initial
injury. For example, mice lacking the A20 deubiquitylating enzyme encoded
by Tnfaip3 die from unchecked inflammation. A20 is thought to switch off
TLR signaling by countering ubiquitylation by TRAF6 or cIAPs (Newton et
al. 2008; Skaug et al. 2009; Shembade et al. 2010). Somatic mutation of
TNFAIP3 occurs frequently in some human B-cell lymphomas, which
suggests that A20 may function as a tumor suppressor, and polymorphisms
within the TNFAIP3 locus have been associated with autoimmune disorders,
including systemic lupus erythematosus, rheumatoid arthritis, Crohn’s
inflammatory bowel disease, and psoriasis (Table 2) (Vereecke et al. 2011).
The tumor suppressor gene CYLD, which is mutated in familial
cylindromatosis, also encodes a deubiquitylating enzyme that limits NF-κB
signaling. CYLD cleaves both linear and K63-linked polyubiquitin efficiently
in vitro (Komander et al. 2009), but mice lacking CYLD do not develop the
multiorgan inflammation seen in A20-deficient mice, perhaps because CYLD
normally is phosphorylated and inactivated by IKK (Reiley et al. 2005).
Another NF-κB target gene that suppresses TLR signaling is Cd200, which
encodes the membrane glycoprotein ligand for CD200R1 (Mukhopadhyay et
al. 2010). TAM (Tyro3, Axl, and Mer) receptor tyrosine kinases are further
examples of receptor systems that negatively regulate TLR signaling (Rothlin
et al. 2007).

3.2 MyD88-Dependent Type I Interferon (IFN) Induction by

TLRs 7 and 9
The MyD88–IRAK4–IRAK1–TRAF6 signaling complex required for
proinflammatory cytokine production by TLR7 and TLR9 also stimulates
synthesis of interferon α (IFNα) and IFNβ in plasmacytoid dendritic cells
(pDCs). The induction of type I IFNs and Ifn-related genes is critical to the
antiviral response and also requires TRAF3, IKKα, and the transcription
factor IRF7. Phosphorylation of IRF7 by IKKα promotes its dimerization and
translocation into the nucleus, where it up-regulates expression of type I Ifn
genes. Differences have been noted in myeloid DCs1; IRF1 is more important
than IRF7, and there does not seem to be a requirement for TRAF3 and
IRAK1 (Hoshino et al. 2010; Takeuchi and Akira 2010).

3.3 TRIF-Dependent Signaling by TLRs 3 and 4

TLR4 is endocytosed following ligand binding and, like TLR3 stimulated
with dsRNA, transduces signals from within the endosomal compartment.
Both receptors recruit the TIR-containing cytoplasmic adaptor TRIF (also
known as TICAM1), which in the case of endocytosed TLR4 occurs via the
bridging adaptor TRAM (also known as TICAM2). TRIF can activate NF-κB
using its RIP homotypic interaction motif (RHIM) to recruit the RHIM-
containing kinase RIP1 (Cusson-Hermance et al. 2005), which is, in turn,
bound and ubiquitylated by the E3 ubiquitin ligase Peli1 (Chang et al. 2009).
K63-linked polyubiquitylation of RIP1 by Peli1 has not been shown, but
could be a mechanism for the recruitment of IKKγ and TAK1. Interaction of
the DD in RIP1 with the DD in the cytoplasmic adaptor TRADD is important
for TRIF-dependent NF-κB and MAPK activation in some cell types (Chen et
al. 2008; Ermolaeva et al. 2008; Pobezinskaya et al. 2008). The TRIF-
containing complex also induces type I IFN, and this is dependent on TRAF3
plus the kinase TBK1, the latter phosphorylating and activating the
transcription factor IRF3 (Takeuchi and Akira 2010). TRAF3 can modify
itself with K63-linked polyubiquitin, and this may be important for its
interaction with TBK1 and subsequent IRF3 activation.

4 RIG-I-LIKE RECEPTORS (RLRs)

The RLR family of PRRs, which comprises RIG-I, MDA5, and LGP2,
signals the production of proinflammatory cytokines and type I IFN in
response to viral and bacterial nucleic acids in the cytoplasm (Fig. 3). These
cytosolic proteins have a central DExD/H-box helicase domain and a
carboxy-terminal regulatory domain (CTD), the latter binding RNA. RIG-I
and MDA5 also have two amino-terminal caspase activation and recruitment
domains (CARDs), which function as protein–protein interaction motifs.
RIG-I recognizes double-stranded RNAs (dsRNAs) that have a 5′
triphosphate and are either viral in origin or generated by RNA polymerase
III from microbial DNA templates (Lu et al. 2010; Wang et al. 2010b).
MDA5 and LGP2 also bind RNA, but MDA5 and RIG-I have non-redundant
functions in sensing certain viruses. LGP2 appears, in most contexts, to act as
a positive regulator of signaling by MDA5 and RIG-I (Venkataraman et al.
2007; Satoh et al. 2010).

Figure 3. Signaling by RIG-I. (A) RIG-I binding to dsRNA that has a 5′ triphosphate and polyubiquitin,
the latter generated by the ubiquitin ligase TRIM25 and E2 ubiquitin-conjugating enzymes Ubc5 and
Ubc13, promotes RIG-I binding to mitochondrial MAVS. Subsequently, a larger complex containing
the adaptor proteins CARD9 and BCL10 is assembled for MAPK and NF-κB activation. TRAF3, the
kinases TBK1 and IKKε, and ER-resident protein STING are required for activation of transcription
factors IRF3 and IRF7. (B) Functional outputs of some of the genes up-regulated by MAVS signaling.

Binding of RIG-I to RNA causes a conformational change (Jiang et al.

2011) that enables its CARDs to bind K63-linked polyubiquitin generated by
the E3 ubiquitin ligase TRIM25. In an ill-defined manner, this interaction
causes RIG-I to engage the mitochondrial CARD-containing protein MAVS
(also called IPS-1, VISA, and Cardif) (Zeng et al. 2010). Subsequently,
MAVS forms large aggregates (Hou et al. 2011) that stimulate MAPK
activation and transcription induced by IRF3, IRF7, and NF-κB. Many of the
components found downstream from TRIF in TLR signaling also are engaged
by MAVS. For example, TRAF3 plus the kinases TBK1 and IKKε mediate
IRF3/7 activation and induction of type I Ifn genes (Takeuchi and Akira
2010). RIG-I, but not MDA5, also requires the transmembrane protein
STING to induce IFN (Ishikawa and Barber 2008). STING is located in the
endoplasmic reticulum (ER), but its precise role in IFN induction by dsRNA
requires further study. Experiments with fibroblasts from gene-targeted mice
also implicate TRAF6, IKKγ, and the DD-containing proteins TRADD,
RIP1, and FADD in IFN induction (Balachandran et al. 2004; Zhao et al.
2007; Michallet et al. 2008; Yoshida et al. 2008). Note that loss of TRADD,
RIP1, or FADD produces a defect less severe than does MAVS or TBK1
deficiency. The extent of IRF3 phosphorylation and dimerization has not
been determined in cells lacking TRADD, RIP1, or FADD; it remains
possible that these proteins, like TRAF6, contribute to type I Ifn gene
expression by activating NF-κB (Wang et al. 2010a). In DCs, MAVS engages
the CARD-containing adaptors CARD9 and BCL10 to activate NF-κB
(Poeck et al. 2010). BCL10 engages TRAF6 in lymphocytes to activate NF-
κB (Sun et al. 2004b), and a similar pathway may operate downstream from
MAVS. A role for FADD, TRADD, and RIP1 in MAVS signaling by DCs
has not been examined.

5 NOD-LIKE RECEPTORS (NLRs)

Members of the Nod-like receptor (NLR) family of cytosolic PRRs are best
known for their ability to signal NF-κB activation (NOD1 and NOD2) or
secretion of the proinflammatory cytokines IL1β and IL18 (NLRP1/NALP1,
NLRP3/NALP3/cryopyrin, and NLRC4/Ipaf) (Fig. 4). These proteins
typically contain a CARD or pyrin domain at the amino terminus, a central
nucleotide-binding oligomerization NACHT domain, and carboxy-terminal
LRRs. NOD2 and NLRP3 have received considerable attention because their
mutation is linked to inflammatory disease. NOD2 mutations are associated
with Crohn’s inflammatory bowel disease and Blau syndrome, whereas
mutations in the CIAS1 gene encoding NLRP3 are associated with familial
cold autoinflammatory syndrome, Muckle–Wells syndrome, and neonatal-
onset multisystem inflammatory disease (Table 2).
Figure 4. Signaling by NLRs. (A) NLRP1, NLRP3, and NLRC4 respond to diverse PAMPS and
DAMPs by engaging the caspase-1 adaptor protein ASC, whereas AIM2 binds ASC in response to
cytoplasmic dsDNA. Activation of caspase 1 within each inflammasome complex results in processing
of pro-IL1β and pro-IL18 and secretion of their biologically active forms. Caspase-1 activation also
triggers a rapid form of cell death termed pyroptosis. NOD1 and NOD2 sense different components of
bacterial peptidoglycan and stimulate either the autophagy machinery or gene transcription via NF-κB
and MAPK activation. The latter outcome requires interaction of NOD1 or NOD2 with the kinase
RIP2, which may be ubiquitylated by cIAPs in order to recruit TAB2/3 and IKKγ for TAK1 and IKK
activation. (B) Functional outputs of some of the genes up-regulated by NOD1 or NOD2 signaling.

NOD1 and NOD2 are sensors of different bacterial peptidoglycan

components, but they both interact with the CARD-containing kinase RIP2 to
activate MAPK and NF-κB signaling (Park et al. 2007). cIAPs are proposed
to bind and ubiquitylate RIP2, and K63-linked polyubiquitylation of RIP2
recruits TAK1 for IKK and MAPK activation (Yang et al. 2007;
Hitotsumatsu et al. 2008; Bertrand et al. 2009). NOD1 and NOD2 also have
been shown to stimulate autophagy2 independently of RIP2 (Travassos et al.
2010).
Distinct PAMPS and DAMPs trigger NLRP1, NLRP3, and NLRC4 to
nucleate signaling complexes termed “inflammasomes” (Table 4). The
CARD- and PYRIN-domain-containing adaptor ASC is a critical
inflammasome component, binding the CARD in the zymogen form of the
aspartate-specific cysteine protease caspase 1 (Mariathasan et al. 2004). The
proximity of caspase-1 zymogens within the inflammasome complex is
believed to facilitate their autocatalytic activation. Caspase-1 substrates
include pro-IL18 and pro-IL1β, the latter being up-regulated transcriptionally
by MyD88-dependent TLR signaling. Inflammasome activation also results
in an extremely rapid form of cell death termed “pyroptosis.” The suicide of
infected macrophages by pyroptosis is important for bacterial clearance
(Miao et al. 2010), but the critical substrates of caspase 1 in this process still
have to be determined.

Table 4. Stimuli detected by inflammasome sensors that activate caspase 1

Sensor Known stimuli
AIM2 Cytoplasmic DNA
NLRP1 Bacillus anthracis lethal toxin
NLRP3 ATP
Ionophore nigericin
Marine toxin maitotoxin
Crystals such as monosodium urate, calcium pyrophosphate dihydrate, silica, and asbestos
Fungi such as Candida albicans
Bacteria such as Staphylococcus aureus, Neisseria gonorrhoeae, Salmonella typhimurium,
and Listeria monocytogenes
Viruses such as sendai, adenovirus, and influenza
NLRC4 Bacterial flagellin
Certain bacterial type III secretion systems

Intriguingly, immunofluorescence microscopy of endogenous

inflammasome components in mouse macrophages infected with Salmonella
typhimurium suggests that inflammasome assembly occurs at a single focus
within a cell (Broz et al. 2010). One question that continues to vex the field is
how NLRP1, NLRP3, and NLRC4 sense PAMPS and DAMPs, because
direct binding has not been shown. Phosphorylation of NLRC4 is critical for
inflammasome activation (Qu et al. 2012) and the diversity of entities that
trigger NLRP3-dependent caspase-1 activation suggests that NLRP3 might
respond to a particular stress-activated signaling pathway. Both potassium
efflux and the generation of reactive oxygen species (ROS) have been
proposed as critical events upstream of NLRP3 activation, but the precise
nature of NLR activation remains obscure.
ASC-dependent caspase-1 activation is also triggered in response to
cytoplasmic DNA that appears during an infection or after tissue injury. The
responsible PRR is not an NLR but the IFN-induced protein AIM2, which
has a HIN200 domain for DNA binding and a pyrin domain to engage ASC.
Cytoplasmic DNA also triggers type I IFN production, but this requires
neither AIM2 nor TLRs. Instead, DNA binding to the enzyme cGAS (cyclic
guanosine monophosphate-adenosine monophosphate synthase) promotes
production of the second messenger cGAMP, which binds to STING and
thereby stimulates activation of IRF3 (Sun et al. 2013; Wu et al. 2013a).
Caspase-1 activation also occurs in a caspase-11-dependent, TLR4-
independent manner in response to intracellular LPS (Kayagaki et al. 2013).
The nature of the intracellular LPS sensor remains to be elucidated.
6 THE PROINFLAMMATORY CYTOKINE TUMOR
NECROSIS FACTOR (TNF)
Induction of the cytokines IL1β and TNF by PRRs serves to amplify the
inflammatory response because they too promote NF-κB and MAPK
activation. Binding of IL1β to IL1R triggers MyD88-dependent signaling
(Muzio et al. 1997), whereas TNF mediates most of its proinflammatory
effects by binding to TNF receptor I (TNFRI) (Peschon et al. 1998).

6.1 NF-κB and MAPK Activation by TNF

TNFRI (also called TNFRSF1A) is a type I transmembrane protein that has
cysteine-rich extracellular domains (CRDs) for TNF binding. Its cytoplasmic
tail contains a DD that recruits the DD-containing adaptor TRADD and
kinase RIP1 (Fig. 5). TRADD facilitates binding of RIP1 to TNF-R1 and
recruits TRAF2, which is an adaptor for the ubiquitin ligases cIAP1 and
cIAP2 (Chen et al. 2008; Ermolaeva et al. 2008; Pobezinskaya et al. 2008).
Analyses of cells lacking cIAPs, RIP1, or TRAF2 indicate that all three
contribute to NF-κB and MAPK activation, but the details of how they do so
continue to be unraveled. Ubiquitylation of RIP1 by the cIAPs and E2 UbcH5
is believed to be important for recruitment of NEMO and TAK1, and
subsequently for IKK activation (Varfolomeev et al. 2008; Xu et al. 2009),
although TRAF2 ubiquitin ligase activity has been invoked recently as well
(Alvarez et al. 2010). In addition, in some cell types, ubiquitylation of
TRAF2 or cIAPs rather than RIP1 may support IKK activation (Li et al.
2009; Wong et al. 2010). Recruitment of LUBAC components sharpin,
HOIL-1, and HOIP to the TNFRI complex is linked to cIAP ubiquitin ligase
activity, and linear ubiquitylation of RIP1 and NEMO may stabilize the
TNFRI signaling complex (Haas et al. 2009; Gerlach et al. 2011; Ikeda et al.
2011; Tokunaga et al. 2011). Underscoring the importance of sharpin in TNF
signaling, mutation of Sharpin in mice causes chronic proliferative dermatitis
that is rescued by TNF deficiency (Gerlach et al. 2011).
Figure 5. Signaling by TNFR1. (A) Binding of TNF to TNFR1 causes the cytoplasmic death domain
(DD) in TNFR1 to bind the DD-containing proteins TRADD and RIP1. TRADD also binds TRAF2,
which serves as an adaptor for the ubiquitin ligases cIAP1 and cIAP2. Ubiquitylation of RIP1, and
potentially other components of the complex, recruits IKKγ and TAK1 for NF-κB and MAPK
activation. Recruitment of LUBAC for linear ubiquitylation of IKKγ may stabilize the signaling
complex. Translocation of TRADD, TRAF2, and RIP1 to the cytoplasm nucleates a second complex
that contains the adaptor protein FADD and caspase 8. If c-FLIP levels are low, activation of caspase 8
and its substrates caspase 3 and caspase 7 causes apoptotic cell death. Inhibition of protein synthesis
and caspases, as might occur in a virus-infected cell, promotes necroptotic cell death that is dependent
on the kinase activities of RIP1 and RIP3. (B) Functional outputs of some of the genes up-regulated by
TNF signaling.

IKKβ activation by TNF triggers not only NF-κB transcription but also
the Tpl2–MEK1–ERK kinase cascade activated by TLRs (see above). In
fibroblasts, but not macrophages or B cells, Tpl2 activation by TNF has been
linked to activation of the MKK4–JNK pathway as well. In addition,
activation of the kinase MSK1 by ERK may enhance NF-κB transcriptional
activity through phosphorylation of RelA (Banerjee and Gerondakis 2007).
Genetic studies indicate that TNF-induced JNK activation is mediated largely
by upstream kinases TAK1 and MKK7, whereas p38 activation requires
TAK1 and MKK3/MKK6 (Brancho et al. 2003; Sato et al. 2005; Shim et al.
2005).

6.2 Apoptosis and Necroptosis Induction by TNF

The TNFRI-associated signaling complex formed in response to TNF is
transient as TRADD, RIP1, and TRAF2 shift to the cytoplasm to form what
is referred to as complex II (Micheau and Tschopp 2003). Here, the DD in
TRADD binds the DD in the adaptor FADD, whereas the death effector
domain (DED) in FADD interacts with the DEDs in the prodomain of
caspase 8 or its catalytically inactive homolog FLIP. If FLIP levels are low,
then stable active dimers of caspase 8 are formed. Caspase-8 substrates
include caspase 3 and caspase 7, which are activated by proteolytic
processing and cleave vital cellular proteins to cause apoptotic cell death (Ch.
19 [Green and Llambi 2014]). Because expression of FLIP is driven by NF-
κB (Micheau et al. 2001) and many viruses have evolved strategies to inhibit
NF-κB activation (Shisler and Jin 2004; Taylor et al. 2009), apoptosis in the
absence of c-FLIP constitutes a host defense mechanism against infection.
Certain viruses encode their own FLIPs, but the infected host cell may yet
prevail in its attempt to die because TNF activates a form of cell death called
necroptosis when protein synthesis and caspases are inhibited. This death is
dependent on the kinase activity of RIP1 and the related kinase RIP3 (Cho et
al. 2009; He et al. 2009; Zhang et al. 2009). These kinases interact through
their RHIMs with RIP3, then phosphorylate the pseudokinase mixed-lineage
kinase domain-like (MLKL) (Sun et al. 2012; Wu et al. 2013b). It is not clear
how phosphorylation of MLKL leads to cell death.

7 SELECTINS AND INTEGRINS

One important output of LPS, TNF, and IL1β signaling in endothelial cells is
increased surface expression of transmembrane proteins involved in cell
adhesion, such as P-selectin, E-selectin, ICAM1, and VCAM1. All four are
induced by NF-κB in mice, whereas up-regulation of human P-selectin relies
on fusion of secretory granules with the plasma membrane. Glycoproteins
including CD44, P-selectin glycoprotein ligand 1 (PSGL1), and E-selectin
ligand (ESL1) on leukocytes interact with the selectins to mediate leukocyte
rolling along vessel walls. Engagement of either PSGL1 or CD44 triggers a
signaling pathway that causes leukocyte β2 integrins LFA1 and MAC1
expressed on the cell surface to adopt a more extended conformation that
increases their affinity for endothelial ICAM1. Interactions between the β2
integrins and ICAM1 then slow leukocyte rolling further. β2 integrin
activation via this “inside-out” signaling (Ch. 8 [Devreotes and Horwitz
2014]) requires the membrane-associated Src family kinases Fgr, Hck, and
Lyn. These tyrosine kinases probably phosphorylate cytoplasmic
immunoreceptor tyrosine-based activation motifs (ITAMs) in the
transmembrane adaptors DAP12 and FcRγ to create docking sites for the
tandem SH2 domains in the tyrosine kinase Syk (Ch. 16 [Cantrell 2014]), but
note that they can also phosphorylate integrin β subunits. Studies with
neutrophils from gene-targeted mice indicate that sequential activation of Syk
and the kinase Btk is essential for slow rolling on E-selectin and ICAM1
(Zarbock et al. 2008; Yago et al. 2010).
Further β2 integrin activation needed for leukocyte arrest before migration
across the endothelial cell barrier is mediated by chemokines and
chemoattractants immobilized on the endothelial cell surface. These factors
engage G-protein-coupled receptors (GPCRs) on the leukocyte surface (see
below). Note that integrin engagement also elicits “outside-in” signaling in
leukocytes, and, similarly to Fc receptor signaling (see below), this activates
leukocyte effector functions (Lowell 2011). Clustering of ICAM1 on
endothelial cells also triggers signals that facilitate leukocyte migration
across the endothelium into the surrounding tissue. Phosphorylation of VE-
cadherin appears to loosen adherens junctions, whereas activation of myosin
light chain kinase (MLCK) mediates endothelial cell contraction (Muller
2011).

8 G-PROTEIN-COUPLED RECEPTORS (GPCRs)

Lipid-based inflammatory mediators such as prostaglandins, leukotrienes,
and platelet-activating factor; vasoactive amines such as histamine and
serotonin; complement fragments C3b, C3a, and C5a; chemokines; proteases;
and bacterial or mitochondrial formylated peptides all activate signaling by
GPCRs linked to heterotrimeric G-proteins composed of α, β, and γ subunits.
Following ligand binding, or cleavage in the case of protease-activated
receptors (PARs), Gα and Gβγ interact with ion channels or enzymes such as
adenylyl cyclase, phospholipase C (PLC), and phosphoinositide 3-kinase
(PI3K). In addition, active GPCRs are phosphorylated by GPCR kinases
(GRKs) to stimulate binding of arrestins, adaptors that stimulate GPCR
endocytosis as well as MAPK activation. To highlight some of the pathways
engaged by GPCRs during inflammation, below we focus on signaling by the
chemoattractants C5a and the prototypical formylated peptide formyl-Met-
Leu-Phe (fMLP) (Fig. 6).
Figure 6. Signaling by GPCRs activated by chemoattractants C5a and fMLP. C5a or fMLP binding to
their respective GPCRs triggers dissociation of Gαi-GTP from Gβγ, the latter interacting with PLCβ,
the p101 regulatory subunit of PI3Kγ, and PAK1. Activated PLCβ generates the second messengers IP3
and DAG to elevate intracellular calcium and activate PKC, respectively. These outcomes regulate JNK
activation, vesicle exocytosis, and superoxide production by the NADPH oxidase. PIP3 generated by
PI3Kγ, whose activation also involves the GTPase Ras, stimulates GEFs (DOCK2 and Prex1) that
activate Rac GTPases. PAK1 interacts with the GEF PIXα for activation of another Rho family GTPase
called Cdc42. Rac1, Rac2, and Cdc42 together regulate chemotaxis by coordinating alterations to the
actin cytoskeleton via mDia1 and the ARP2/3 complex. Rac2 is also an essential component of the
NADPH oxidase. The signaling components regulating gene transcription are less defined.

8.1 C5a Receptor (C5aR) and Formyl Peptide Receptors

(FPRs)
Complement protein C5a is produced by complement plasma proteases
activated by IgM- and IgG-containing antibody complexes (the classical
pathway), pathogens coated with host mannose-binding lectin or C-reactive
protein (the lectin pathway), or pathogens in isolation (the alternative
pathway). C5a can also be generated by noncomplement proteases such as
thrombin and kallikrein, which are components of the clotting system
activated in response to endothelial cell injury. C5a and formyl peptides, the
latter of bacterial or mitochondrial origin, stimulate leukocyte chemotaxis,
degranulation, superoxide production for microbe killing, and, as mentioned
above, activation of integrins for cell adhesion. Similarly to TNF, C5a
stimulates endothelial cells to increase expression of cytokines, chemokines,
and cell adhesion molecules such as E-selectin, ICAM1, and VCAM1
(Albrecht et al. 2004).
C5a and fMLP activate predominantly pertussis toxin-sensitive Gi
proteins. The Gβγ dimer that is released activates several enzymes, including
PLCβ (Camps et al. 1992). Hydrolysis of phosphatidylinositol 4,5-
bisphosphate in the plasma membrane by PLC yields the second messengers
inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (p. 95 [Bootman
2012]). Binding of IP3 to its receptor causes depletion of calcium stores
within the ER and relocation of the calcium-binding type-I transmembrane
protein STIM1 from the ER to structures near the plasma membrane
(Brechard et al. 2009). STIM1 then activates the plasma membrane calcium-
release-activated calcium channel (CRAC) to cause an influx of calcium into
the cell. Elevated intracellular calcium together with protein kinase C (PKC)
activation by DAG is important for vesicle exocytosis, superoxide production
by the NADPH oxidase, and JNK activation (Li et al. 2000). Calcium
stimulates lysosome exocytosis by activating the synaptotagmin regulator of
vesicle fusion SYT7 (Colvin et al. 2010).
GTP-bound Ras, activated by a mechanism that is unclear, and Gβγ
dimers activate PI3Kγ by binding to its p101 regulatory subunit and p110γ
catalytic subunit, respectively. Phosphatidylinositol 3,4,5-trisphosphate
(PIP3) produced by PI3Kγ activates Rac guanine-nucleotide exchange factors
(GEFs), such as Prex1 and DOCK2 (Welch et al. 2002; Kunisaki et al. 2006),
and contributes to superoxide production and chemokinesis3 (Suire et al.
2006; Ferguson et al. 2007; Nishio et al. 2007). The GTPase RhoG also has a
role in superoxide production but is dispensable for neutrophil migration
(Condliffe et al. 2006). The GTPase Rac2 appears to be essential for the
assembly of filamentous actin (F-actin) and the NADPH oxidase, whereas
Rac1 localizes F-actin to the leading edge of the cell facing the
chemoattractant (Sun et al. 2004a). This asymmetrical polymerization of F-
actin drives membrane protusions in the direction of migration. Active Rac is
thought to exert its effect on the actin cytoskeleton by interacting with the
adaptor Cyfip1 (also called Sra1), which, in combination with several
proteins, stimulates the ARP2/3 actin nucleation complex (Ch. 8 [Devreotes
and Horwitz 2014]).
The GEF PIXα also is required for F-actin assembly at the leading edge in
C5a-stimulated neutrophils. It is recruited to Gβγ via the kinase PAK1 and
appears to function by interacting with the GTPase Cdc42 and the GTPase-
activating (GAP) protein GIT2 (Li et al. 2003; Mazaki et al. 2006). Activated
Cdc42 interacts with the adaptor WASP to engage the ARP2/3 actin
nucleation complex. WASP appears to work in concert with the actin-
nucleating protein mDia1, because neutrophils lacking both WASP and
mDia1 show a profound defect in chemotaxis (Shi et al. 2009).

9 Fc RECEPTORS
Repeated exposure to a polyvalent foreign substance can elicit an
inflammatory response called a hypersensitivity reaction if the host makes
antibodies against the substance. Immune complexes containing the antigen
and IgG or IgM antibodies activate complement proteases, culminating in the
generation of C3a and C5a, which signal leukocyte recruitment and activation
(see above); the opsonin C3b, which coats and promotes phagocytosis of
bacteria; and the membrane attack complex for bacterial cell lysis (C5b-9). In
addition, complexes containing IgG or IgE antibodies engage Fc receptors on
leukocytes. Members of the Fc receptor family are type I transmembrane
proteins (with the exception of human GPI-anchored FcγRIIIB) that produce
activating (human FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIIIB, and
FcεRI) or inhibitory (human FcγRIIB) signals. Mast cells expressing the
high-affinity receptor for IgE, FcεRI, play a central role in allergic reactions.
FcεRI engagement causes intracellular granules to fuse with the plasma
membrane such that preformed inflammatory mediators including histamine,
serotonin, and proteases are released into the extracellular environment.
Activated mast cells also secrete proinflammatory prostaglandins,
leukotrienes, and cytokines, but these are synthesized de novo.

9.1 FcεRI
FcεRI is an αβγ2 heterotetramer. Its α-chain contains extracellular Ig-like
domains for binding the heavy-chain constant region of IgE, whereas the β-
chain and a γ-chain homodimer transduce signals via cytoplasmic ITAMs
(Fig. 7) (Ch. 16 [Cantrell 2014]). IgE-induced clustering of FcεRI promotes
activation of Src family kinases Lyn and Fyn. Lyn substrates include both
positive and negative regulators of mast cell activation, which fine-tune the
magnitude and duration of the response. Lyn stimulates activation by
phosphorylating the FcRγ ITAM, which recruits the SH2 domains in Syk.
Subsequent Syk-dependent phosphorylation of the transmembrane adaptors
LAT1 and LAT2 recruits additional SH2-containing signaling components,
such as PLCγ and the adaptors Grb2 and Gads. SH3 domains in Grb2 and
Gads bind proline-rich regions in additional proteins such as the adaptors
SLP76 and Gab2. SLP76 interacts with the Rho/Rac GEF VAV1, which
contributes to PLCγ and JNK activation. Fyn-dependent phosphorylation of
Gab2 recruits the SH2-containing p85 regulatory subunit of PI3Kδ. PIP3
produced by PI3Kδ retains proteins containing plextrin homology (PH)
domains at the plasma membrane, such as PLCγ, Gab2, Akt, and Btk. The
kinase Btk phosphorylates and enhances the activity of PLCγ (Alvarez-Errico
et al. 2009).
Figure 7. Signaling by FcεRI. (A) Binding of the Fc region of antigen-bound IgE to FcεRI activates the
Src family kinases Lyn and Fyn. Tyrosine phosphorylation of the FcRγ ITAM recruits the tyrosine
kinase Syk, which is required for phosphorylation of LAT transmembrane adaptor proteins.
Phosphorylated LAT1 binds PLCγ and the adaptors Gads and Grb2. Gads recruits the adaptor SLP76,
which regulates activation of PLCγ and the GEF Vav1. Grb2 binds Gab2, which is phosphorylated by
Fyn and binds the p85 regulatory subunit of PI3Kδ. PIP3 generated by PI3Kδ retains signaling
components such as Gab2, PLCγ, and Btk at the plasma membrane. IP3 generated by PLCγ depletes
ER calcium stores, which causes a STIM1-dependent influx of calcium that promotes mast cell
degranulation. Elevated intracellular calcium also activates the phosphatase calcineurin, stimulates
NFAT-dependent gene expression, and triggers the translocation of cPLA2 and 5-lipoxygenase (5-LO)
to the nuclear envelope, cytoplasmic lipid bodies, or ER. cPLA2 releases arachidonic acid from
membrane phospholipids. COX enzymes and downstream synthases metabolize arachidonic acid into
prostaglandins and thromboxane, whereas leukotriene (LT) synthesis from arachidonic acid involves
five-lipoxygenase-activating protein (FLAP), 5-LO, and downstream LTC4 synthase or LTA4
hydrolase. DAG generated by PLCγ activates PKC, which is important for IKK activation via MALT1,
BCl10, and TRAF6, as well as subsequent NF-κB-dependent gene transcription. IKKβ has also been
implicated in mast cell degranulation independent of NF-κB activation. (B) Functional outputs of some
of the genes up-regulated by FcεRI signaling.

PLC-γ signaling triggers STIM1-dependent calcium influx (p. 95

[Bootman 2012]), which is essential for normal mast cell degranulation,
leukotriene synthesis, and activation of NFAT transcription factors via the
calcium-dependent phosphatase calcineurin (Baba et al. 2008; Vig et al.
2008). NFAT promotes expression of the cytokines TNF and IL13.
Eicosanoids including leukotrienes and prostaglandins are derived from
arachidonic acid, which is liberated from phospholipids by cytosolic
phospholipase A2 in response to elevated intracellular calcium and MAPK
activation (Fujishima et al. 1999).
PKC activation by DAG is required for degranulation and activation of
NF-κB, the latter contributing to the induction of TNF and IL6. BCL10,
MALT1 (also called paracaspase), and TRAF6 regulate NF-κB activation but
are dispensable for degranulation (Klemm et al. 2006; Chen et al. 2007; Yang
et al. 2008), whereas IKKβ is required for both functions (Suzuki and Verma
2008). The mechanism by which IKKβ is activated for degranulation remains
unclear, but, once activated, IKKβ appears to promote exocytosis by
phosphorylating the SNARE receptor SNAP23.

9.2 Fcγ Receptors

Activating Fcγ receptors, in common with FcεRI, contain cytoplasmic
ITAMs and stimulate Src family kinases plus Syk. The downstream signaling
events that promote phagocytosis, degranulation, cytokine production, and
superoxide production are less well defined but probably involve many of the
components engaged by FcεRI. Superoxide production by the NADPH
oxidase that assembles on phagosomal membranes requires Vav-mediated
activation of Rac GTPases, the putative Rac adaptor CAPRI, and PLCγ.
Depending on the cell type and context, CAPRI, VAV, and Rac also
contribute to remodeling of the actin cytoskeleton for phagocytosis, along
with PI3K and its adaptor Gab2 (Gu et al. 2003; Zhang et al. 2005; Hall et al.
2006; Utomo et al. 2006; Jakus et al. 2009). FcγRIIB is unique among Fc
receptors in that it suppresses ITAM signaling through an immunoreceptor
tyrosine-based inhibitory motif (ITIM). Lyn-dependent ITIM
phosphorylation recruits the SH2-containing inositol 5′-phosphatase (SHIP),
which hydrolyzes PIP3 and thereby limits recruitment of PH-domain-
containing proteins such as PLC-γ, Btk, and VAV (Lowell 2011).

10 INFLAMMATION AS A RISK FACTOR

FOR CANCER
Bacteria and viruses that establish persistent infections are linked to certain
cancers. For example, Helicobacter pylori bacteria increase the risk of gastric
cancer and MALT lymphoma, whereas Hepatitis B and Hepatitis C viruses
increase the risk of hepatocellular carcinoma. Similarly, pancreatitis is a risk
factor for pancreatic ductal adenocarcinoma, and this has been modeled
successfully in mice expressing oncogenic K-Ras in adult pancreatic acinar
cells (Guerra et al. 2011). Studies with gene-targeted mice have confirmed
that inflammatory signaling pathways promote tumor development. For
example, deletion of FcRγ suppresses squamous cell carcinoma driven by
keratinocyte-specific expression of the human papilloma virus oncogene
HPV16 (Andreu et al. 2010). Similarly, IL6 deficiency in hematopoietic cells
suppresses colon tumors that develop in response to procarcinogen
azoxymethane plus colitis-inducing dextran sulfate sodium (DSS)
(Grivennikov et al. 2009). IL6 enhances proliferation and survival of
intestinal epithelial cells through activation of the transcription factor STAT3.
In another mouse model, the ability of tobacco smoke to promote lung
tumors driven by oncogenic K-Ras is reduced by IKKβ deletion in myeloid
cells (Takahashi et al. 2010). Finally, IL6 or TNFR1 deficiency protects
obese mice from hepatocellular carcinomas that form in response to the
procarcinogen diethylnitrosamine by limiting lipid accumulation and
inflammatory infiltrates in the liver (Park et al. 2010). Note, however, that in
all of these tumor models, inflammation alone is insufficient for tumor
development, which implies that carcinogen-induced mutations are needed.

11 CONCLUDING REMARKS
Many of the major players in inflammatory signaling have been identified,
but the importance and complexity of posttranslational modifications such as
ubiquitylation in these pathways continue to be unraveled. Binding of TLRs,
TNF and IL1 receptors, GPCRs, integrins, selectins, and Fc receptors to their
ligands triggers the formation of multisubunit signaling complexes, but it
remains to be seen how diverse inflammatory stimuli can activate
intracellular PRRs such as NLRP3 and NLRC4. An attractive hypothesis is
that posttranslational modifications to NLR family members are key to their
activation. Once activated, both surface and intracellular PRRs stimulate
transcription of inflammatory genes; TLRs, RLRs, and some NLRs (e.g.,
NOD1 and NOD2) engage common downstream signaling pathways to
stimulate transcription factors such as NF-κB, AP1, CREB, and c/EBPβ,
whereas caspase-1-activating PRRs (e.g., AIM2, NLRP3, and NLRC4)
stimulate similar pathways indirectly via the secretion of IL1β and IL18.
Going forward, it will be important to understand how innate immune cells
exposed to multiple inflammatory mediators and stimuli in vivo integrate
signals from diverse receptors, because this will offer insight into what
critical components might be targeted for therapeutic benefit in inflammatory
disorders.

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1DCs are very heterogeneous. pDCs acquire DC morphology and secrete large amounts of IFN during
virus infections. They can be distinguished from other DC subsets by their cell surface markers. The
myeloid DCs referenced were derived in vitro from bone marrow cells with granulocyte/macrophage
colony-stimulating factor (GM-CSF).
2Autophagy is the process by which cytoplasmic components, including organelles and invading
bacteria, are sequestered inside double-membrane vesicles and then delivered to the lysosome for
degradation.
3Chemokinesis refers to random cell migration, whereas chemotaxis is directed cell migration along a
chemical gradient.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006049
CHAPTER 16

Signaling in Lymphocyte Activation

Doreen Cantrell
College of Life Sciences, Wellcome Trust Biocentre, University of Dundee,
Dundee DD1 5EH, Scotland, United Kingdom
Correspondence: d.a.cantrell@dundee.ac.uk

SUMMARY

The fate of T and B lymphocytes, the key cells that direct the adaptive
immune response, is regulated by a diverse network of signal
transduction pathways. The T- and B-cell antigen receptors are coupled
to intracellular tyrosine kinases and adaptor molecules to control the
metabolism of inositol phospholipids and calcium release. The
production of inositol polyphosphates and lipid second messengers
directs the activity of downstream guanine-nucleotide-binding proteins
and protein and lipid kinases/phosphatases that control lymphocyte
transcriptional and metabolic programs. Lymphocyte activation is
modulated by costimulatory molecules and cytokines that elicit
intracellular signaling that is integrated with the antigen-receptor-
controlled pathways.

Outline
1 Introduction
 2 Antigen-receptor structure and function
3 Immunoreceptor tyrosine-based activation motifs
4 Adaptor molecules for antigen receptors
5 Calcium and diacylglycerol signaling
6 Downstream from calcium signaling in lymphocytes
7 Diacylglycerol signaling in lymphocytes
8 PKC and lymphocytes
9 Ras signaling and lymphocytes
10 Costimulatory molecules, cytokines, and lymphocyte activation
11 Cytokine signaling in lymphocytes
12 PI3K-mediated signaling in lymphocytes
13 Inhibitory signals and lymphocyte activation
14 Concluding remarks
References

1 INTRODUCTION
The adaptive immune response is directed by B and T lymphocytes. These
cells express specific receptors that recognize pathogen-derived antigens: the
B-cell antigen receptor (BCR) and the T-cell antigen receptor (TCR),
respectively. B lymphocytes have two principal roles: to produce and secrete
specific antibodies/immunoglobulins, and to function as antigen-presenting
cells (APCs). T cells have multiple roles in adaptive immune responses. In
this context, peripheral T cells can be subdivided on the basis of whether they
express CD8 or CD4, receptors that recognize class I and class II major
histocompatibility complex (MHC) molecules, respectively. CD8+ T cells
differentiate to cytolytic effectors that directly kill virus- or bacteria-infected
cells. CD4+ T cells are referred to as “helper” T cells because they produce
regulatory cytokines and chemokines that mediate autocrine or paracrine
control of T-cell differentiation and/or regulate the differentiation of B cells
and/or direct the activity of macrophages and neutrophils (O’Shea and Paul
2010). At least five major subpopulations of mature CD4+ cells exist with
distinct functions that are tailored to deal with different pathogens. Th1 cells,
characterized by interferon (IFN)γ production; Th2 cells, characterized by
interleukin 4 (IL4) and IL13 production; Th17 cells, which produce the
proinflammatory cytokines IL17 and IL22; regulatory T (Treg) cells that
function to restrain autoimmunity and strong inflammatory responses; and
follicular helper T (Tfh) cells, a class of effector CD4+ T cells that regulate
the development of antigen-specific B-cell immunity.
The paradigm of the adaptive immune response is that a primary response
to an antigen causes clonal expansion of antigen-reactive T or B cells and
produces a large number of effector lymphocytes that cause clearance of the
pathogen. Once the pathogen is cleared there is a contraction phase of the
immune response characterized by loss of effector lymphocytes and the
emergence of long-lived memory cells capable of mounting rapid secondary
responses to reinfection with the original pathogen.
The proliferation and differentiation of mature lymphocytes in adaptive
immune responses are directed by antigen receptors, costimulatory
molecules, adhesion molecules, cytokines, and chemokines. These extrinsic
stimuli are coupled to a diverse network of signal transduction pathways that
control the transcriptional and metabolic programs that determine lymphocyte
function. At the core of lymphocyte signal transduction is the regulated
metabolism of inositol phospholipids and the resultant production of inositol
polyphosphates and lipids such as polyunsaturated diacylglycerols (DAGs).
These second messengers direct the activity of protein and lipid kinases and
guanine-nucleotide-binding proteins that control lymphocyte proliferation,
differentiation, and effector function. Below, I outline both the unique and
the conserved aspects of signaling in lymphocytes, focusing on signaling
pathways controlled by antigen receptors and how these responses are
subsequently shaped and modulated by cytokines and chemokines.

2 ANTIGEN-RECEPTOR STRUCTURE AND FUNCTION

The TCR and BCR are multiprotein complexes comprising subunits
containing highly variable antigen-binding regions linked noncovalently to
invariant signal transduction subunits. In both cases, rearrangements of the
DNA sequences that encode the antigen-binding region create a diversity in
antigen-receptor structures. A key feature of T- and B-cell populations is that
each individual lymphocyte will express multiple copies of a unique antigen
receptor with a single antigen specificity (defined by three complementarity-
determining regions [CDRs]). It is the selectivity of antigen receptors that
underpins immune specificity by ensuring that only those lymphocytes that
recognize a specific pathogen are activated by it.
The BCR is composed of a highly variable membrane-bound
immunoglobulin of either the IgM or IgD subclass in a complex with the
invariant also known as Igα and Igβ (CD79a and CD79b) heterodimer (Tolar
et al. 2009). Immunoglobulin subunits are highly variable because the genes
that encode these proteins undergo rearrangements and somatic
hypermutation during B-cell development, which produces a high degree of
protein diversity (≥1011 different receptors) (Schatz and Ji 2011).
The TCR is also characterized by highly variable antigen-binding
subunits, either an αβ or a γδ dimer (Davis 2004; Krogsgaard and Davis
2005; Xiong and Raulet 2007). These are coupled to the invariant CD3
subunits γε, δε, and ζζ, which are essential for trafficking and stability of the
γδ and αβ subunits at the plasma membrane. CD3 antigens also transmit
signals into the cell across the plasma membrane. Like the BCR
immunoglobulin sequences, the TCR-αβ or γδ dimers are highly variable
because the genes that encode them undergo rearrangements (but not
hypermutation) during their development. Indeed, there is potential for the
production of ∼1018 different TCR-αβ receptor complexes. This is compared
with to a minimal estimate of 1011 BCR complexes. The salient feature is that
each T cell only expresses an αβ or a γδ receptor complex with a single
specificity.
T cells that express TCR-γδ complexes are found predominantly at
epithelial barriers (e.g., in the skin and gut epithelia). The ligands for TCR-γδ
complexes are not well defined but can be bacterial phosphoantigens,
alkylamines, and aminobisphosphonates (Hayday 2009). T cells that express
TCR-αβ complexes typically recirculate between the blood, secondary
lymphoid organs (spleen and lymph nodes), and the lymphatic system. The
ligands for TCR-αβ complexes are not antigens per se but rather pathogen-
(or transplantation-antigen)-derived peptides bound to MHC molecules, a
group of molecules that display the short, approximately nine-residue
peptides on the surface of APCs. TCR-αβ-expressing T cells are thus not
triggered by soluble pathogen-derived peptides but only by peptide-MHC
complexes on the surface of dendritic cells, B cells, and other cells that can
function as APCs (Krogsgaard and Davis 2005).

3 IMMUNORECEPTOR TYROSINE-BASED
ACTIVATION MOTIFS
The antigen-receptor subunits that mediate signal transduction are the
invariant chains CD3γ, δ, ε, ζ in T cells, Igα and Igβ in B lymphocytes, and
the FcRγ chain in mast cells (see below). These signaling subunits have no
intrinsic signaling capacity, but all contain a YxxL/I-X6–8-YxxL/I motif
referred to as an immunoreceptor tyrosine-based activation motif (ITAM)
(Abram and Lowell 2007; Love and Hayes 2010). The CD3γ, δ, and ε
subunits each contain a single ITAM, and there are three ITAMs in the CD3ζ
chain. The minimal TCR complex thus has 10 ITAMs. These couple the TCR
to intracellular tyrosine kinases (see below). ITAM motifs are a defining
feature of antigen-receptor complexes. Igα and Igβ, the signaling subunits of
the BCR, both have a single ITAM.
ITAM motifs are not restricted to the TCR and BCR. For example, mast
cells comprise an important group of lymphocytes whose fate is determined
by antigen-specific immunoglobulin. These cells respond to antigen because
they express a high-affinity receptor for IgE. This receptor, termed FcεR1,
binds to the immunoglobulin IgE with high affinity. When FcεR1-IgE
complexes are cross-linked by polyvalent antigen they can trigger mast cell
degranulation and the release of cytokines and allergic mediators. The FcεR1
is assembled from three subunits: the α subunit that binds to the Fc region of
IgE, a β subunit that provides important accessory signaling, and the FcRγ
chain, which is a signaling subunit that contains a single ITAM (Beaven and
Metzger 1993; Abram and Lowell 2007; p. 125 [Samelson 2011]).
TCR/BCR/FcεR1 signaling is initiated by the tyrosine phosphorylation of
ITAMs by Src-family tyrosine kinases such as Lck and Fyn in T cells, Lyn in
B cells, and Fyn in mast cells (Salmond et al. 2009). When both tyrosine
residues are phosphorylated, the ITAM forms a high-affinity binding site for
Syk-family tyrosine kinases; generally in T cells this is Zap-70 (Wang et al.
2010), whereas in B cells and mast cells Syk is recruited (Chu et al. 1998).
Zap-70 and Syk contain tandem SH2 domains that bind with high affinity to
the doubly phosphorylated ITAM (Chu et al. 1998). The activation of Zap-70
or Syk is initiated by binding to phosphorylated ITAMs. This is proposed to
release Syk/Zap-70 from an autoinhibited conformation and expose
regulatory tyrosine residues for phosphorylation by Src-family kinases (Au-
Yeung et al. 2009). The phosphorylation of tyrosine residues in the activation
loop in the Zap-70/Syk catalytic domain, as well as two residues in the
adjacent linker region, then further stimulates their catalytic activity.
Antigen-receptor control of Syk-family tyrosine kinases is fundamental for
lymphocyte activation and underpins the ability of antigen receptors to
transduce signals from pathogen-derived antigens to the interior of
lymphocytes (Mocsai et al. 2010; Wang et al. 2010).
How the Src-family kinases such as Lck are regulated is central to
antigen-receptor signal transduction (Salmond et al. 2009). The activity of
Lck is regulated by phosphorylation and dephosphorylation of a carboxy-
terminal tyrosine (Y505) by the ubiquitously expressed kinase carboxy-
terminal Src kinase (CSK), as well as autophosphorylation of the activation
loop tyrosine residue, Y394. Phosphorylated Y505 forms an intramolecular
binding site for the Lck SH2 domain, thereby locking the kinase into an
autoinhibited state. The key to initiating the activation of Lck and its relatives
is to dephosphorylate the carboxy-terminal tyrosine and relieve autoinhibition
of the kinase. This is mediated by transmembrane-receptor-like tyrosine
phosphatases, such as CD45 and CD148 (Hermiston et al. 2009; Zikherman
et al. 2010). Hence in T cells, the Lck activation threshold is set by the
balanced activity of the kinase-phosphatase pair CSK, which phosphorylates
Y505, and CD45, which dephosphorylates this residue (Zikherman et al.
2010).
 It is frequently assumed that triggering antigen receptors stimulates Src
kinase family activity, and antigen receptors are often depicted as molecular
switches that are either on or off. In reality, antigen receptors are always
signaling and it is the intensity of the signal that changes. The assembly of
antigen receptors at the plasma membrane is thus proposed to mediate low-
level signaling and the engagement with high-affinity ligands (antigen or
antigen–MHC) increases the intensity. Indeed Src-family kinases such as Lck
are constitutively active before antigen-receptor engagement and cause low-
level ITAM phosphorylation (Nika et al. 2010). The levels of ITAM
phosphorylation are limited by tyrosine phosphatases, and the increases in
ITAM phosphorylation that follow antigen-receptor engagement probably
result from spatial constraints on the ITAM-phosphatase interaction (van der
Merwe and Dushek 2011).
How are these spatial constraints regulated to explain how ligand
occupancy triggers TCR signaling? Surprisingly, we do not know, although
there is no shortage of theories. Current models range from the ligand-
induced conformational change to the idea that the TCR is a mechanosensor
that converts the mechanical energy generated by antigen binding into a
biochemical signal (Kim et al. 2009). One other idea well supported by
experimental data is that binding of the TCR to peptide-MHC complexes on
the surface ofAPCs causes spatial segregation of TCR complexes (which
have small ectodomains) away from receptor tyrosine phosphatases such as
CD45 and CD148 (which have very large ectodomains). This might locally
perturb the kinase–phosphatase balance sufficiently to favor ITAM
phosphorylation and Zap-70 recruitment (van der Merwe and Davis 2003;
van der Merwe and Dushek 2011). Note that the MHC-binding coreceptors
CD4 and CD8 are also thought to play a role in perturbing the kinase-
phosphatase balance in localized areas of the T-cell membrane. CD4 and
CD8 can thus promote TCR signaling by stabilizing interactions between the
TCR and peptide-MHC ligands. However, the cytoplasmic domains of CD4
and CD8 constitutively bind Lck and hence facilitate the recruitment of this
kinase to ligand-engaged TCR complexes (Artyomov et al. 2010).
What about the BCR and FcεR1? In quiescent B cells, the BCR may exist
in an oligomeric autoinhibited state, and ligand occupancy could drive the
dissociation of these oligomers into monomers that interact more effectively
with downstream tyrosine kinases (Yang and Reth 2010a,b). For the FcεR1,
the opposite is probably the case. This receptor binds IgE but is only
effectively triggered when antigen oligomerizes the receptor (Beaven and
Metzger 1993).

4 ADAPTOR MOLECULES FOR ANTIGEN RECEPTORS

The immediate substrates for tyrosine kinases activated by
TCRs/BCRs/FcεR1s are specialized adaptor proteins that coordinate the
localization and activation of key effector enzymes. In T cells and mast cells,
the adaptors LAT and SLP76 are substrates for Zap-70 and Syk, respectively
(Jordan and Koretzky 2010; p. 125 [Samelson 2011]). In B cells, the adaptor
coupling Syk to effector enzymes is B-cell linker protein (BLNK), also
known as SLP65 (Koretzky et al. 2006).
LAT is an integral membrane protein with a cytoplasmic tail containing
nine tyrosine residues. When phosphorylated, these act as docking sites for
effector enzymes containing SH2 domains. For example, phosphorylated
Y132 of LAT recruits phospholipase Cγ (PLCγ), a critical molecule for
lymphocyte activation. The subsequent tyrosine phosphorylation of PLCγ
activates the enzyme, resulting in the hydrolysis of its substrate
phosphatidylinositol 4,5-bisphosphate (PIP2). LAT not only recruits PLCγ
but also plays a complex role as a scaffold that ensures PLCγ activation.
Phosphorylated Y171, Y191, and Y226 in LAT can thus bind to the SH2
domain of Grb2 family members such as Gads, which recruits SLP76 to the
LAT complex.
SLP-76 contains three key tyrosine residues, a central SH3-binding
proline-rich domain and a carboxy-terminal SH2 domain. The SLP76
proline-rich domain binds to the SH3 domain of Gads; the SLP76-Gads
complex is then recruited to LAT via binding of the Gads SH2 domain
binding to tyrosine-phosphorylated LAT.
Tyrosine-phosphorylated SLP76 can recruit a number of effector
molecules into the LAT complex, notably the Tec-family tyrosine kinase Itk,
which phosphorylates PLCγ, leading to its activation. The SH2 domain of
SLP76 is also important because it binds to the cytosolic adaptor ADAP,
which links SLP76 to the regulation of integrin-mediated cell adhesion. The
LAT-SLP76 complex thus nucleates and organizes multiple TCR-dependent
signaling pathways in T cells. Indeed, LAT and SLP76 are essential for TCR
function: there are multiple defects in thymus T-cell development and
peripheral T-cell function in the absence of these adaptors.
LAT and SLP-76 are equally important for mast cell function, coupling
Syk to signaling pathways downstream from the FcεR1 (Alvarez-Errico et al.
2009; Kambayashi et al. 2009). However, neither LAT nor SLP76 is
expressed in B cells; there, the predominant adaptor molecule is BLNK
(Kurosaki and Hikida 2009). BLNK is a Syk substrate and contains nine
tyrosine residues that are rapidly phosphorylated following BCR triggering.
Its recruitment to the plasma membrane requires association with CIN85, and
the BLNK-CIN85 complex coordinates recruitment of effectors such as PLCγ
and Grb2-family adaptors (Oellerich et al. 2011). BLNK is essential for
normal B-cell development and for peripheral B-cell function (see Fig. 1).
Figure 1. Signaling downstream from immune receptors bearing immunoreceptor tyrosine-based
activation motifs (ITAMs; yellow rectangles). T-cell receptors, B-cell receptors, and FcεR1s all
contained ITAMs that can be tyrosine phosphorylated (red circles) by Src-family kinases such as Fyn
and Lck. This creates docking sites for the recruitment and activation of the tyrosine kinases Zap-70
and Syk. These in turn phosphorylate adaptor complexes that recruit numerous additional signaling
molecules that control phospholipid, calcium, small G protein, and kinase signaling.

5 CALCIUM AND DIACYLGLYCEROL SIGNALING

A major function for antigen-receptor-coupled tyrosine kinases and adaptors
is to regulate intracellular calcium levels and control DAG-mediated
signaling (Oh-hora and Rao 2008; Matthews and Cantrell 2009). Inositol
1,4,5-trisphosphate (IP3) produced by PLCγ binds to IP3 receptors on
endoplasmic reticulum (ER) membranes, initiating release of calcium from
stores and an increase in cytosolic calcium concentration (p. 95 [Bootman
2012]). This in turn triggers calcium entry across the plasma membrane via
activation of highly selective store-operated calcium-release-activated
calcium (CRAC) channels. Stromal interaction molecules 1 and 2 (STIM1
and STIM2) sense depletion of the ER stores and relocate to ER–plasma-
membrane junctions. There they bind to the CRAC channel protein Orai1,
which activates the channels to allow entry of extracellular calcium to
promote a sustained increase in intracellular calcium levels. This coupling of
antigen receptors to CRAC channels allows lymphocytes to sustain high
levels of intracellular calcium concentrations during an immune response
(Hogan et al. 2010).

6 DOWNSTREAM FROM CALCIUM SIGNALING IN

LYMPHOCYTES
Increases in intracellular calcium concentration in lymphocytes initiate
signaling by the calcium/calmodulin-dependent protein kinase kinases
(CaMKKs) (Matthews and Cantrell 2009). The best-studied role for calcium
signaling in both B and T lymphocytes, however, is control of calcineurin
(also known as protein phosphatase 2B, PP2B), a protein phosphatase that
controls the intracellular localization of members of the NFAT (nuclear factor
of activated T cells) family of transcription factors (Im and Rao 2004; Muller
and Rao 2010). These are key regulators of cytokine gene expression in B
and T lymphocytes, in which they control expression of IL2, IL4, TNF, and
IFNγ. In quiescent lymphocytes, before antigen-receptor engagement, NFATs
are constitutively phosphorylated via the actions of NFAT kinases that
include CK1 and GSK3. This phosphorylation of NFATs causes their nuclear
exclusion as a result of binding to 14-3-3 proteins, thus maintaining them
inactive in the cytosol. NFATs remain inactive until triggering of antigen
receptors raises intracellular free calcium levels, which activates calcineurin,
which then dephosphorylates NFATs, allowing their translocation to the
nucleus.
In the nucleus, NFATs form complexes with other transcription factors,
bind to target genes and modulate gene transcription. In the context of IL2
expression, NFAT–AP1 complexes act as positive regulators of IL2
production, whereas complexes containing NFAT with the Foxp3
transcription factor appear to repress cytokine gene expression (Im and Rao
2004; Muller and Rao 2010). The impact of NFAT translocation to the
nucleus on the T-cell transcriptional program thus depends on cellular
context and the available NFAT-binding partners. Nevertheless, the rate-
limiting step for NFAT activation is antigen-receptor-regulated increases in
intracellular calcium and the resultant activation of calcineurin.
The importance of calcium/calcineurin signaling for T-cell activation is
emphasized by the clinical efficacy of drugs based on the compound
cyclosporin A or FK506 that prevent calcineurin activation and NFAT
dephosphorylation (Gallo et al. 2006). These are potent T-cell
immunosuppressants used for the prevention of organ transplant rejection and
for the treatment of chronic T-cell-mediated autoimmune diseases, such as
ectopic eczema.

7 DIACYLGLYCEROL SIGNALING IN LYMPHOCYTES

Multiple species of DAG are produced as intermediates in phospholipid
resynthesis pathways. Consequently, quiescent lymphocytes have high levels
of DAG before immune activation. However, antigen-receptor stimulation
induces further production of polyunsaturated DAG by triggering PLCγ-
mediated hydrolysis of PIP2; in particular, triggering localized increases in
DAG levels in membrane microdomains (Spitaler et al. 2006; Quann et al.
2009). DAG binds with high affinity to proteins that contain a conserved
cysteine-rich domain (CRD) (H-X12-C-X2-C-X13/14-C-X2-C-X4-H-X2-C-X7-
C). In lymphocytes, these proteins include the Ras/Rap guanyl-releasing
protein (GRP) family of guanine nucleotide exchange factors (GEFs), which
activate Ras and Rap GTPases, and the serine/threonine kinases protein
kinase C (PKC) and protein kinase D (PKD).

8 PKC AND LYMPHOCYTES

Lymphocytes express multiple PKC isoforms, including α, βI, βII, δ, ε, η, and
θ, and these have key roles in lymphocyte activation (Matthews and Cantrell
2009). They are important regulators of lymphocyte transcriptional programs
and, in particular, control expression of genes encoding cytokines and
cytokine receptors. DAG/PKC signaling also plays a key role in controlling
integrin-mediated cell adhesion and lymphocyte polarity. The direct
substrates for PKCs include PKDs (Matthews et al. 2010). Lymphocytes
predominantly express PKD2 and activation of this kinase requires trans-
phosphorylation of conserved serine residues within the enzyme’s catalytic
domain (S701 and S711). These sites are substrates for both conventional and
novel PKCs, and their phosphorylation is essential for efficient TCR-induced
cytokine production and for optimal antibody production by B lymphocytes.
Other PKC substrates include scaffolding proteins such as Carma1 and GEFs
for the GTPases Ras and Rap1 (Matthews and Cantrell 2009). In particular,
PKC-mediated phosphorylation of RapGEF2 is critical for activation of the
GTPase Rap1, which controls the activity of the integrin LFA1 (also known
as integrin αLβ2) and hence lymphocyte adhesion (Kinashi 2005).
The coordination of integrin-mediated cell adhesion by PKC and GTPases
is essential to allow T cells, B cells, and natural killer (NK) cells to form tight
contacts with APCs or target cells via a structure known as the
immunological synapse (Dustin et al. 2010; Springer and Dustin 2011).
These are formed between naïve T cells and APCs or effector cytolytic T
cells and pathogen-infected target cells. B cells can also form immunological
synapses with APCs in a process that potentiates antigen binding and
processing of even membrane-tethered antigens (Harwood and Batista 2011).
Immunological synapses are highly ordered structures characterized by the
segregation of receptors and signaling molecules into distinct areas known as
supramolecular activation clusters (SMACs). Stable immune synapses are
arranged in concentric zones: antigen receptors accumulate in the center
(cSMAC), whereas integrins segregate to the periphery (pSMAC). One
common misconception is that the immune synapse is involved in the
initiation of antigen-receptor signaling. The reality is that immune synapses
are formed as a downstream consequence of antigen-receptor engagement.
Immunological synapses provide a focus for DAG signaling following
antigen-receptor engagement (Spitaler et al. 2006). Moreover, formation of
immunological synapses is associated with the polarization of the
microtubule-organizing center (MTOC) toward the target cell. This MTOC
polarization is coordinated by calcium and DAG signaling pathways, with
PKC family members playing a crucial role. The reorientation of the MTOC
controls the ability of lymphocytes to direct cytokine secretion and to direct
the exocytosis of secretory or lytic granules. For example, in cytotoxic T cells
the immunological synapse directs the secretion of the granules that contain
cytolytic effector molecules such as perforin and granzymes toward the target
cell (Jenkins and Griffiths 2010).
One of the best characterized roles for PKCs in lymphocytes is the control
of gene expression via the transcription factor NF-κB1 (also known as p50)
(Oeckinghaus et al. 2011; Gerondakis and Siebenlist 2012). In quiescent
lymphocytes, NF-κB1 is sequestered in the cytosol in a complex with
inhibitor of NF-κB (IκB). The activation of PKC results in the assembly of a
complex comprising the scaffolding protein Carma1, Bcl10, and MALT1
(Blonska and Lin 2009). This PKC-induced Carma1-Bcl10-MALT1 complex
subsequently binds to and activates the IκB kinase (IKK) complex, which
then phosphorylates IκB, triggering its rapid ubiquitylation by the E3 ligase
SCF-βTrCP and degradation by the proteasome. The removal of IκB unmasks
the nuclear localization sequence of NF-κB1 and permits its translocation to
the nucleus, where it stimulates the transcription of target genes. This
mechanism is common to all lymphocytes but there is redundancy between
PKC isoforms: in B lymphocytes, PKCβ isoforms are involved, whereas in T
cells PKCε and θ are essential.

9 Ras SIGNALING AND LYMPHOCYTES

In quiescent lymphocytes Ras GTPases are predominantly inactive.
Engagement of antigen receptors stimulates Ras proteins to accumulate in a
GTP-bound state. This allows Ras to bind to the serine/threonine kinase Raf1,
which in turn activates the kinase MEK1 that phosphorylates and activates
the MAP kinases (MAPKs) ERK1 and ERK2 (p. 81 [Morrison 2012]). Two
major classes of GEFs couple antigen receptors to Ras activation: the Ras
GRPs and SOS. Ras GRPs are activated by DAG and PKC-mediated
phosphorylation. RasGRP1 acts downstream from antigen receptors in T
cells, whereas RasGRP1 and RasGRP3 function in B cells, and RasGRP4
functions in mast cells. SOS is activated independently of DAG/PKC via a
tyrosine-kinase-dependent pathway. It thus binds constitutively to the SH3
domains of the adaptor Grb2 and is recruited to the plasma membrane when
the SH2 domain of Grb2 binds to tyrosine-phosphorylated adaptors such as
LAT in T cells or Shc in B cells. Note that Ras is also activated by members
of the common cytokine-receptor γ chain (γc) family of cytokines (see
below), such as IL2. Receptors for these cytokines recruit SOS to the plasma
membrane via Grb2 and the adaptor Shc (p. 117 [Harrison 2012]).
The prototypical role for Ras in lymphocytes is to control gene
transcription via ERK1 and ERK2 (Matthews and Cantrell 2009). These
phosphorylate and regulate a number of key substrates, including the ternary
complex factor (TCF) subfamily of ETS-domain transcription factors. They
also control the activity of the RSK serine/threonine kinases that are known
to have important functions in lymphocyte development and peripheral
lymphocyte function. The initiating step for RSK activation is thus ERK1/2-
mediated phosphorylation of S369, T365, and T577 in the carboxy-terminal
catalytic domain of the kinase. The activated carboxy-terminal catalytic
domain of RSK then phosphorylates S386 intramolecularly to create a
docking site for the kinase PDK1, which then phosphorylates S227 in the
amino-terminal RSK kinase domain, thereby activating the enzyme (Finlay
and Cantrell 2011a).
A full list of ERK1/2 substrates in lymphocytes is beyond our scope here
but there have been some unexpected insights into the complexity of ERK
signaling pathways in lymphocytes that warrant discussion. Flow cytometric-
based assays that assess ERK activity at the single-cell level have shown that,
when lymphocytes respond to an increasing strength of antigen-receptor
stimulus, ERK activation is a digital (all or nothing) rather than an analog
response (Chakraborty et al. 2009; Das et al. 2009). In this digital response,
the frequency of cells within a population that activate ERK changes, each
cell activating it to an equivalent level. This means, in practice, that even a
strong antigen-receptor stimulus can only trigger a proportion of lymphocytes
to activate ERKs at any one time. The digital nature of this ERK response
creates signaling heterogeneity within the responding lymphocyte population.

10 COSTIMULATORY MOLECULES,
CYTOKINES, AND LYMPHOCYTE
ACTIVATION
Lymphocyte responses both prior and subsequent to antigen-receptor
engagement are modulated by multiple costimulatory and coinhibitory
receptors. Signaling via Toll-like receptors (TLRs) is also a major factor
influencing the fate of lymphocytes during an immune response. Because T
and B lymphocytes respond to antigens presented to them by APCs,
lymphocyte activation can be regulated by the adhesion molecules and
costimulatory molecules expressed by the APC. Note also that many of the
cytokines that control lymphocyte fate are produced in response to TLR-
mediated activation of dendritic cells and macrophages (Ch. 15 [Newton and
Dixit 2012]). Hence, the nature of the pathogen challenge to the innate
immune system, and the resultant cytokine milieu modulate the adaptive
immune response.
For T cells, key coreceptor molecules include the MHC receptors CD4
and CD8, and proteins such as CD28 (a positive coregulator) and CTLA4 and
PD-1 (negative coregulators) (Artyomov et al. 2010; Francisco et al. 2010;
Bour-Jordan et al. 2011; Walker and Sansom 2011). In B cells, molecules
such as CD19 and the CD21 receptor for complement component C3d are
essential (Carter and Fearon 1992; Depoil et al. 2008; Elgueta et al. 2009;
Mackay et al. 2010) as are the TNF receptor family members CD40 and
receptor for B-cell-activating factor (BAFFR) (Watts 2005; Elgueta et al.
2009; Karin and Gallagher 2009).
A full review of lymphocyte regulation by costimulatory factors is beyond
our scope here but there are some general themes. Costimulatory molecules
frequently work as adaptors to recruit signaling molecules to the plasma
membrane and hence amplify antigen-receptor-mediated signaling. For
example, CD4 and CD8 in T cells recruit Lck to the plasma membrane.
Similarly, CD28 in T cells and CD19 in B cells both have cytoplasmic
domains that can be tyrosine phosphorylated and thus can act as docking sites
for SH2-domain-containing adaptors and enzymes. The CD19 cytoplasmic
tail contains nine tyrosine residues with the potential to be phosphorylated
and interact with signaling molecules including lipid kinases, Vav-family
GEFs, and adaptor proteins such as Grb2. Other important examples of
molecules that recruit key adaptor molecules to the plasma membrane are the
lymphocytic activation molecule (SLAM) family of receptors and associated
intracellular adaptors of the SLAM-associated protein (SAP) family (Veillette
2010).
The engagement of CD40 by its ligand (CD40L) leads to signals via
adaptor proteins known as TNFR-associated factors (TRAFs), which activate
signaling pathways, including MAPKs and NF-κB (p. 121 [Lim and Staudt
2012]).
The plethora of costimulatory molecules that can contribute to
lymphocyte activation can be confusing, particularly because all seem to
activate similar signal transduction pathways. The key message is that these
receptors function at different times and in different contexts. For example,
CD28 binds to the B7 family members CD80 and CD86, which are mainly
expressed on APCs responding to TLR signaling. The ligand for CD40 is
produced transiently by antigen-activated T cells and plays a key role in
promoting specific T cell “help” to B cells by ensuring integration of signals
between CD40-expressing B cells and antigen-primed T cells. In contrast,
BAFF is mainly produced by neutrophils, monocytes, and macrophages and
hence allows cross talk between B cells and these cells of the innate immune
system.

11 CYTOKINE SIGNALING IN
LYMPHOCYTES
Cytokines that signal via the Janus tyrosine kinases (JAKs) (p. 117 [Harrison
2012]), such as the γc family of cytokines, IFNs, and cytokines such as IL12
and IL23, are particularly important to the adaptive immune system
(Rochman et al. 2009). For example, CD4-expressing αβ T cells differentiate
during immune responses to produce distinct effector subpopulations
(O’Shea and Paul 2010) and the specification of these CD4+ T-cell subsets is
controlled by cytokines that direct the combinatorial action of multiple
chromatin regulators and key lineage-specifying transcription factors. For
example, IL12 drives Th1 T-cell differentiation and IL6, IL21, and IL23
drive Th17 cell differentiation. Moreover, cytokines have pleotropic roles.
IL2 is important for the differentiation of antigen-primed CD8+ T cells to
effector cytotoxic T cells (CTLs) but is also required for optimal Th1 T-cell
differentiation and for the development of Treg cells.
One striking feature of lymphocyte biology is that the ability of cells to
respond to cytokines (i.e., to express particular cytokine receptors) can be
shaped by antigen-receptor triggering. Cytokine production by cells of the
immune system is, in turn, controlled by triggering of antigen receptors in T
and B cells or by receptors of the innate immune system. A prototypical
example is IL2, which is only produced by antigen-receptor-activated T cells
and B cells or pathogen-triggered dendritic cells. Moreover, expression of the
IL2 receptor (IL2R) is tightly controlled by immune activation. The ILR2
receptor complex consists of a γc, a β subunit (CD122), and an α subunit
(CD25). The expression of CD25 is rate limiting as it determines the ability
of the receptor to bind IL2 with high affinity. Importantly, CD25 is not
expressed on naïve CD4 and CD8 T cells but only on activated T cells. In
addition, the expression of CD25 is transient and its sustained expression
requires constant immune stimulation. IL2 responsiveness is thus tightly
linked to antigen-receptor triggering to ensure the tight control of T cells by
IL2. IL12 receptors are similar: these are only expressed on activated T cells.
Furthermore, IL12 receptor expression needs to be sustained by IL2 and there
is tight control of IL12 secretion by pathogen-activated dendritic cells and
macrophages. Such dynamic regulation of cytokine and cytokine-receptor
expression during immune activation ensures the immune specificity of
cytokine action (i.e., only lymphocytes that have been primed by antigen-
receptor triggering can respond to IL12). Note the production of cytokines is
also limited to either pathogen-activated innate immune cells or antigen-
activated lymphocytes (Fig. 2).
Figure 2. Signaling by interleukin (IL) receptors. Many cytokines signal via receptors linked to Janus
tyrosine kinases (JAKs), which regulate the SH2-domain-containing transcription factors STATs. The
different ILs produced by different cell types activate receptors coupled to different combinations of
JAKs and STATs.

Cytokines that activate JAKs regulate the function of SH2-domain-

containing transcription factors known as STATs (signal transducers and
activators of transcription) (Ghoreschi et al. 2009; p. 117 [Harrison 2012]).
There are four JAKs (JAK1, JAK2, JAK3, and Tyk2) and 7 STATs (STAT1,
STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6). A single JAK, or
combination of JAKs, associates selectively with the cytoplasmic domains of
the cytokine receptors. The model for JAK activation is that ligand
occupancy of cytokine-receptor dimers results in JAK transphosphorylation
and activation. The type I IFN receptors signal via JAK1 and Tyk2; IL12 and
IL23 receptors signal via JAK2 and Tyk2. The IFNγ receptor activates JAK1
and JAK2, whereas γc-containing receptors, which include the receptors for
IL2, IL4, IL7, IL9, IL15, and IL21, use JAK1 and JAK3. JAK activation
results in phosphorylation of tyrosine residues within the cytoplasmic tails of
cytokine-receptor subunits that act as docking sites for the SH2 domains of
the STATs. The recruitment of STATs leads to their phosphorylation by the
JAKs. The STATs then form homodimers via SH2 domain interactions and
translocate to the nucleus to bind STAT-response elements in DNA. STATs
control lymphocyte transcriptional programs by working as transcriptional
activators but they can also function as gene repressors (O’Shea and Paul
2010).
The specificity of STAT activation is determined by the selectivity of
STAT SH2 domains for the STAT-recruitment motifs in the different
cytokine-receptor subunits. For example, IL2 predominantly activates
STAT5, because tyrosine-phosphorylated IL2Rβ subunits contain a high-
affinity binding site for STAT5. The IL4 receptor, which comprises γc and a
unique IL4 receptor α chain, activates STAT6 because tyrosine-
phosphorylated IL4 receptors selectively bind STAT6. Figure 2 summarizes
current information about the JAK/STAT signaling combinations that
function downstream from the major cytokine receptors.
It should be stressed that although the activation of STATs is pivotal for
cytokine actions it is usually not sufficient to mimic the effects of cytokines.
Indeed, cytokines can regulate other signal transduction pathways, some of
which are shared with other receptors (e.g., IL2 and IL15 also activate
Ras/ERK signaling) (Cantrell 2003). Moreover, many cytokines induce
accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of
phosphoinositide 3-kinases (PI3Ks) (Okkenhaug and Fruman 2010; Finlay
and Cantrell 2011).

12 PI3K-MEDIATED SIGNALING IN
LYMPHOCYTES
PI3K signaling is important for lymphocyte activation and integrates multiple
receptor inputs. For example, in naïve T cells, low basal levels of PIP3 are
maintained by IL7 signaling; these increase strikingly in response to
triggering of the antigen-receptor complex and are then sustained by stimuli
from costimulatory molecules such as CD28. Cytokines such as IL2 and IL15
can then further sustain intracellular concentrations of PIP3. Similarly, in B
cells, cytokines such as BAFF and low-level signaling by non-antigen-
engaged BCRs maintain a low level of PIP3 (Srinivasan et al. 2009). The
levels of PIP3 increase following BCR activation, and costimulatory
molecules such as CD19 and cytokines such as IL4 can also sustain levels of
this lipid.
Antigen receptor and cytokines control PIP3 metabolism in lymphocytes
via class I PI3Ks, which typically exist in a complex comprising a p110
catalytic subunit and an 85-kDa SH2-domain-containing regulatory/adaptor
subunit. Four p110 isoforms exist (α, β, γ, and δ) and two p85 subunits (α and
β) exist. These different isoforms function in distinct pathways in
lymphocytes, and expression of p110δ is restricted to hematopoietic cells.
p110δ produces the PIP3 that is generated in response to many antigen
receptors and cytokines, whereas p110γ, which heterodimerizes with the
p101 regulatory subunit rather than a p85-type subunit, is involved in
chemokine receptor signaling (Okkenhaug and Fruman 2010).
The production of PIP3 requires recruitment of PI3K to the plasma
membrane. There are two possible mechanisms: binding of the SH2 domain
of p85 to phosphorylated tyrosine residues in receptor cytoplasmic domains
or membrane-localized adaptors; and direct recruitment of p110 by Ras. In
the case of the BCR, CD19 recruits PI3K to the plasma membrane via
binding of p85 to its tyrosine-phosphorylated cytoplasmic domain. Tyrosine-
phosphorylated cytokine receptors similarly recruit PI3K by binding p85.
Surprisingly, how TCR and CD28 signaling induces PIP3 accumulation is not
known, but direct recruitment to tyrosine-phosphorylated CD28 does not
occur, and it is more likely that adaptors such as LAT or SLP76 are
important.
 PIP3 binds to pleckstrin homology (PH) domains in other signaling
proteins to control their activity and subcellular localization. In lymphocytes,
these include Tec-family tyrosine kinases such as Itk and Btk, GEFs for Rho
family GTPases, and the kinases PDK1 and Akt (also known as PKB) (p. 87
[Hemmings and Restuccia 2012]). Akt is activated by PDK1-mediated
phosphorylation of T308 within its catalytic domain. This is PIP3 dependent
probably because the binding of PIP3 to the Akt PH domain causes a
conformational change that allows PDK1 to phosphorylate T308. PDK1 also
has a PIP3-binding PH domain, but this promotes translocation of the enzyme
to the plasma membrane (where it can colocalize with Akt) rather than
enzyme activation (Finlay and Cantrell 2011).
Once activated, Akt phosphorylates a number of critical signaling
molecules. For example, it phosphorylates and inactivates the Rheb GAP
TSC2, causing accumulation of Rheb-GTP complexes, which play a role in
activating the mTORC1 complex (mammalian target of rapamycin complex
1) (p. 91 [Laplante and Sabatini 2012]). Akt also phosphorylates the
transcription factors Foxo1/3 and Fox4A. These Foxo family transcription
factors are nuclear and active in quiescent cells but, when phosphorylated,
they exit the nucleus and form a complex with 14-3-3 proteins in the cytosol,
which terminates their transcriptional activity.
Akt is fundamentally important in many cells because it controls nutrient
uptake and cellular metabolism. In particular, activated lymphocytes up-
regulate glucose, amino acid and iron uptake, and switch their metabolism to
glycolysis (see Ch. 7 [Ward and Thompson 2012]). This increases cellular
energy production and nutrient uptake to support the increased biosynthetic
demands of rapid cell proliferation. Note, however, that it is difficult to
ascribe a universal function for Akt that holds for all lymphocyte
subpopulations. For example, Akt is important for metabolism and cell
survival in peripheral B lymphocytes (Srinivasan et al. 2009) and in T
lymphocyte progenitors in the thymus, but is not essential for metabolism or
for the survival of peripheral or effector cytotoxic T cells (Finlay and Cantrell
2011). Moreover, the Akt/Foxo pathway has a critical role controlling
expression of the recombinase genes responsible for antigen-receptor
diversity in B cells (Kuo and Schlissel 2009) but there is no evidence for such
a role in T cells. The molecular basis for these differences is not understood
but probably reflects redundancies with other kinases that have similar
substrate specificities (e.g., SGK1).
Akt/Foxo signaling is also uniquely linked to the regulation of the
expression of key cytokine and chemokine receptors and adhesion molecules
in lymphocytes (Hedrick 2009; Lorenz 2009; Macintyre et al. 2011). Hence,
when Akt is inactive in quiescent lymphocytes, nonphosphorylated Foxo1,
Foxo3, Foxo3A, and Foxo4 are found in the nucleus, where they drive
transcription of genes encoding the receptor for IL7, an essential homeostatic
cytokine for lymphocytes. Moreover, Foxo transcription factors also drive
expression of the transcription factor KLF2; this directly regulates
transcription of adhesion molecules and chemokine receptors that together
control lymphocyte entry and egress from secondary lymphoid tissues and
lymphocyte positioning in lymphoid tissue. The activation of Akt thus causes
lymphocytes to change their trafficking program around the body. Akt
activation also changes the cytokine-receptor profile of T cells and hence the
ability of cytokines to determine T-cell fate.
In many cells, a key role for Akt is to control the activity of the
mammalian target of rapamycin complex 1 (mTORC1) (p. 91 [Laplante and
Sabatini 2012]). Rapamycin is a powerful immunosuppressant that is used in
the clinic to prevent rejection of organ transplants. mTORC1 coordinates
inputs from nutrients and antigen and cytokine receptors to control T-cell
differentiation (Powell and Delgoffe 2010). The molecular mechanisms used
by mTORC1 to control T-cell differentiation are not fully understood; neither
are the signaling processes that activate mTORC1. There is, however,
evidence that mTORC1 controls expression of genes encoding effector
cytokines and cytolytic molecules. Moreover, mTORC1 directs the tissue-
homing properties of T cells by regulating the expression of chemokine and
adhesion receptors (Sinclair et al. 2008).

13 INHIBITORY SIGNALS AND

LYMPHOCYTE ACTIVATION
Signals from APCs and other immune cells can also deliver inhibitory signals
to lymphocytes to ensure immune homeostasis. Indeed these are vital for a
balanced immune response because a failure to limit immune responses
results in excessive inflammation and potentially autoimmunity. Examples of
signaling molecules that mediate key negative-feedback pathways in
lymphocytes include SHIP, a lipid phosphatase with specificity for the 5′
position of PIP3 (Parry et al. 2010). SHIP is recruited to the plasma
membrane by the binding of its SH2 domain to a tyrosine-phosphorylated
immune cell tyrosine-based inhibitory motif (ITIM) located in the cytosolic
domain of cell-surface receptors and dampens production of PIP3. A
prototypical example of this feedback process occurs in B cells when
coligation of the BCR with the FcγRIIB by antigen-antibody complexes
results in tyrosine phosphorylation of the ITIM in FcγRIIB (Daëron and
Lesourne 2006). SHIP binds to the phosphorylated ITIM, thereby recruiting
this inositol 5′ phosphatase into the BCR-FcγRIIB complex. SHIP
dephosphorylates PIP3 to produce PI(3,4)P2 and, accordingly, diminishes the
BCR-dependent elevation of intracellular PIP3 levels. There are many other
examples of ITIM-containing receptors that play an important role in immune
homeostasis. For example, an extensive family of sialic-acid-binding
immunoglobulin-like lectins, siglecs, responds to sialylated glycans to
regulate lymphocyte function (Nitschke 2009; Cao and Crocker 2011).
Siglecs are key regulators of B cell, NK cell, and macrophage biology.
In T cells, transmembrane receptors such CTLA4 and PD1 are critical for
limiting T-cell function during immunity and tolerance (Veillette et al. 2002;
Francisco et al. 2010; Bour-Jordan et al. 2011). The purpose of PD1 signaling
is to limit the expansion of effector T cells during an immune response and
hence to limit the pathology and tissue damage associated with effector CD8+
T-cell-mediated tissue destruction. However, the failure to control chronic
viral infections such as HIV results from inhibitory-receptor-driven
exhaustion of antigen-specific T cells, demonstrating how the balancing of
positive- and negative-feedback signaling needs to be finely tuned to ensure a
favorable outcome. The impact of any imbalance of these pathways on
human health is enormous: a failure of feedback control leads to
autoimmunity; too much feedback control can limit the ability of the immune
system to clear the pathogen.
B lymphocytes express the siglec family member CD22 (also known as
siglec2), which inhibits B-cell signaling and B-cell-mediated autoimmunity
by recruiting SHP1 (Lorenz 2009; Nitschke 2009). CD22 interacts with
ligands carrying α2–6-linked sialic acids both in cis and in trans to modulate
the BCR signaling threshold. The importance of SHP1 is strikingly illustrated
by the phenotype of the moth-eaten (me/me) mouse, which lacks SHP1
tyrosine phosphatase activity and displays a variety of hematopoietic and
immune disorders that result in death two or three weeks after birth (Lorenz
2009).
There are additional key negative regulator receptors in which there is
either no classical ITIM or controversy as to the importance of the
recruitment of phosphatases. CTLA4 is an example. It is an essential negative
regulator of T-cell-mediated immune responses: CTLA4-deficient mice show
a fatal lymphoproliferative disorder. CTLA4 binds the same two ligands
(CD80 and CD86) the costimulatory molecule CD28 binds. The engagement
of CD28 by CD80 or CD86 results in T-cell costimulation, whereas CTLA4
engagement results in inhibition of T-cell activation. CTLA4 might deliver a
negative signal to the T cell by recruiting tyrosine phosphatases to the plasma
membrane. However, two other models exist. One proposes that CTLA4
activates T cells to increase their motility and that this prevents T cells from
making stable contacts with APCs (Rudd 2008). The other proposes that
CTLA4 competes with CD28 for ligand but binds to CD80/86 with higher
avidity than does CD28. Indeed CTLA4 has now been shown to capture its
ligands CD80 and CD86 by trans-endocytosis (Qureshi et al. 2011). It could
thus inhibit CD28 costimulation by depleting CD28 ligands. These models
are not necessarily mutually exclusive, and how CTLA4 and the other
inhibitory molecules exert essential feedback control is still the subject of
much debate.

14 CONCLUDING REMARKS
In lymphocytes, signal inputs generated by specific pathogens regulate the
activity of evolutionarily conserved signaling pathways. Antigen receptors
direct the immune response but lymphocyte signaling is also controlled by
cytokines and chemokines that are not antigen specific. These antigen-
specific and -nonspecific elements of lymphocyte signal transduction are
tightly coupled because antigen-receptor signaling controls the repertoire of
cytokine and chemokine receptors and adhesion molecules expressed by
lymphocytes. Antigen receptors also direct lymphocyte trafficking between
the blood, peripheral tissues, and secondary lymphoid organs and hence
control the cytokine milieu available to these cells. This coordination of
antigen receptor and cytokine signaling ensures the immune specificity of
lymphocyte activation and is fundamental for adaptive immune responses.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a018788
CHAPTER 17

Vertebrate Reproduction

Sally Kornbluth1 and Rafael Fissore2

1Duke University School of Medicine, Durham, North Carolina 27710
2University of Massachusetts, Amherst, Veterinary and Animal Sciences, Amherst, Massachusetts
01003
Correspondence: sally.kornbluth@duke.edu

SUMMARY

Vertebrate reproduction requires a myriad of precisely orchestrated

events—in particular, the maternal production of oocytes, the paternal
production of sperm, successful fertilization, and initiation of early
embryonic cell divisions. These processes are governed by a host of
signaling pathways. Protein kinase and phosphatase signaling pathways
involving Mos, CDK1, RSK, and PP2A regulate meiosis during
maturation of the oocyte. Steroid signals—specifically testosterone—
regulate spermatogenesis, as does signaling by G-protein-coupled
hormone receptors. Finally, calcium signaling is essential for both sperm
motility and fertilization. Altogether, this signaling symphony ensures
the production of viable offspring, offering a chance of genetic
immortality.

Outline
 1 Introduction
2 Oocyte maturation
3 Sperm maturation
4 Fertilization
5 From egg to zygote
6 Concluding remarks
References

1 INTRODUCTION
Mammalian reproduction depends on the proper development and maturation
of both the female egg and the male sperm. These gametes fuse through a
complex series of events, known as fertilization, that ensure the highest
quality of offspring. Both gamete development and fertilization depend on
numerous connected signaling pathways, a flaw in any of which can lead to
infertility or birth defects.
The egg and sperm are haploid germ cells that, upon fertilization,
reconstitute a diploid cell—the embryo. Production of haploid gametes from
diploid precursors requires a modified cell cycle known as meiosis. Before
meiosis, the full complement of parental chromosomes is first duplicated in S
phase, to produce so-called sister chromatids (i.e., four copies of each
chromosome per cell) and paternal and maternal chromosomes pair up. The
homologous chromosomes from each chromosome pair are then separated in
the first meiotic M phase (meiosis I, also known as MI). Subsequently,
without further replication, the cells reenter M phase (meiosis II, also known
as MII) to divide the sister chromatids equally into four haploid daughter
cells. For male gametes, four mature sperm are generated, whereas in the
female, a single final gamete (the egg) is produced together with three polar
bodies. Movement through these stages of meiosis is carefully controlled by
kinases, phosphatases, ubiquitin-dependent degradation of key regulators,
and calcium flux.
 Before acquiring the capacity to fertilize eggs, a sperm must reside in the
female reproductive tract and undergo physiological changes that render it
fertilization competent (Bedford 1970). The acquisition of fertilization
competence and the biochemical, membrane, and enzymatic changes that
underlie it are collectively known as capacitation (Austin 1951; Chang 1951).
As with gamete development and maturation, capacitation and fertilization
depend on careful regulation through signaling pathways. These include
pathways involving gonadotropins, G-protein-coupled receptors (GPCRs),
kinases, and calcium signaling (Salicioni et al. 2007).

2 OOCYTE MATURATION
Oocyte maturation has been most extensively studied in the frog Xenopus
laevis, because its very large oocytes allow both physical manipulation of the
cell (microinjection of proteins, RNAs, and antisense oligonucleotides) and
observation of the progression through meiosis with the naked eye. Although
some notable differences have been observed, genetic studies in mammals
(primarily mouse) have revealed similar overall regulation of meiotic
progression (Fig. 1).
Figure 1. Regulation of oocyte maturation. Immature oocytes are held in G2 arrest through the activity
of PKA, which is stimulated by GPR3-dependent production of cAMP. Progesterone signaling leads to
a loss of PKA activity, leading to disinhibition of the CDK1 activator Cdc25 and stimulation of the
CDK1 inhibitor Wee1. Additionally, progesterone stimulates translation of both Mos and cyclin B
proteins (the former also stimulating translation of the latter) through induction of Eg2, which
phosphorylates and activates CPEB to unmask the messages and promote their polyadenylation. In
parallel, progesterone stimulates translation of RINGO, which can activate CDK1 independently of
cyclin and make it phosphorylate and inhibit the CDK1 inhibitor Myt1. All of these events result in
activation of MPF, which drives the oocyte into MI. Maturation continues via the Mos-MEK-ERK
pathway as shown. Blockade of APC (anaphase-promoting complex) activity by CSF and Emi2 holds
the mature oocyte at a second arrest until the time of fertilization.

Oocytes and sperm both begin life as primordial germ cells (PGCs) that
migrate to the nascent gonads (ovaries in females, testes in males) in early
embryonic development. Under the influence of a variety of cytokines and
growth factors, PGCs that will become oocytes continue dividing mitotically
within cell clusters. In oogenesis, the premeiotic S phase is followed by a
prolonged arrest in prophase I of meiosis until sexual maturity. During this
phase, the oocyte is maintained in a G2-phase-arrested state through G-
protein-coupled signaling (see below). When mitosis ceases, these oocytes
each become surrounded by somatic granulosa and theca cells, which form
the primordial follicles that serve as repositories of dormant oocytes for later
ovulation. Oocytes nestled within the follicles grow and stockpile nutrients
until they become competent to undergo maturation; upon receipt of
appropriate hormonal signals, one follicle from the larger pool will mature
fully during each menstrual cycle in the mammal. Stimulated by pituitary
hormones (gonadotropins) and as a consequence of maturation-inducing
steroid hormones (e.g., progesterone) synthesized by the ovarian follicle
cells, the oocyte exits prophase arrest, and progresses through MI,
transitioning promptly to MII without any intervening DNA replication. At
MII, the oocyte arrests again awaiting fertilization.
The end product of oocyte maturation is a haploid egg capable of being
fertilized. A strong MII arrest helps to prevent parthenogenesis, which is the
aberrant entry of the haploid egg into the mitotic cell cycle in the absence of
fertilization. In an effort to define the factors responsible for MII arrest,
Masui and colleagues injected extract prepared from a mature M-phase-
arrested frog egg into blastomeres formed after the first embryonic cell
division (Masui and Markert 1971); injected cells remained arrested in M
phase, whereas the uninjected cells continued to divide. These experiments
helped to identify both maturation promoting factor (MPF), which drives
entry into both MI and MII during oocyte development, and the cytostatic
factor (CSF), which maintains MII arrest. We now know that MPF is
equivalent to the complex of cyclin B and cyclin-dependent kinase 1 (cyclin-
B–CDK1) that drives entry into mitosis in the somatic cell cycle (Dunphy et
al. 1988; Labbe et al. 1989; Ch. 6 [Rhind and Russell 2012]). CSF was shown
to be the kinase Mos, the cellular counterpart of the viral oncoprotein v-Mos,
which is expressed primarily in germ cells (Propst et al. 1987; Sagata et al.
1989). Proper maturation from MI entry through MII arrest depends on
tightly controlled temporal regulation of both cyclin-B–CDK1 and Mos
activity.

2.1 Meiosis I
During MI, oocytes are maintained in the G2-arrested state by high levels of
cytosolic cAMP. Constitutive signaling by the GPCR GPR3 probably
stimulates adenylyl cyclase to keep cAMP levels high. Indeed,
overexpression of GPR3 in frog oocytes makes them resistant to the
maturation effects of progesterone, and in mice lacking GPR3, oocytes
mature in the absence of additional stimuli (Freudzon et al. 2005; Hinckley et
al. 2005; Mehlmann 2005; Deng et al. 2008). Although sphingosine 1-
phosphate and sphingosylphosphorylcholine have been proposed as GPR3
ligands, unliganded GPR3 appears to be able to stimulate adenylyl cyclase
and thus the role of GPR3 in maintenance of G2 arrest is not entirely clear
(Eggerickx et al. 1995; Uhlenbrock et al. 2002; Hinckley et al. 2005).
Progesterone stimulation seems to antagonize the GPR3 signal, triggering a
decrease in cAMP levels that is at least partly mediated by stimulation of
phosphodiesterases that degrade cAMP (primarily PDE3 in the oocytes)
(Tsafriri et al. 1996). This leads to a diminution of protein kinase A (PKA)
activity. If PDE3 is artificially inhibited or cAMP synthesis is artificially
stimulated, progesterone-induced maturation can be blocked. Conversely,
injection of oocytes with the PKA inhibitor PKI can promote resumption of
meiosis even without progesterone stimulation (Stanford et al. 2003).
As in the mitotic cell cycle, cyclin-B–CDK1 activity is controlled by
phosphorylation of CDK1 on Y15 by Wee1-family kinases, which is opposed
by the Cdc25 phosphatase (Watanabe et al. 1995; Berry and Gould 1996).
The Wee1 relative Myt1 contributes to suppressing CDK1 phosphorylation,
and the Cdc25c isoform mediates its subsequent dephosphorylation. In mice,
this process is mediated by oocyte-specific isoforms: WEE1B and CDC25B.
Loss of WEE1B irreversibly arrests oocytes in prophase (Han et al. 2005). In
the G2-arrested oocyte, PKA directly phosphorylates Cdc25 on S287
(Xenopus numbering), promoting the binding of the small acidic protein 14-
3-3 (Duckworth et al. 2002). 14-3-3 interferes with the ability of Cdc25 to
interact with and dephosphorylate cyclin-B–CDK1 and prevents its
translocation into the nucleus, where it would promote rapid cyclin-B–CDK1
activation (Kumagai and Dunphy 1999; Lopez-Girona et al. 1999; Yang et al.
1999). Thus, the drop in PKA activity required for maturation promotes
Cdc25 activation. PKA also phosphorylates and activates Wee1/Myt1
(Stanford and Ruderman 2005); so the drop in PKA also promotes CDK1
activation by alleviating its suppression by these kinases.
The formation and activity of MPF is also regulated by translation of
cyclin B, whose mRNA is translationally dormant before the induction of
oocyte maturation owing to its very short poly-A tail. At the time of oocyte
maturation, cis-acting sequences within the 3′ UTR of the cyclin B mRNA
promote cytoplasmic polyadenylation, elongating the tail more than 100
nucleotides. These cytoplasmic polyadenylation elements (CPEs) within the
3′ UTR of the mRNA are bound by CPE-binding protein (CPEB) (Hake and
Richter, 1994). Through a process that is not entirely clear, the drop in PKA
activity that heralds the onset of oocyte maturation also induces activation of
a kinase, Eg2, which phosphorylates CPEB, activating it to both unmask the
mRNA and recruit a poly(A) polymerase to elongate the poly(A) tail
(Andresson and Ruderman 1998; Frank-Vaillant et al. 2000; Hodgman et al.
2001).
Note that in every species there is at least some preformed cyclin-B–
CDK1 complex (known as pre-MPF) whose activity is suppressed by
phosphorylation of CDK1 at T14 and Y15. Indeed, the initial discovery of
MPF relied on the ability of the injected MPF to mobilize the pre-MPF pool
through autoamplification (Masui and Markert 1971; Drury and Schorderet-
Slatkine 1975; Wasserman and Masui 1975). Phosphorylation by cyclin-B–
CDK1 suppresses Wee1/Myt1 and activates Cdc25, which promotes more
conversion of pre-MPF to MPF. In species in which most of the CDK1 is
bound to cyclin B in pre-MPF complexes, new cyclin B translation is not
absolutely required for induction of oocyte maturation; however, in those
species that have low amounts of pre-MPF and high levels of free CDK1,
cyclin B synthesis is an obligate step in maturation (Jagiello 1969; Fulka et
al. 1986; Moor and Crosby 1986; Hunter and Moor 1987; Gautier and Maller
1991; Mattioli et al. 1991).
In mammals, the signal emanating from a loss of PKA activity may be
conveyed directly to CDK1 via Wee1B, as this appears to be a direct PKA
target. For Myt1, there is evidence for indirect pathways of inhibition. First,
soon after progesterone treatment, a non-cyclin alternative activator of CDK1
known as RINGO is translated (Ferby et al. 1999). This protein can bind to
and activate CDK1, causing it to phosphorylate and suppress Myt1 (Ruiz et
al. 2008). This, in turn, leads to activation of cyclin-B–CDK1 complexes. A
second pathway is an oocyte-specific MAP kinase (MAPK) cascade
involving the MAPKKK Mos, the MAPKK MEK, and the MAPK ERK. In
frogs, the terminal effector in this pathway is RSK, which can phosphorylate
and inhibit Myt1 (Palmer et al. 1998). RSK can also phosphorylate Cdc25,
contributing to its activation. Accordingly, injection of activated RSK into
Xenopus oocytes can induce meiotic maturation and RSK inhibition interferes
with progesterone-induced maturation. In mice, alternative pathways must
operate (e.g., the direct inhibition of WEE1B by PKA, as described above),
because mice lacking all known isoforms of RSK do not show defects in
oocyte maturation (Dumont et al. 2005).
In some species, including Xenopus, activation of the Mos-ERK pathway
precedes completion of MI and breakdown of the nuclear envelope (known as
germinal vesicle breakdown [GVBD] in oocytes). In others, it occurs after
GVBD (because cyclin-B–CDK1 can actually activate ERK). Whether the
Mos-MEK-ERK-RSK pathway is involved at MI depends on the organism,
and Mos accumulation is controlled by multiple mechanisms (reviewed in
Fan and Sun 2004). The 3′ end of the Mos mRNA in the immature oocyte has
a short poly(A) tail whose elongation (necessary for efficient translation) is
masked through binding of CPEB. In addition to the Eg2-induced
phosphorylation of CPEB, which unmasks and enhances the translation of
Mos (Mendez et al. 2000), the stability of Mos protein is greatly enhanced by
phosphorylation at S3 as both dephosphorylation of this residue and the
presence of a proline at residue 2 are required for recognition by the
ubiquitin-proteasome degradation system (Nishizawa et al. 1993). This site is
phosphorylated in a positive-feedback loop by ERK, which stabilizes Mos.
Where Mos accumulates only after GVBD, it is phosphorylated at the same
site by cyclin-B–CDK1. Together, increased translation and stabilization
promote Mos accumulation during maturation. Note that, in Xenopus,
redundant pathways allow MI progression even when Mos is ablated; yet, the
kinetics are delayed, indicating that Mos normally enhances meiotic
progression in this species. Indeed, when either cyclin B synthesis or Mos
synthesis is impaired, progesterone-induced GVBD can proceed, but ablation
of both abolishes this.

2.2 MI-MII Transition

Mos appears to be more widely important for MII. For example, unlike RSK-
knockout mice, Mos-knockout mice are sterile and oocytes fail to mature
properly (Colledge et al. 1994; Hashimoto et al. 1994). To understand the
role of Mos, we must first consider cyclin B dynamics in meiosis. At the time
of exit from MI, cyclin B must be degraded by the anaphase-promoting
complex (APC), a multisubunit E3 ubiquitin ligase also known as the
cyclosome. However, because cyclin-B–CDK1 inhibits formation of
prereplicative complexes necessary for DNA replication, complete loss of
cyclin-B–CDK1 kinase activity (as occurs in a somatic mitosis) would result
in reinitiation of S phase. Thus, cyclin B translation is ramped up
immediately after GVBD. Moreover, there must be sufficiently rapid
reaccumulation of cyclin B to drive MII. Mos participates in two ways: it
helps to drive cyclin B synthesis, and it helps control cyclin B degradation.
This allows partial but not complete loss of cyclin B at MI exit—a decrease
sufficient to exit MI but not initiate S phase—followed by unimpeded cyclin
B accumulation to drive MII.
A key effector of this pathway is an inhibitor of the APC known as Emi2
(reviewed in Wu and Kornbluth 2008). Emi2 binds directly to the APC,
inhibiting its ability to ubiquitylate substrates and so cause their proteasomal
degradation (e.g., cyclin B). Emi2 is also regulated at the level of protein
stability and is a substrate of another multisubunit E3 ubiquitin ligase, SCFβ–
TrCP (Liu and Maller 2005; Rauh et al. 2005; Hansen et al. 2006).
Recognition by this E3 ligase, which requires a phosphodegron in its targets,
depends on phosphorylation of Emi2 at critical sites within the amino-
terminal half of the protein, catalyzed by cyclin-B–CDK1. It is this feedback
phosphorylation of Emi2 that helps to precisely control cyclin B levels. As
cyclin B is synthesized, it binds to and activates its partner CDK1. Active
cyclin-B–CDK1 complexes phosphorylate Emi2, promoting its degradation,
thereby alleviating suppression of the APC to allow cyclin B degradation and
MI exit. If Emi2 is artificially stabilized at this transition, it causes MI arrest
by preventing cyclin B degradation (Fig. 2).

Figure 2. Regulation of Emi2 and the APC during the MI–MII transition. Phosphorylation controls
Emi2 stability during oocyte maturation. At MI, CDK1 phosphorylates four amino-terminal sites
(S213, T239, T252, and T267) on Emi2; this triggers Emi2 degradation, required for MI exit. At MI
anaphase, cyclin B is degraded, leading to a drop in CDK1 activity. Emi2 is stabilized by
dephosphorylation triggered by Mos signaling. Emi accumulates, resulting in APC inhibition, critical
for S phase block and MII entry. CDK1 activity is low in MII relative to MI. Emi2 is stable in MII, as
required for CSF arrest. At fertilization, Emi2 is quickly degraded through a CaMKII-mediated
pathway, allowing activation of the APC and exit from MII. At the onset of MI anaphase, APC-
mediated cyclin B degradation results in decreased CDK1 activity. With the Mos-PP2A pathway
predominant, dephosphorylated and stabilized Emi2 protein prevents complete ubiquitylation of cyclin
B by the APC. This is essential for the inhibition of S phase between MI and MII. (From Tang et al.
2008; adapted, with permission.)

If cyclin-B–CDK1 phosphorylation of Emi2 were unopposed, then

ultimately accumulation of cyclin B would eradicate Emi2, allowing
complete, rather than the required partial, cyclin B degradation. However, the
Mos-ERK pathway interferes at this point. Emi2 is phosphorylated directly
by RSK, the kinase downstream from ERK. RSK-mediated phosphorylation
of Emi2 promotes docking of the protein phosphatase PP2A on Emi2 (Wu et
al. 2007b). PP2A, in turn, dephosphorylates the sites on Emi2 that are
phosphorylated by cyclin-B–CDK1, thereby stabilizing Emi2 and restoring
its inhibitory binding to the APC (Tang et al. 2008). This allows the
accumulation of cyclin B necessary for blocking S phase and, ultimately, for
entry into MII. Mos may also promote inhibition of Myt1 and consequently
T14 and Y15 dephosphorylation and activation of CDK1. This could help to
activate cyclin-B–CDK1 as cyclin B accumulates. As MI is completed, cyclin
B synthesis is markedly enhanced; eventually, this exceeds the ability of the
APC to keep pace (even without Emi2 inhibition), cyclin B is degraded, and
MII ensues. Although the general role of Emi2 appears to be conserved in
mammals, the pathway appears to be somewhat different from that in frogs;
because RSK is dispensable, another downstream target of Mos, MEK or
ERK, probably phosphorylates Emi2 and recruits PP2A.

2.3 MII Arrest

Once MII is initiated, the oocyte arrests again, this time as a mature egg
awaiting fertilization. Mos and Emi2 are also central to the activity that
maintains this arrest (Kanki and Donoghue 1991; Hashimoto et al. 1994;
Dupre et al. 2002; Madgwick et al. 2006; Ohe et al. 2007). The critical nature
of Mos is clear because removal of Mos from egg extracts by
immunodepletion destroys CSF activity (Daar et al. 1991), and eggs from
mice or frogs lacking Mos fail to arrest in MII (Colledge et al. 1994;
Hashimoto et al. 1994; Araki et al. 1996). The target of the Mos-MAPK
cascade that produces the MII arrest is again the APC. Indeed, when
radiolabeled cyclin B is injected into Xenopus eggs, treatment with the MEK
inhibitor UO126 promotes cyclin B degradation because the APC inhibition
mediated by the ERK MAPK pathway is lifted (Gross et al. 2000). Inhibition
of the APC in a CSF-arrested egg requires Emi2, which is targeted by the
ERK MAPK pathway during MI and MII (Tung et al. 2005). Loss of Emi2 in
either frog or mouse eggs prevents MII arrest and allows parthenogenetic
divisions.
Accumulation of cyclin B must be carefully regulated to maintain MII
arrest. Cyclin B is synthesized continuously. If unopposed, this would make
it difficult to achieve the rapid degradation of cyclin B required for a sharp
cell-cycle transition upon fertilization. Again, tight regulation of Emi2 occurs
through a negative-feedback loop involving its phosphorylation by cyclin-B–
CDK1 and RSK. The carboxy-terminal Emi2 phosphorylations impede
association of Emi2 with the APC; although the precise manner in which
Emi2 inhibits the APC is not yet clear, the physical association of Emi2 with
the APC is critical for APC inhibition and the amino-terminal
phosphorylations dissociate Emi2 from the APC, allowing cyclin B
degradation (Wu et al. 2007a). Thus, the Mos-ERK-RSK pathway maintains
the CSF arrest, restricting cyclin B levels within a narrow limit. Although the
precise sites of phosphorylation do not appear to be conserved from Xenopus
to mammals, the overall mode of regulation may be conserved through
phosphorylation of alternative sites.

3 SPERM MATURATION
Spermatogenesis occurs over the course of several weeks and encompasses
three successive phases (Sharpe 1994): proliferation, meiosis, and
differentiation. During proliferation, spermatogonial stem cells (SSCs)
differentiate into spermatogonia. These undergo several mitotic divisions,
giving rise to spermatocytes. After two meiotic divisions, spermatocytes form
haploid spermatids. The final transformation of spermatids into mature sperm
entails a major physical and structural reorganization of the cell that is known
as spermiogenesis (Fig. 3).
Figure 3. Regulation of sperm maturation. (A) Spermatogenesis is a continuous process that starts after
puberty. This is possible because a subset of spermatogonium, spermatogonial stem cells (SSCs), are
capable of self-renewal. Glial-cell-line-derived neurotrophic factor (GDNF) and the receptor complex
composed of Ret protooncoprotein and the GDNF family receptor α1 (GFRα1) are key signaling events
for self-renewal. (From Hofmann 2008; adapted, with permission.) (B) Spermatocytes undergo a series
of maturation steps before differentiating into sperm. These are tightly regulated by interaction with
Sertoli cells and testis factors such as GDNF, ERM, KitL, and retinoic acid (RA). (C) Sperm must
additionally undergo capacitation before they are capable of fertilization. This includes both fast and
slow events. The fast events stimulate flagellar activity through a calcium flux that is controlled by the
adenylyl cyclase SACY and mediated by the CatSper and sodium/bicarbonate (NBC) channels. Slow
events increase motility and introduce changes that prepare sperm for fertilization. These include
increases in both intracellular calcium levels and tyrosine phosphorylation of PKA substrates. cAMP
levels are also controlled by endogenous phosphodiesterase (PDE). Unknown cholesterol acceptors
present in uterine/oviduct fluids mediate cholesterol efflux during capacitation. (From Visconti 2009;
adapted, with permission.)

As oocyte maturation is supported by follicular cells, so is

spermatogenesis supported by nongermline cells in and around the
seminiferous tubules. These include Sertoli, Leydig, and myoid cells (Hermo
et al. 2010). The Sertoli cells perform a plethora of functions to sustain germ
cells through all stages of development. They express receptors for growth
factors and hormones and secrete many essential regulatory factors required
for spermatogenesis. Lying outside the tubules in the interstitial space and in
close proximity to blood vessels, Leydig cells are responsible for the
regulated production of androgens in the testis. Lastly, in the peritubular
tissue, along with extracellular matrix, myoid cells support the seminiferous
tubules (Yoshida et al. 2007; de Rooij 2009). These cells create a
microenvironment that allows SSCs to self-renew and/or differentiate, as is
required for continual fertility (Nalam and Matzuk 2010).
Complete spermatogenesis requires the coordinated action of peptide and
steroid hormones, which are important regulators of seminiferous tubule
function (McLachlan et al. 2002). Only somatic cells in the testis express
hormone receptors; therefore these cells (i.e., Sertoli cells) are the exclusive
mediators of hormone activity in spermatogenesis. Of the two gonadotropins,
follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which
also play important roles during oogenesis (Richards and Pangas 2010), FSH
has broader involvement in spermatogenesis whereas LH functions primarily
in the regulation of testosterone production by Leydig cells (Ruwanpura et al.
2010). However, both gonadotropins exert their biological effects by
activating cognate GPCRs. In mice, following formation of the seminiferous
cords, gonocytes, the precursors of SSCs, proliferate until day E15–16, when
they become quiescent, which is coincident with changes in the expression of
several cell-cycle proteins (van den Ham et al. 2003). Formation and
proliferation of SSCs is observed during the first postnatal week and
continues with the synchronous first wave of spermatogenesis that extends in
the prepubertal testis over the first 35 days after birth (Itman et al. 2006).

3.1 Stem Cell Proliferation and Maintenance

The predominant role of FSH is regulating Sertoli cell proliferation during
prepubertal development (Holdcraft and Braun 2004). Stimulation of the FSH
receptor on Sertoli cells activates several downstream signaling cascades,
including those involving cAMP, calcium release, ERK, PI3K, and
phospholipases A2 and C. These activate the cAMP-responsive transcription
factor CREB, leading to stimulation of gene expression (Ruwanpura et al.
2010). Testosterone produced in response to LH mostly functions via the
nuclear androgen receptor (AR) and its subsequent stimulation of gene
expression (Wang et al. 2009; p. 129 [Sever and Glass 2013]). It may also act
via alternative mechanisms, which also result in phosphorylation of CREB.
These “nongenomic” pathways are thought to involve the activation of
several kinases, including Src (Cheng et al. 2007). Importantly for
proliferation of both Sertoli cells and SSCs, FSH stimulation increases
expression and secretion of glial-cell-line-derived neurotrophic factor
(GDNF) (Hu et al. 1999), a distant member of the transforming growth factor
β (TGFβ) family superfamily that regulates the proliferation of uncommitted
SSCs (Meng et al. 2000; Hermo et al. 2010).
GDNF promotes the activation and expression of many signaling
molecules, including the transcription factors Fos and BCL6. GDNF also
stimulates Ras, Akt, and Src-family kinases; Akt activation, specifically, is
important for preventing apoptosis of SSCs (Sariola and Saarma 2003;
Hermo et al. 2010; Oatley and Brinster 2012). These signals are initiated
following its engagement of a receptor complex composed of the Ret proto-
oncoprotein (McGuinness et al. 1996) and the GDNF family receptor α1
(GFRα1) protein (Meng et al. 2000). GFRA1 appears to be expressed only by
a subpopulation of SSCs, possibly a marker of true “stemness.” This is
consistent with the effects of its down-regulation, which causes widespread
inhibition of SSC proliferation and differentiation (reviewed in Nalam and
Matzuk 2010). SSCs lacking GFRα1 display reduced phosphorylation of Ret
at Y1062, a known binding site for several of the downstream targets of this
pathway. Additionally, loss of either member of the receptor complex leads
to defective SSC proliferation and differentiation (Naughton et al. 2006). The
GDNF pathway is thus essential for the maintenance of uncommitted SSCs.
SSC renewal requires the transcription factor Ets variant gene 5 (ERM) a
product of the Sertoli cells. Deletion of ERM in mice results in inhibition of
spermatogenesis following the first wave (Chen et al. 2005). Thus, although
GDNF plays a critical role in spermatogenesis during the perinatal period,
ERM does so at puberty. Expression of ERM and GDNF is increased in
Sertoli cells by addition of the fibroblast growth factor (FGF) 2 and
activation of the FGF2 receptor, which stimulates ERK and PI3K signaling.
Therefore, FGFs may also play an important role in regulating the functions
of Sertoli cells that control the establishment of the SSC pool (Simon et al.
2007).
Another factor regulating proliferation and differentiation of SSCs is the
protooncoprotein Kit, a receptor tyrosine kinase expressed at high levels in
differentiating spermatogonia and early spermatocytes (Sorrentino et al.
1991). In undifferentiated spermatogonia, Kit expression is suppressed by the
transcription factor Plzf. Plzf is necessary for the maintenance of
undifferentiated SSCs. Plzf-null mice display accumulation of Kit-positive
cells and depletion of germ cells (Buaas et al. 2004; Costoya et al. 2004), and
dominant white spotting (W) mutants affecting the Kit locus inhibit
spermatogonia differentiation without affecting either mitosis of
undifferentiated SSCs or initiation of meiosis in spermatocytes (Yoshinaga et
al. 1991). Furthermore, in the postnatal testis, expression of the Kit ligand
(KitL, also known as stem cell factor [SCF]), in Sertoli cells is critical for
spermatogenesis (Flanagan et al. 1991), and mutations in KitL phenocopy the
spermatogenic defects observed in W mutant mice (Bedell and Mahakali
Zama 2004). The binding of KitL to Kit causes receptor dimerization and
phosphorylation of cytoplasmic tyrosine residues. These phosphorylated
residues become anchoring sites for SH2-domain containing proteins such as
phospholipase Cγ (PLCγ), Src, and PI3K. Consequent induction of calcium
release, phosphorylation of target proteins such as p70S6K (Feng et al. 2000),
and gene expression leads to proliferation and differentiation of
spermatogonia (Sette et al. 2000). Note that spermatogonia from Kit-null
testes can nevertheless undergo spermatogenesis, which indicates other
parallel pathways must also operate (Nalam and Matzuk 2010).

3.2 Spermatocyte Meiosis and Release

After differentiation, spermatogonia undergo a few mitotic divisions before
entering meiosis, which is delayed until puberty. Retinoic acid (RA) (see p.
129 [Sever and Glass 2013]) is a key signaling molecule in meiotic initiation
(Vernet et al. 2006), and Sertoli cells are believed to be the main source of
RA in the postnatal testes. Importantly, although RA is generated in Sertoli
cells by the enzyme aldehyde dehydrogenase family 1, subfamily A1
(ALDH1A1), male gonads also express an enzyme responsible for RA
degradation, cytochrome P450, family 26, subfamily b polypeptide 1
(CYP26B1) (Bowles et al. 2006). This limits the availability of RA and
postpones meiosis in the male.
Hormonal signaling plays an important role in control of meiosis and
production of functional spermatids. Suppression of FSH production and
signaling leads to reduced numbers of pachytene spermatocytes (Matthiesson
et al. 2006) and compromises the release of sperm, which suggests that FSH
regulates adhesion between Sertoli cells and spermatids (Saito et al. 2000;
Ruwanpura et al. 2010). Spermatocyte meiosis is arguably even more
dependent on testosterone signaling. In mice lacking AR in Sertoli cells
spermatogenesis is arrested at the late spermatocyte stage and spermiation,
which is the release of the mature spermatids into the lumen of the
seminiferous tubules, fails (Chang et al. 2004; De Gendt et al. 2004).
Moreover, in adult mice that lack both gonadotropins owing to a defect in the
GnRH gene, testosterone supplementation alone can restore full
spermatogenesis (Haywood et al. 2003). The processes modulated by
testosterone during spermiogenesis include adhesion between Sertoli cells
and different stage spermatids. Testosterone is thought to regulate expression
and/or function of proteins required for the assembly and/or disassembly of
adhesion junctions, including α and β integrins, focal adhesion kinases, and
Src kinases (Ruwanpura et al. 2008, 2010; Shupe et al. 2011).

3.3 Sperm Capacitation and Calcium Channels

After entering the oviduct, most mammalian sperm associate with epithelial
cells in the isthmus creating a sperm reservoir. Sperm are sporadically
released from this reservoir and migrate to the ampulla, which is the site of
fertilization (Suarez 2008b). The mechanisms that facilitate this release are
unclear but they involve the loss of BSPs (originally isolated from the cow,
so named bovine seminal plasma proteins) and other extrinsic proteins from
the sperm; these events are seemingly precipitated by the progression of
capacitation events, including hyperactivation (a change in sperm motility)
(Suarez 2008a). Unfolding over several hours, sperm capacitation begins as
sperm come in contact with the female reproductive tract and involves a
series of sequential and simultaneous processes. Sperm acquire motility
immediately after their release from the cauda epididymis (for reviews see
Yanagimachi 1994; Salicioni et al. 2007). These subsequent changes involve
both modification of the plasma membrane phospholipid composition and
increases in intracellular calcium concentration. Early in capacitation,
activation of soluble adenylyl cyclase (SACY) (Chen et al. 2000) causes an
increase in cAMP levels, which activates PKA containing a unique catalytic
subunit (Cα2 or Cs). These events are initiated by an increase in the
intracellular concentration of bicarbonate, which activates the enzyme by
promoting closure of the catalytic active site and metal recruitment
(Steegborn et al. 2005). This is itself triggered by the high concentrations of
bicarbonate in the seminal fluid, which enters via the Na+/HCO3−
cotransporter. PKA activation coincides with phosphorylation of a number of
proteins, although its targets remain mostly unknown (Signorelli et al. 2012)
except for FSCB, a 270-kDa protein involved in the biogenesis of the fibrous
sheath (Li et al. 2007), a cytoskeletal structure in the mammalian sperm
flagellum. The ongoing alkalinization, which activates the downstream
sperm-specific plasma membrane calcium channel CatSper (Ren et al. 2001),
also increases intracellular calcium levels (Carlson et al. 2003). Together, this
increase and the PKA-regulated phosphorylation of proteins on tyrosine
residues, including A-kinase-anchoring proteins (AKAPs) (Ficarro et al.
2003), drive the activation of flagellar motility that is necessary for both
sperm migration and residence in the female reproductive tract.
Additional aspects of capacitation that are more protracted are required
for sperm to fertilize the egg. These occur closer to the site of the ovulated
egg(s) and include a general increase in protein phosphorylation, loss of
cholesterol from the plasma membrane, hyperactivation (acquisition of an
asymmetrical, nonlinear motility pattern), and increased susceptibility to
stimuli that promote the acrosome reaction. These slow processes are also
regulated by bicarbonate; however, they additionally require the transfer of
cholesterol from the plasma membrane to unknown cholesterol acceptors
present in the uterine/oviduct fluids. Also, despite the dominant role of PKA
in sperm capacitation, research suggests that other kinases, such as Src-
family kinases and PKC, and phosphatases such as PP1α, PP1γ2, PP2A, and
PP2B may play a role regulating the rapid and slow events of capacitation
(reviewed in Visconti et al. 2011; Signorelli et al. 2012).
Hyperactivated sperm show increased flagellar bend amplitudes
(Yanagimachi 1970), which is most often observed in the oviduct. This
amplification generates enough power for the sperm to make its way through
the oviductal mucus, cumulus matrix, and the zona pellucida (ZP)
surrounding the egg (Chang and Suarez 2010; Hung and Suarez 2010).
CATSPER channels and calcium are critical for hyperactivation; either the
absence of external calcium or the presence of mutations in any of the four
CATSPER subunits results in a failure to hyperactivate (Carlson et al. 2003),
and CATSPER-defective sperm cannot leave the oviduct sperm reservoir and
are incapable of penetrating the ZP. The mechanism of CATSPER regulation
in the oviduct remains unclear. Progesterone is a possible regulator (Lishko et
al. 2011; Strunker et al. 2011) produced by cumulus cells and is present in the
follicular fluid (Sun et al. 2005). It may act as the long-sought
chemoattractant. In this regard, hyperactivation could promote sperm
chemotaxis (Chang and Suarez 2010). Indeed, progesterone can induce
increases in intracellular calcium near the base of the sperm head—the site
where the calcium stores are located (Fukami et al. 2001). Note that in marine
species such as the sea urchin Arbacia punctulata, in which sperm migrate
toward the oocyte along a chemoattractant gradient, motility is regulated by
incremental increases in intracellular calcium concentration caused by the
binding of the chemoattractant resact to the sperm (Bohmer et al. 2005).
Progesterone could act similarly in mammals.

4 FERTILIZATION
4.1 The Acrosome Reaction in Sperm
Before fusing with the egg’s plasma membrane, sperm must undergo the
acrosome reaction (Yanagimachi and Mahi 1976). The acrosome is a Golgi-
derived organelle that lies above the tip of the sperm head. Its contents are
released following fusion between the outer acrosomal membrane and the
plasma membrane (Kim et al. 2011), and this reaction is critical for
interaction with the ZP of the egg. Only capacitated sperm are capable of
undergoing the acrosome reaction. Progesterone produced by the cumulus
cells surrounding the oocyte has been proposed as the possible inducer of this
reaction (Osman et al. 1989; Jin et al. 2011). Upon breaching the egg’s
plasma membrane, the sperm induces the initiation of embryonic
development by evoking an increase in the intracellular concentration of free
calcium, a signaling mechanism that regulates numerous cellular processes
(Fig. 4) (Berridge et al. 2000b; p. 95 [Bootman 2012]).
Figure 4. Fertilization events. Both egg and sperm must undergo complex changes before fertilization
can occur. Upon interaction with the zona pelucida, sperm undergo a calcium-flux-driven
reorganization of SNARE fusion proteins, called the acrosome reaction. Fusion with the egg membrane
releases factors from the sperm into the cytoplasm. These factors stimulate calcium release from the
egg ER and subsequent activation of CaMKII and calcineurin (CN). The consequences of CaMKII
activation include phosphorylation of Emi2, which promotes its Plx1 and SCFβ-TrCP-dependent
degradation and consequent reactivation of the APC. CN activation promotes dephosphorylation of
Cdc20 and also the Cdc27 subunit of the APC. At the same time, dephosphorylation of M phase
phosphoproteins is promoted by inactivation of the Greatwall kinase (Gwl), thereby alleviating
inhibition of PP2A, allowing it to dephosphorylate M phase CDK1 substrates.

The signaling mechanisms leading to the acrosome reaction are well

characterized (Arnoult et al. 1996; Evans and Florman 2002). Activation of
PLCδ4 by a calcium influx and the consequent generation of inositol 1,4,5-
trisphosphate (IP3) (Distelhorst and Bootman 2011) causes a second influx of
calcium (Fukami et al. 2001). This promotes exocytosis by stimulating the
reorganization of a SNARE protein in the sperm membranes (Mayorga et al.
2007). This reorganization is facilitated by the NSF and α-SNAP proteins and
the calcium sensor synaptotagmin. Release of proteolytic enzymes and
hyaluronidase from the acrosome aids the penetration of the cumulus cells
and ZP by the sperm (Kim et al. 2008)
How the sperm interacts with ZP has been a point of some dispute. Early
studies implicated several sperm-surface enzymes as critical for the
interaction of the sperm with the egg coat (Lu and Shur 1997; Ikawa et al.
2010), but more recent work supports the involvement of a disintegrin and
metalloproteinase family member, ADAM3 (Shamsadin et al. 1999). On the
egg side, recent studies point to the amino-terminal domain of ZP2, which is
cleaved after fertilization by the egg cortical granule component ovastacin, as
the ZP ligand (reviewed in Avella et al. 2013). After binding, sperm must
penetrate the ZP, probably using sperm-associated serine proteases such as
acrosin and testisin (also known as PRSS21 or TESP5) (Baba et al. 1994;
Yamashita et al. 2008).

4.2 Fusion and Egg Activation Leading to Fertilization

Soon after negotiating the ZP, the sperm fuses with the egg plasma
membrane (Evans and Florman 2002). Early studies implicated the proteases
ADAM1b and ADAM2, together known as fertilin, but gene knockout
studies showed that fertilin is not required for fusion (Inoue et al. 2007).
Subsequent work identified IZUMO, a member of the immunoglobulin
superfamily, as the protein that appears to mediate fusion (Inoue et al. 2005;
Sosnik et al. 2009). In the egg, the tetraspanin protein CD9 has been shown to
be critical for fusion (Le Naour et al. 2000; Miyado et al. 2000). Whether
CD9 and IZUMO directly interact is not clear.
Fusion of the gametes produces a calcium signal in the egg. A variety of
calcium signals are required for egg activation; these reflect both the
plasticity of the signaling machinery as well as the distinct requirements for
egg activation in different species. Species typically either display a single
increase in calcium concentration (e.g., in sea urchins, starfish, frogs, and
fish) or show calcium oscillations (e.g., in nemertian worms, ascidians, and
mammals) (Stricker 1999; Stricker and Whitaker 1999; Miyazaki and Ito
2006). The mechanism(s) that mediate calcium influx remains poorly
characterized in mammalian eggs. Cells use several calcium influx
mechanisms, including receptor-operated channels (ROCs) and voltage-
operated calcium channels (VOCCs) (Berridge et al. 2000a; Tosti and Boni
2004; Smyth et al. 2006). Calcium influx may be attained, at least in part, by
store-operated calcium entry (SOCE), a mechanism regulated by ER calcium
levels and driven by store-operated calcium channels (SOCs) (Park et al.
2009). Stromal interaction molecule (STIM1) in the ER acts as a calcium
sensor (Liou et al. 2005; Roos et al. 2005) and causes opening of Orai1, a
channel partner protein in the plasma membrane to replenish stores (Feske et
al. 2006; Vig et al. 2006).
The IP3 receptor (IP3R) on the ER is the main intracellular calcium-
release channel in many mammalian cell types (reviewed in Berridge et al.
2000b; Bootman et al. 2001; p. 95 [Bootman 2012]). Although mammalian
oocytes, eggs, and the surrounding cells express all three IP3R isoforms
(reviewed in Fissore et al. 1999a; Berridge et al. 2000b; Díaz-Muñoz et al.
2008), oocytes and eggs overwhelmingly express the type I IP3R isoform
(Kume et al. 1997; Fissore et al. 1999b; Jellerette et al. 2000; Tokmakov et
al. 2002), which requires binding by both calcium and IP3 for activation and
is stimulated at low calcium levels and inhibited at high calcium levels (Iino
1990a,b; Finch et al. 1991). This makes it especially suited to support long-
lasting oscillations. The importance of IP3R1 in fertilization is supported by
studies showing injection of an anti-IP3R1 antibody blocks sperm-initiated
calcium oscillations (Miyazaki et al. 1992) and egg activation in mice (Xu et
al. 1994) and works in the same manner in other vertebrates (Parys et al.
1994; Thomas et al. 1998; Yoshida et al. 1998; Runft et al. 1999; Goud et al.
2002; Iwasaki et al. 2002).
The signals that initiate the calcium increase at fertilization have proven
elusive (Whitaker 2006; Parrington et al. 2007). Nonetheless, in some
species, especially those with a single calcium transient at fertilization, Src-
family kinases and PLCγ have been shown to lead to production of IP3 during
fertilization (Giusti et al. 1999; Sato et al. 2000). The receptor responsible for
recruiting and activating the kinases remains to be identified (Mahbub Hasan
et al. 2005). Similarly, it is unclear how sperm induce calcium oscillations in
mammals. Calcium responses can be initiated in eggs from various species,
including mammals by the same agonists that cause calcium release in
somatic cells (Katayama et al. 1993; Miyazaki and Ito 2006). However, these
fail to replicate the pattern of calcium oscillations associated with
fertilization; other mechanism(s) might, therefore, be at play. Studies of sea
urchin, ascidian eggs, and later, mammalian eggs showed that injection of
sperm extracts or whole sperm can replicate fertilization-like responses in
these species (Stice and Robl 1990; Swann 1990; Tesarik and Testart 1994;
Nakano et al. 1997; Stricker 1997; Swann and Lai 1997; Wu et al. 1997;
Kurokawa and Fissore 2003; Malcuit et al. 2006; Swann et al. 2006)
strengthening the notion of a sperm cytosolic factor (SF). The mechanism
may involve the release of SF into the ooplasm after fusion of the gametes.
Importantly, the SF is not IP3 or calcium but an uncharacterized protein
moiety (Swann 1990; Wu et al. 1997; Kyozuka et al. 1998; Harada et al.
2007). Once initiated, calcium oscillations trigger all events of egg activation
(Schultz and Kopf 1995). The presence of oscillations ensures the persistent
degradation of Emi2 and the stepwise progression of activation, as events
such as cortical granule exocytosis exit require fewer calcium increases than
exit from MII or recruitment of maternal RNAs (Ducibella et al. 2002).
Cytosolic preparations from mammalian sperm possess high PLC activity
(Parrington et al. 1999; Jones et al. 2000; Rice et al. 2000), and this is highly
sensitive to calcium and could therefore be (Rice et al. 2000) the oscillation
initiator. Indeed, a novel sperm-specific PLCζ (Saunders et al. 2002) has been
identified (Fujimoto et al. 2004; Kouchi et al. 2004) and can evoke
appropriate oscillations in mouse (Saunders et al. 2002), rat (Ito et al. 2008),
human (Rogers et al. 2004), bovine (Malcuit et al. 2005; Ross et al. 2008),
porcine (Yoneda et al. 2006), and equine (Bedford-Guaus et al. 2008) eggs.
The findings that aberrant PLCζ expression is associated with infertility, and
sperm from patients with repeated fertilization failure after intracytoplasmic
sperm injection (ICSI), which also fail to initiate calcium oscillations (Yoon
et al. 2008; Heytens et al. 2009), supports the idea that PLCζ released from
the sperm triggers oscillations in fertilized eggs. Nevertheless, questions
remain regarding its expression during spermatogenesis and storage, the
means by which release into the ooplasm occurs, and how activation occurs
upon egg entry.

5 FROM EGG TO ZYGOTE

Once fertilization has occurred, interphase nuclei form separately around the
paternal (sperm) and maternal (Eggerickx et al. 1995) genomes in structures
known as pronuclei. The paternal genome undergoes a series of
transformations that result in both exchange of spermatic chromatin
packaging proteins (protamines) for maternal histones and demethylation of
many repressed paternal genes. Upon completion of DNA replication, the
pronuclear envelopes break down and the maternal and paternal
chromosomes comingle on a common mitotic spindle in the zygote. In this
transition to the zygote, maternal mRNA transcripts are largely degraded;
many are replaced by zygote-specific transcripts both to take over the
function of maternal housekeeping messages and to allow expression of new
proteins that will build the early embryo.
At fertilization, the egg exits its MII arrest, in part as a consequence of
events set in motion by the calcium response, which inactivates CSF through
degradation of Mos and reactivates the APC, leading to cyclin degradation.
Reactivation of the APC appears to be mediated in part by the calcium-
dependent phosphatase calcineurin (also known as PP2B). Inhibition of
calcineurin by cyclosporine A halts destruction of cyclin B. Not only has a
core component of the APC, Cdc27, been shown to serve as a calcineurin
substrate, but the APC-activating subunit Cdc20 is also a substrate of
calcineurin. To exit meiosis, a host of CDK1–cyclin-B substrates must be
dephosphorylated. This is accomplished largely by PP2A (as described
above). Premature inactivation of CDK1–cyclin-B substrates would prohibit
entry into or maintenance of MII. Inappropriate PP2A activation is therefore
prevented through binding of a small peptide called Arpp19 and α
endosulfine. Phosphorylation of Arpp19 by the conserved mitotic kinase
Greatwall, which is activated by CDK1 at M phase entry, allows it to inhibit
the PP2A isoform B55δ. At M phase exit, Greatwall is inactivated, which
alleviates inhibition of PP2A and consequently allows the requisite
dephosphorylation of M phase substrates. Additionally, rapid destruction of
Emi2 alleviates inhibition of the APC, leading to loss of CDK1 activity
(through degradation of cyclin B). In Xenopus eggs, this is triggered by
calcium via a calcium-calmodulin-dependent kinase (CaMKII). CaMKII
phosphorylates Emi2 protein on T195. This creates a docking site on Emi2
for another kinase, Plx1 in Xenopus, which then phosphorylates Emi2 within
a sequence (the phosphodegron) required for recognition by the SCFβ-TrCP
ubiquitin ligase. This series of events leads to ubiquitylation and degradation
of Emi2, liberation of the APC, and degradation of substrates required for M
phase exit. Note the Plx1-phosphorylation site does not appear to be
conserved in mammals, and it is not known whether the ability of CaMKII to
trigger Emi2 degradation in mammalian eggs is also triggered by recruitment
Plk1 (the mammalian Plk1 ortholog). The culmination of these events allows
the fertilized egg to exit arrest and proceed with cell division.

6 CONCLUDING REMARKS
In gametes of both sexes, development, maturation, and fertilization require
the careful coordination of complex processes. From hormone-initiated and
kinase/phosphatase-controlled maturation, to calcium-induced capacitation
and fertilization, regulatory mechanisms ensure that reproduction occurs only
under conditions in which they are best poised for success. For males, the
signals that regulate spermatogenesis are at first contained within the testis,
but following spermiation and ejaculation, proper sperm function depends on
factors outside of the male reproductive tract: for external fertilizers, factors
in the outside environment; and for internal fertilizers, the milieu of the
female reproductive tract. Taking into account these potentially harsh
environments, male reproduction relies on the production of large quantities
of sperm, with progenitor cells retaining mitotic capacity into adulthood. For
female internal fertilizers, the production of eggs is more contained, restricted
to the follicle until ovulation and to the female ducts until fertilization and
beyond, relying on the production of few gametes; in contrast, externally
fertilizing females, like their male counterparts, produce large numbers of
gametes. Even for these organisms, though, oocytes and eggs are self-
contained developmental units capable of sustaining the early stages of
development whose progenitors have generally entered meiosis and have lost
the capacity to regenerate.
In every species, regardless of the site of fertilization, cascades of protein
modifications regulate cell-cycle transitions to guarantee that oocytes can be
fertilized only at an appropriate time. Hormonal regulation and calcium
signaling promote capacitation of sperm only in the correct environment for
fertilization. Furthermore, multiple overlapping pathways provide the checks
and balances that are necessary to prevent defective reproduction.
Although great progress has been made over the last 50 years in detailing
the molecular events underlying most aspects of vertebrate fertilization, there
are still aspects of gamete development and fertilization whose precise
regulation by cell signaling events remain to be determined. Elucidation of
these events is likely to have important implications for the continued
development of reproductive technologies and for maximizing the health of
gametes, and thus of progeny.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006064
CHAPTER 18

Cell Signaling and Stress Responses

Gökhan S. Hotamisligil1 and Roger J. Davis2

1Department of Genetics and Complex Diseases, Broad Institute of Harvard-MIT, Harvard School of
Public Health, Boston, Massachusetts 02115
2Howard Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
Correspondence: roger.davis@umassmed.edu

SUMMARY

Stress-signaling pathways are evolutionarily conserved and play an

important role in the maintenance of homeostasis. These pathways are
also critical for adaptation to new cellular environments. The
endoplasmic reticulum (ER) unfolded protein response (UPR) is
activated by biosynthetic stress and leads to a compensatory increase in
ER function. The JNK and p38 MAPK signaling pathways control
adaptive responses to intracellular and extracellular stresses, including
environmental changes such as UV light, heat, and hyperosmotic
conditions, and exposure to inflammatory cytokines. Metabolic stress
caused by a high-fat diet represents an example of a stimulus that
coordinately activates both the UPR and JNK/p38 signaling pathways.
Chronic activation of these stress-response pathways ultimately causes
metabolic changes associated with obesity and altered insulin sensitivity.
Stress-signaling pathways, therefore, represent potential targets for
therapeutic intervention in the metabolic stress response and other
 disease processes.

Outline
1 Introduction
2 The unfolded protein response
3 Stress signaling by MAP kinases
4 Conclusions
References

1 INTRODUCTION
An important aspect of cellular physiology is the maintenance of
homeostasis. Evolutionarily conserved biochemical mechanisms play a key
role in this process. Thus, exposure to extra- or intracellular stress disrupts
cellular homeostasis and causes the engagement of signaling pathways that
serve to rebalance biochemical processes within the cell. One example is the
AMP-activated protein kinase signaling pathway, which responds to
increased AMP and ADP concentrations within the cell by dampening
anabolic pathways and promoting catabolic pathways that replenish the ATP
supply (Ch. 14 [Hardie 2012]). A second example is the cellular response to
DNA damage that engages the ataxia telangiectasia mutated (ATM) stress-
signaling pathway to induce growth arrest mediated by the p53 tumor
suppressor protein and promote DNA repair before reentry into the cell cycle
(Ch. 6 [Rhind and Russell 2012]). A third example is the regulation of
receptor ligand sensitivity to control the amplitude of signal transduction.
This type of stress response maintains, for example, the dynamic range of
vision following exposure to high- and low-intensity light sources (see Ch. 11
[Julius and Nathans 2012]). Similarly, signaling by the metabolic hormones
leptin and insulin is dynamically regulated by stress-signaling pathways to
control feeding behavior and biosynthetic processes (Ch. 14 [Hardie 2012]).
Such pathways are critical for normal cellular homeostasis and adaptive
changes in cell physiology that benefit the organism.
In addition to their contributions to normal physiology, stress-activated
signaling pathways play roles in establishing dysfunctional states associated
with stress exposure and the development of disease. Here, we focus on two
different mammalian stress-activated-signaling pathways to illustrate these
concepts: the unfolded protein response (UPR) and stress-activated MAP
kinase (MAPK) pathways. The UPR is engaged within the endoplasmic
reticulum (ER) during biosynthetic stress and leads to a coordinated
inhibition of general protein translation and specific up-regulation of ER
functional capacity. The pathways involved therefore serve to maintain
cellular homeostasis. Stress-activated MAPK pathways, in contrast, are
regulated by a diverse array of intra- and extracellular stresses, including
environmental physical/chemical changes and exposure to inflammatory
cytokines. These stress pathways cause phosphorylation of nuclear and
cytoplasmic substrates, leading to a network response and adaptation to the
new cellular environment.
The UPR and MAPK pathways can function separately or cooperatively.
For example, increased saturated fatty acid levels, caused by a high-fat diet,
induce both the UPR and stress-activated MAPK pathways. Together, these
pathways cause adaptation to the new diet by regulating insulin signaling,
blood glucose concentration, and obesity (Fig. 1).
Figure 1. Stress-signaling pathways activated in response to metabolic stress. Feeding mice a high-fat
diet causes metabolic stress that leads to the UPR and activation of stress-activated MAP kinases.
These signaling pathways result in an adaptive response associated with obesity and altered insulin
sensitivity.

2 THE UNFOLDED PROTEIN RESPONSE

The ER is a multifunctional organelle that comprises reticular and tubular
structures spanning the cell and has numerous fundamental functions in all
cells. It has two major domains: smooth ER, which is devoid of ribosomes;
and rough ER, which is studded by attached ribosomes (Palade and Porter
1954; Lynes and Simmen 2011). The rough ER is responsible for the
synthesis and trafficking of secreted and integral membrane proteins (Gething
and Sambrook 1992; Ellgaard and Helenius 2003), whereas smooth ER is
associated with lipid synthesis and metabolism, and calcium storage. The
functional specialization within the ER is more complex and various
subregions support distinct pathways involved in homeostasis and survival.
For example, rough ER is involved in quality control and protein degradation
and harbors oxidoreductases (Gething and Sambrook 1992; Ellgaard and
Helenius 2003; Kostova and Wolf 2003; Rutkowski and Kaufman 2004;
Meusser et al. 2005) and there are additional ER domains devoted to
specialized functions including the mitochondria-associated ER membrane
(MAM), the nuclear envelope, peroxisomal components, Russell bodies, and
lipid droplets (Lynes and Simmen 2011). Similarly, the smooth ER has
specialized domains, such as the plasma membrane-associated ER and
regions that can also form MAM, autophagosomes, and lipid droplets
(English et al. 2009; Hayashi et al. 2009).
Adaptation of the ER to a variety of metabolic and stress conditions is
critical for cell function and survival, as well as organismal health (Walter
and Ron 2011). Given the demands fluctuating conditions place on the
functional capacity of the ER, a potent adaptive response, the UPR (Mori
2000; Marciniak and Ron 2006; Zhao and Ackerman 2006), has evolved to
maintain the functional integrity of this organelle (Fig. 2). In eukaryotic cells,
UPR signaling is initiated by three ER-membrane-associated proteins: PERK
(PKR-like eukaryotic initiation factor 2α [eIF2α] kinase), IRE1 (inositol-
requiring enzyme 1), and ATF6 (activating transcription factor 6). Acting in
concert, signaling through these branches controls protein synthesis,
facilitates protein degradation, and produces the molecules necessary for the
ER to restore equilibrium (Mori 2000; Marciniak and Ron 2006; Zhao and
Ackerman 2006).
Figure 2. The canonical UPR. (A) In the canonical model of the UPR, unfolded or misfolded proteins
activate the three major sensing molecules (IRE1, PERK, and ATF6) at the ER membrane by recruiting
the ER chaperone BiP away from the lumenal domains of these proteins. IRE1 is a kinase and
ribonuclease that on autophosphorylation activates splicing and produces the active transcription factor
XBP1, which induces the expression of ER chaperones, degradation components, and lipid synthesis
enzymes. PERK is a kinase that is also activated through dimerization and autophosphorylation and
phosphorylates eIF2α to attenuate general protein synthesis. ATF6 is a transcription factor that once
released from the ER will move to the Golgi. After processing at this site, it translocates to the nucleus
to activate the transcription of chaperone genes. Together, these pathways reduce entry of proteins into
the ER, facilitate disposal of the misfolding proteins, and produce the components for the ER to adapt
its folding capacity to reach equilibrium. When these pathways fail to reach homeostasis, they can also
trigger death. Under severe stress conditions, the synthesis of ATF4 is enhanced in an eIF2α-
phosphorylation-independent manner that promotes apoptosis. (B) Domain structure of the ER stress
sensors IRE1, PERK, and ATF6. SP, signal peptide; TM, transmembrane domain; TAD, transcriptional
activation domain; bZIP, basic leucine zipper; GLS1 and GLS2, Golgi localization sequences 1 and 2.
Dark gray bars represent regions of limited sequence similarity between IRE1 and PERK.

Early studies of adaptive ER responses showed that the levels of two ER-
localized chaperones, 78- and 94-kDa glucose-regulated proteins (GRP78 and
GRP94), are increased on glucose starvation (Shiu et al. 1977) and protein N-
glycosylation inhibitors and calcium ionophores enhance their expression
(Welch et al. 1983; Resendez et al. 1985; Kim et al 1987). Sambrook and
colleagues subsequently observed increased levels of ER chaperones on
overexpression of mutant influenza virus hemagglutinin and were the first to
propose that malfolded proteins in the lumen of the ER are detected and
invoke a response (Kozutsumi et al. 1988). Subsequently, genetic screens in
yeast identified IRE1 as an ER-localized receptor-like kinase and
ribonuclease required for the ER-to-nucleus signaling that activates
chaperone expression under ER stress conditions (Nikawa and Yamashita
1992; Cox et al. 1993; Mori et al. 1993). Hac1 was shown to be the leucine-
zipper transcription factor that functions downstream from IRE1 to induce
transcription by binding to a defined DNA sequence, the UPR element
(UPRE) (Cox and Walter 1996; Nikawa et al. 1996) in promoters turned on
by the UPR in yeast. The equivalent of Hac1 in multicellular organisms is
XBP1 (Shen et al. 2001; Calfon et al. 2002).
An additional cis-acting element (ER stress element, ERSE) was later
identified in promoter regions of mammalian ER chaperones and led to the
description of another ER-resident leucine-zipper transcription factor, ATF6
(Yoshida et al. 1998). Together, ATF6 and XBP1 stimulate the expression of
a broad array of genes involved in protein folding, secretion, and degradation
to clear misfolded proteins from the ER (Walter and Ron 2011). Finally, the
third molecule activated during the UPR was identified as PERK, one of the
four known eIF2α kinases (Baird and Wek 2012; Donnelly et al. 2013). It is
involved in translational attenuation, temporarily halting arrival of new
proteins in the ER (Harding et al. 1999). These three branches are now
considered as mediators of the canonical UPR (Fig. 2).
In the canonical model, the intraluminal domains of these initiators (i.e.,
the amino termini of IRE1 and PERK, and the carboxyl terminus of ATF6)
are bound by the chaperone Grp78 (also called BiP) in the absence of stress
and rendered inactive (Bertolotti et al. 2000; Shen et al. 2002). Accumulation
of improperly folded proteins in the ER lumen results in the recruitment of
BiP away from these UPR sensors. Stripping off BiP allows for
oligomerization and activation of PERK and IRE1, and translocation of
ATF6 to the Golgi cisternae, which lead to a cascade of downstream
signaling events (Shamu and Walter 1996; Bertolotti et al. 2000). Recent
studies support the view that more complex luminal events underlie mounting
of the UPR. For example, IRE1 can form higher-order oligomers on
activation in vitro (Li et al. 2010) and directly interact with unfolded proteins
(Gardner and Walter 2011). Dynamic regulation of IRE1 by BiP may thus
adjust the magnitude of activation as opposed to providing an “on-or-off”
switch (Pincus et al. 2010). Hence, it is likely that stress responses emanating
from the ER are more complex than the canonical UPR model.
Activation of the ATF6 branch of the UPR requires translocation of ATF6
to the Golgi body and processing by the serine protease site-1 protease and
the metalloprotease site-2 protease to release an active transcription factor
(Chen et al. 2002). This branch also responds to signals other than BiP
sequestration—for example, the redox status of the ER. Active ATF6 moves
to the nucleus to stimulate the expression of genes containing the ERSE1,
ERSE2, UPRE, and cAMP-response elements (p. 99 [Sassone-Corsi 2012])
in their promoters (Yoshida et al. 1998). Genes required for ER-associated
degradation and the gene encoding the ER degradation-enhancing α-
mannosidase-like protein (EDEM) contain UPREs and, when induced,
facilitate clearance and degradation of misfolded proteins from the ER lumen
(Yoshida et al. 1998; Friedlander et al. 2000; Kokame et al. 2001).
The oldest branch of the UPR is mediated by IRE1, which is conserved
from yeast to humans (Patil and Walter 2001; Calfon et al. 2002). IRE1 has
two known isoforms, α and β, the latter being restricted primarily to the
intestine (Wang et al. 1998). IRE1 harbors two distinct catalytic activities: a
serine/threonine kinase for which the only known substrate is IRE1 itself, and
an endoribonuclease activity (Sidrauski and Walter 1997). The
endoribonuclease activity is activated on dimerization and
autophosphorylation and cleaves a 26-nucleotide intron from the XBP1
messenger RNA (mRNA), generating an mRNA whose translation produces
functional XBP1 (so-called XBP1s) (Shamu and Walter 1996; Sidrauski and
Walter 1997). XBP1s, alone or in conjunction with ATF6α, launches a
transcriptional program that induces many ER chaperones (including BiP),
proteins involved in ER biogenesis, and secretion (for example, EDEM,
ERdj4, protein disulfide isomerase [PDI], and other ER proteins) (Yoshida et
al. 2001, 2003; Lee et al. 2003). The endonuclease activity of IRE1 can also
degrade other mRNAs, preventing their translation and thereby providing an
additional way to reduce the translational burden and thus relieve ER stress
(Hollien and Weissman 2006). This mechanism has been termed regulated
IRE1-dependent degradation.
GTP-bound eIF2 is essential for loading of the initiator Met-tRNA
(tRNA) onto an mRNA-charged 40S ribosomal subunit for translation
initiation (Hinnebusch and Lorsch 2012). Phosphorylation of its GTP-binding
subunit, eIF2α, at S51 by PERK is another important aspect of the UPR. This
converts eIF2α into a competitive inhibitor of eIF2B (the GTP exchange
factor for eIF2α). This sequesters eIF2B and reduces the rate of regeneration
of the eIF2-GTP-tRNAiMet ternary complex, which, in turn, results in lower
rates of global protein synthesis, thereby reducing the ER workload (Shi et al.
1998; Harding et al. 1999). At least three other kinases can phosphorylate
eIF2α at S51: double-stranded RNA-dependent kinase (PKR), general control
nonderepressible 2, and heme-regulated inhibitor kinase (Baird and Wek
2012; Donnelly et al. 2013). The PERK branch of the UPR is also linked to
transcriptional regulation through several distinct mechanisms, which
increase the level of and/or activate the transcription factors ATF2, ATF4,
C/EBP (Harding et al. 2000; Ma et al. 2002; Ron and Walter 2007), NRF2
(Cullinan et al. 2003), and NF-κB (Jiang et al. 2003; Deng et al. 2004).
Generation of the protein products of the induced transcripts is achieved in
the context of general translational attenuation through features in the
mRNAs that permit their preferential translation. For example, the 5′-end of
the ATF4 transcript has two upstream open reading frames (uORFs) that
prevent translation under normal circumstances (Somers et al. 2013).
However, under stressed conditions, ribosome capacitation is delayed, the
uORFs are skipped, and functional ribosome complexes are assembled at the
bona fide start codon (Harding et al. 2000). The synthesis of functional ATF4
consequently activates the expression of genes involved in apoptosis, ER
redox control, glucose metabolism, and the relief of eIF2α inhibition
(Harding et al. 2000; Ma et al. 2002; Jiang et al. 2004).

2.1 Noncanonical Aspects of the UPR and Other Stress

Signals
The ER has developed additional strategies to ensure its proper function
under stress conditions. For example, the Golgi reassembly stacking protein 1
facilitates the exit of mutant or misfolded proteins from the ER lumen
through the activation of an unconventional secretory pathway (Gee et al.
2011). In addition, chaperone-mediated autophagy assists the disposal of
misfolded proteins in the ER, and ER-phagy (selective autophagy of the ER)
promotes the turnover of damaged ER in bulk (Klionsky 2010; Arias and
Cuervo 2011). Moreover, during cytokinesis, alternative surveillance
mechanisms other than the UPR are also activated to monitor the “fitness” of
the ER and ensure its proper transmission into daughter cells (Babour et al.
2010). ER stress also generates oxidative stress that needs to be alleviated
during recovery (Cullinan and Diehl 2006). Reactive oxygen species (ROS)
are produced in the ER owing to UPR-stimulated up-regulation of protein
chaperones involved in disulfide bond formation as well as oxidative
phosphorylation in the mitochondria (Sevier et al. 2001). During disulfide
bond formation in the ER, electrons are passed through a series of thiol-
disulfide exchange reactions from the thiols of the substrate protein to PDI,
then to ERO1, and finally to molecular oxygen (Tu and Weissman 2002). As
a byproduct of these reactions, ROS (hydrogen peroxide and other peroxides,
superoxide radical, hydroperoxyl radical, and hydroxyl radical) accumulate
during the UPR-increased protein folding and can produce sufficiently high
levels of ROS to be toxic to the cell (Harding et al. 2003; Sevier and Kaiser
2008). Finally, there are also numerous metabolic adaptations integrated into
the UPR (see below).

2.2 Physiological Roles of the UPR

In addition to the pathways discussed above, the UPR also engages many
other processes that are critical for normal cellular and organismal
adaptations (Hotamisligil 2010). Three specific examples are discussed
below: (1) survival pathways, apoptosis, and autophagy; (2) immune
responses and inflammation; and (3) nutrient sensing and metabolic
regulation. In each case, components of the UPR interact with other signaling
pathways to control processes beyond simply protein folding and ER stress,
which can have effects both within and beyond the cell in which they are
activated. A challenging aspect of ER biology is to understand the
mechanisms leading to adaptive versus maladaptive/apoptotic responses in
the face of stress. It is likely that defective activity or disproportionate
(prolonged or imbalanced) engagement of distinct signaling networks
stimulated by each branch of the UPR is a critical determinant of detrimental
outcomes (Hotamisligil 2010).

2.2.1 Cell Survival and Death Responses of the ER

Under ER stress conditions, activation of the UPR reduces unfolded protein
load through several prosurvival mechanisms, including the expansion of the
ER membrane, selective synthesis of key components of the protein folding
and quality control machinery, and attenuation of the influx of proteins into
the ER. In conditions in which ER homeostasis cannot be established owing
to severe or prolonged stress, or unusual challenges (such as those presented
by energy or nutrient overload and inflammation), the responses triggered by
ER stress result in a maladaptive set of events leading to various cellular or
systemic pathologies, including death by apoptosis (Rao et al. 2004; Holcik
and Sonenberg 2005; Szegezdi et al. 2006; Scull and Tabas 2011).
When cells are subject to irreparable ER stress, the UPR drives
proapoptotic signals to eliminate the damaged material (Ch. 19 [Green and
Llambi 2014]). Both PERK and ATF6 induce expression of the transcription
factor C/EBP homologous protein (CHOP), which in turn leads to reduced
expression of the antiapoptotic gene Bcl2 and increased expression of a
number of proapoptotic genes (McCullough et al. 2001; Ma et al. 2002;
Marciniak et al. 2004). The proteins p58IPK, GADD34, and TRB3 are also
involved in the PERK-mediated apoptotic pathway. These targets have
individually been linked to the promotion of apoptosis; GADD34 and p58IPK
both negatively regulate eIF2α signaling and downstream adaptive responses,
and TRBP inhibits signaling by the kinase Akt (Novoa et al. 2001; Ladiges et
al. 2005; Bromati et al. 2011). IRE1α activation is linked to apoptosis
through its ability to activate the JNK MAPK (Urano et al. 2000) and
subsequent downstream phosphorylation of the Bcl2 family members Bim
and Bmf, which in turn cause Bax/Bak-dependent apoptosis (Lei and Davis
2003). IRE1 may also regulate the activation of ER-localized caspase-12
through the modulation of a TRAF2–caspase-12 complex (Yoneda et al.
2001; Walter and Ron 2011). Autophagosome formation is accelerated in
cells under ER stress (Kroemer et al. 2010), and disturbance of autophagy
can render them vulnerable to ER stress and, consequently, lead to death
(Ogata et al. 2006). IRE1α–JNK signaling can trigger autophagy by leading
to phosphorylation of Bcl2, which disrupts Bcl2–beclin-1 binding. This
results in the activation of beclin 1, an essential autophagy regulator (see Wei
et al. 2008; Ch. 19 [Green and Llambi 2014]).
In several different contexts, nutrient-sensing pathways are coupled to ER
function, in particular, to the IRE1 axis. This is best illustrated by the
coordinated regulation of a major cellular nutrient sensor, the mTORC1
complex (see p. 91 [Laplante and Sabatini 2012]), and the UPR. Loss of the
mTOR inhibitors TSC1 or TSC2 in cell lines and mouse or human tumors
hyperactivates mTORC1 and its downstream network (Yecies and Manning
2011). A key function of mTORC1 is to stimulate overall translational
initiation (Proud 2009), which causes ER stress (Ozcan et al. 2008). This can
promote mTORC1-mediated negative-feedback inhibition of insulin action
and further increase the vulnerability of cells to apoptosis (Ozcan et al. 2008).
The UPR is sensitive to the nutritional status of the cell, just like the
mTORC1 complex, responding to glucose deprivation, exposure to excess
fatty acids, hypoxia, and growth stimuli (Appenzeller-Herzog and Hall 2012).
In pancreatic β cells, for example, glucose can regulate IRE1 activation by
promoting the assembly of an IRE1α-RACK1-PP2A complex. RACK1 is a
β-propeller protein that binds to the 40S ribosomal subunit (Coyle et al. 2009;
Sharma et al. 2013), linking cell regulation and translation. In response to an
acute increase in glucose levels, RACK1 directs PP2A to IRE1α, promoting
its dephosphorylation (Qiu et al. 2010). Conversely, ER stress or prolonged
exposure to high glucose levels causes RACK1 to dissociate from PP2A,
resulting in disruption of this tripartite regulatory module (Qiu et al. 2010). In
this scenario, RACK1-associated phosphorylated IRE1α may have altered
functional outputs. The balance in IRE1 signaling is, thus, vital to the
survival of these cells and insulin biosynthesis.

2.2.2 The UPR and Inflammation

The UPR and other stress-signaling networks are highly integrated with
immune signaling (Gregor and Hotamisligil 2011). For example, all three
main arms of the UPR regulate NF-κB signaling during ER stress through
distinct mechanisms (Jiang et al. 2003; Kaneko et al. 2003; Deng et al. 2004;
Hu et al. 2006; Yamazaki et al. 2009). Moreover, signaling through Toll-like
receptors (TLRs) (on p. 121 [Lim and Staudt 2013]) can activate IRE1, via
NOX2-mediated production of ROS, resulting in production of inflammatory
cytokines (Martinon et al. 2010). TLRs engage IRE1α, but not the other
branches of the UPR, to promote cytosolic splicing and activation of XBP1,
which occur in the absence of an ER stress response and do not seem to
contribute to the induction of ER-stress-induced genes. Instead, activation of
XBP1 by IRE1 promotes sustained production of inflammatory mediators,
including interleukin (IL) 6, in certain contexts (Martinon et al. 2010). It is
important to emphasize that these responses may not necessarily be related to
ER stress per se, despite using IRE1. This distinction and the duration and
context of the signaling are critical to determining signaling outcome.
Links between the immune response and ER stress can be even more
complex and involve both innate and adaptive immunity. This is exemplified
by the secretory cells of the gut (McGuckin et al. 2011). In intestinal goblets
cells, a mutation in mucin 2 (a component of mucus) causes a disease similar
to ulcerative colitis in humans with a complex pattern of inflammation
involving many immune mediators (Heazlewood et al. 2008). In addition,
mutation of mucin 2 results in ER vacuolization and activation of GRP78 and
XBP1. Similarly, experimentally triggered ER stress in cultured cells can
cause increased expression of many inflammatory molecules, such as IL8,
IL6, MCP1, and tumor necrosis factor (TNF) (Li et al. 2005). As mentioned
above, ER stress and autophagy are linked, and autophagy is a critical
regulator of innate immune responses (Levine et al. 2011). Importantly,
cytokines and the pathways they activate influence the function of ER (Zhang
et al. 2008; Jiao et al. 2011). Activation of certain pathways, such as those
involving JNK and IκB kinase (IKK) (Deng et al. 2004; Hu et al. 2006), and
production of certain mediators, such as ROS, all have negative effects on the
function of ER (Cullinan and Diehl 2006; Gotoh and Mori 2006; Uehara et
al. 2006). The extent of oxidative stress and the levels, duration, and
magnitude of ROS and/or NO production can tip the balance in ER responses
toward a maladaptive profile (Chan et al. 2011). The metabolic status of the
ER (or the metabolic environment within which it has to operate) is thus a
key determinant of the balance between adaptive and maladaptive responses.
The kinase PKR, although not known to be physically associated with the
ER, is also activated during ER stress (Shimazawa et al. 2007; Nakamura et
al. 2010). Once activated, it brings together several key stress and
inflammatory signaling molecules, including JNK and insulin-signaling
components such as IRS1, with eIF2α. PKR interacts with and directly
phosphorylates IRS1 through which it links ER stress to suppression of
insulin action. Inflammatory cytokines and toxic lipids, such as palmitate,
induce phosphorylation of IRS by PKR, leading to inhibition of insulin
signaling. In the absence of PKR activity, neither inflammatory cytokines nor
toxic lipids can interfere with insulin action, and deletion of PKR in mice
results in significantly improved glucose metabolism.
PKR also contributes to the activation of the NLRP3 inflammasome and
HMGB1 (high-mobility group protein B1) production (Lu et al. 2012), which
are responsible for activation of inflammatory processes (Lamkanfi and Dixit
2012) in response to stimuli such as double-stranded RNA, adjuvant alum
and Escherichia coli (Ch. 15 [Newton and Dixit 2013]). PKR also physically
interacts with NLRP3, and activates the inflammasome in a cell-free system
with recombinant NLRP3, ASC, and procaspase-1 (Lu et al. 2012). Recent
reports also suggest direct regulation of the inflammasome by IRE1 itself via
the thioredoxin-interacting protein (TXNIP) (Lerner et al. 2012; Oslowski et
al. 2012). Under irremediable ER stress conditions, activation of IRE1α
results in elevated expression of TXNIP, leading to a terminal UPR featuring
activation of the inflammasome. Hence, the UPR, through PKR and IRE1,
appears to act at the interface of ER stress signaling, inflammatory responses,
and metabolic regulation. However, other studies indicate that TXNIP is
involved in the endocytosis of the glucose transporter GLUT1 (Wu et al.
2013); so, the connection to inflammasome function may be indirect.

2.2.3 Metabolic Responses Emanating from the ER

The ER is critical for regulation of metabolic homeostasis and its dysfunction
plays an important role in the emergence of metabolic disease. The ER is
sensitive to the metabolic status of cells, responding to feeding–fasting
cycles, acute nutrient exposure, and circadian rhythms, and is equipped with
direct and indirect means to alter metabolic responses (Cretenet et al. 2010;
Hotamisligil 2010; Pfaffenbach et al. 2010; Boden et al. 2011; Hatori et al.
2011). These aspects of ER function are critical in a number of metabolic
diseases, such as obesity and type 2 diabetes. Many of the signals generated
through the UPR are directly linked to metabolic responses and also engage
inflammatory and stress-signaling pathways (Fig. 3), which are also linked to
metabolic regulation. Integration of these pathways is critical for glucose
metabolism, insulin secretion, insulin action, and the metabolic activities of
the ER (Marciniak and Ron 2006; Hotamisligil 2010; Fu et al. 2012).
Figure 3. The UPR in stress signaling, inflammation, and metabolism. The UPR contributes to both
inflammatory/stress signaling and metabolic regulation, as exemplified in chronic metabolic diseases. It
activates several stress-related kinases including ERK, p38, JNK, and IKK. The resulting signals can
impair signaling by insulin or other endocrine hormones and disrupt metabolism. The UPR can also
modulate glucose and lipid metabolism directly. Nuclear ATF6 inhibits gluconeogenesis and
lipogenesis by directly binding to TORC2 and SREBP2 proteins, respectively. This mechanism is
defective in metabolic disease. The spliced form of XBP1 (XBP1s) can directly or indirectly (through
SREBP1) activate the lipogenesis program while inhibiting gluconeogenesis. eIF2α phosphorylation
leads to the synthesis of CHOP and the activation of lipogenesis programs via CEBPα/β. (Note that the
opposing effects of the UPR on gluconeogenesis and lipogenesis are context dependent.) Activation of
lipogenesis alters the membrane lipid composition of the ER, inhibits SERCA calcium pumps, and
propagates ER stress, which in turn disrupts metabolism further. Hence, inflammatory, stress, and
metabolic responses generate a vicious cycle if the ER dysfunction cannot be remedied.

Insulin is the master regulator of glucose metabolism in mammals and

defective insulin action and/or production results in diabetes (Ch. 7 [Ward
and Thompson 2012] and Ch. 14 [Hardie 2012]). Obesity and diabetes cause
ER dysfunction and/or stress in animal models (Ozcan et al. 2004; Nakatani
et al. 2005) and humans (Boden et al. 2008; Sharma et al. 2008; Gregor et al.
2009), and chemicals that reduce ER stress and improve ER function improve
glucose metabolism in animal models (Ozawa et al. 2005; Ozcan et al. 2006;
Kammoun et al. 2009) and in humans (Kars et al. 2010; Xiao et al. 2011). ER
stress responses intersect with insulin action at several points, including the
stimulation of JNK by PKR (Nakamura et al. 2010), CaM kinase II (Li et al.
2009; Ozcan and Tabas 2012; Ozcan et al. 2012), and IRE1-dependent
interaction between TRAF2 and ASK1 (a MAP kinase kinase kinase,
MAPKKK) (Urano et al. 2000). This inhibits insulin signaling by uncoupling
the insulin receptor from the substrate, IRS1, that mediates its metabolic
actions (Hirosumi et al. 2002; Ozcan et al. 2004; Sabio and Davis 2010;
Ozcan et al. 2012).
ER stress and inflammation in the central nervous system and gut can also
indirectly regulate glucose metabolism in peripheral tissues, particularly the
liver (Caricilli et al. 2011; Purkayastha et al. 2011; Milanski et al. 2012).
Chronic ER stress affects hypothalamic neuroendocrine pathways that
regulate satiety, body weight, and metabolism. Acute ER stress in the brain
can induce glucose intolerance and systemic and hepatic insulin resistance,
and blocking brain TLR4 or TNF action in obese individuals improves
insulin sensitivity and glucose homeostasis in the liver (Mighiu et al. 2012;
Milanski et al. 2012). In the gut, alterations in the composition of the gut
microbiota seen in TLR2-deficiency are accompanied by ER stress, JNK
activation, and impaired insulin signaling in the liver (Caricilli et al. 2011).
Hence, ER function is part of the inter-organ communication network that
ensures metabolic homeostasis.
Insulin is produced by pancreatic β cells, which, as professional secretory
cells, are heavily dependent on a healthy, functioning ER. Mutations in the
PERK branch (Wolcott-Rallison syndrome) of the UPR (Delepine et al. 2000;
Harding et al. 2001; Zhang et al. 2002) or the WFS1 gene (for Wolfram
syndrome 1), which encodes wolframin, result in death and dysfunction of β
cells and cause diabetes (Ishihara et al. 2004; Fonseca et al. 2005; Yamada et
al. 2006). Wolframin targets ATF6 for proteasomal degradation, and its loss
results in ER stress signaling mediated by increased levels of ATF6α
(Fonseca et al. 2010). XBP1 deficiency in mice also compromises β-cell
function and survival (Lee et al. 2011a). Moreover, in type 2 diabetes,
pancreatic β cells also suffer from a vicious cycle of inflammatory
alterations, which can compromise the folding capacity of the ER and normal
functioning of β cells, which progressively lose their ability to produce
insulin (Eguchi et al. 2012). This is a challenging problem for therapeutic
intervention because both deficiency and hyperactivity of each one of the
three UPR branches can be detrimental for β cells (Nozaki et al. 2004; Seo et
al. 2008; Trusina et al. 2008) and potentially for other cells.
The UPR is thus a critical determinant of metabolic homeostasis. Lipid,
carbohydrate, and protein metabolism are all regulated by the UPR, and both
the synthesis of and sensitivity to key metabolic hormones, such as insulin,
are regulated by these pathways. The survival and function of key cell types
critical to metabolism, including pancreatic β cells, gut epithelium, and
hypothalamic neurons, are controlled by the UPR. Furthermore, the link
between inflammation and metabolism is intimately related to ER function.

3 STRESS SIGNALING BY MAP KINASES

MAPK pathways are universally conserved eukaryotic-signaling modules
that transduce extracellular and intracellular signals to regulatory networks
within the cell by phosphorylation of key protein targets (p. 81 [Morrison
2012]). MAPKs are activated by dual phosphorylation of tyrosine and
threonine residues in a partially unstructured segment (activation loop)
between the amino and carboxy-terminal lobes of the catalytic domain (Payne
et al. 1991). Phosphorylation on the threonine residue improves the geometry
of the active site catalytic residues, leading to formation of hydrogen bonds
on the surface of the MAPK that connect the amino-terminal domain to the
activation loop and promote closure of the active site cleft. Phosphorylation
on the tyrosine residue leads to the creation of new hydrogen bonds and
refolding of the activation loop to form a structure that contributes to the
substrate-interaction sites (Canagarajah et al. 1997; Rodriguez Limardo et al.
2011). The ERK family of MAPKs are primarily activated by exposure of
cells to cytokines and growth factors (Robinson and Cobb 1997). In contrast,
the p38 and JNK MAPK families respond primarily to the exposure of cells
to extracellular and intracellular stress (Davis 2000; Cuadrado and Nebreda
2010). Consequently, the JNK and p38 MAP kinases are often termed stress-
activated MAPKs. They respond to inflammatory cytokines and many
changes in the physical/chemical environment, as well as DNA damage and
redox imbalance (Fig. 4).

Figure 4. Stress-activated MAPK-signaling pathways. The p38 MAP kinases are primarily activated by
the MAPKK isoforms MKK3 and MKK6, but a minor contribution of MKK4 can be detected. All p38
MAPK isoforms are activated by MKK3 and MKK6, although p38δ is activated by MKK3
significantly more potently than MKK6. The JNK group of MAPKs is activated by the MAPKK
isoforms MKK4 and MKK7.

The JNKs are encoded by three genes (JNK1, JNK2, and JNK3), which
are alternatively spliced to yield 10 different isoforms (Gupta et al. 1996).
JNK1 and JNK2 are ubiquitously expressed, but JNK3 is expressed primarily
in the brain (Davis 2000). Gene disruption studies in mice show that these
JNK isoforms can mediate different biological responses (Davis 2000). The
p38 MAPKs are encoded by four genes that can be divided into two
subgroups (p38α/β and p38γ/δ). These p38 isoforms show nonredundant
functions and different sensitivities to small molecule inhibitors (Cuenda and
Rousseau 2007; Cuadrado and Nebreda 2010).
The minimal consensus sequence for target protein phosphorylation by
MAPKs is -S/T-P-. However, the presence of this consensus motif is not
sufficient for phosphorylation by a MAPK, which frequently requires a
docking interaction between the MAPK and another region of the substrate
(Enslen and Davis 2001; Tanoue and Nishida 2003; Akella et al. 2008). Two
types of MAPK docking motifs have been identified in substrates: (1) the
FXFP motif, and (2) the D domain, comprising a hydrophobic motif (LXL)
plus a basic region (Bardwell and Thorner 1996; Jacobs et al. 1999; Enslen
and Davis 2001). Structural analysis of proteins docked to p38 MAPK
(Chang et al. 2002) and JNK3 (Heo et al. 2004) show extensive interactions
between the docked proteins and regions of the MAPK outside the active site.
The sites of D domain and FXFP interaction on MAPKs are different (Akella
et al. 2008). In addition to these conserved interactions, the carboxy-terminal
sequence of p38γ MAPK can dock directly to proteins that have PDZ
domains (e.g., α1 syntrophin, PSD95 [also known as SAP90], and DLG [also
known as SAP97]) to direct their phosphorylation (Hasegawa et al. 1999;
Hou et al. 2010).
Bioinformatic analyses and protein interaction screens (e.g., two-hybrid
assays) have identified many MAPK substrates. More recently, chemical
genetic methods and mass spectroscopy have enabled a more comprehensive
analysis of MAPK substrates in specific tissues (Allen et al. 2007; Carlson et
al. 2011). These include membrane, cytosolic, and nuclear proteins that
participate in many biological processes, and especially many transcription
factors, such as ATF2 (activated by JNK1/2/3 and p38α/β MAPK), Jun
(activated by JNK1/2/3), MEF2C (activated by p38α/β MAPK and ERK5),
and Elk1 (activated by JNK1/2/3, p38α/β MAPK, and ERK1/2) (Whitmarsh
and Davis 2000). Other MAPK targets include protein kinases
phosphorylated and activated by MAPKs, including eEF2K (activated by
p38γ/δ MAPK), MK2/3 (activated by p38α/β MAPK), MK5 (activated by
ERK3/4), MNK1/2 and MSK1/2 (activated by ERK1/2 and p38α/β MAPK),
and RSK1/2/3 (activated by ERK1/2) (Cargnello and Roux 2011). Protein
phosphatases are also MAPK targets, including nuclear DUSP1 (protected
against proteasomal degradation by ERK phosphorylation) and cytoplasmic
DUSP6 (proteasomal degradation is promoted by ERK phosphorylation)
(Caunt and Keyse 2013). A complete understanding of MAPK function will
require a systems-level approach to define the network of interactions that
mediate MAPK signaling (Ch. 4 [Azeloglu and Iyengar 2014]). Nevertheless,
we already have a good understanding of the roles of these kinases in stress
signaling from biochemical and genetic experiments in multiple organisms.

3.1 MAPK Activation by Stress Signals

Canonical phosphorylation-dependent activation of a MAPK is mediated by a
MAPKK. Different MAPKK isoforms selectively activate particular MAPKs:
MKK1 and MKK2 activate ERK1 and ERK2, MKK3 activates p38, MKK4
activates JNK and p38, MKK6 activates p38, and MKK7 activates JNK (Fig.
4). Gene disruption studies in mice have shown that MKK3 and MKK6 are
the main MAPKKs responsible for p38 activation in vivo, although a minor
role of MKK4 can be detected (Brancho et al. 2003). This role is supported
by biochemical studies that show phosphorylation of p38 on threonine and
tyrosine by both MKK3 and MKK6. Similar gene disruption studies show
that MKK4 and MKK7 collaborate in JNK activation (Fleming et al. 2000;
Tournier et al. 2001). JNK is preferentially phosphorylated on tyrosine by
MKK4 and threonine by MKK7.
Although MAPK activation by MAPKK isoforms represents the major
mechanism of MAPK regulation in vivo, noncanonical mechanisms of
MAPK activation can be detected under specific circumstances. In yeast,
association of the MAPK Fus3 with the scaffold protein Ste5 promotes
autophosphorylation of the tyrosine in its activation loop (Bhattacharyya et
al. 2006; Good et al. 2009) and autophosphorylation on the activation loop
tyrosine of the Smk1 MAPK is driven by its association with the meiosis-
specific protein Ssp2 (Whinston et al. 2013). In T cells, the tyrosine kinase
Zap70 phosphorylates p38α on tyrosine and causes subsequent
autophosphorylation of the dual phosphorylation motif in the activation loop
(Salvador et al. 2005). Similarly, ERK7 is activated by autophosphorylation
(Abe et al. 2001). Another noncanonical pathway is represented by the
atypical MAPK isoforms ERK3 and ERK4, which can be activated by the
protein kinase PAK1 (Deleris et al. 2011).
MAPKKs are themselves activated by phosphorylation of their activation
loop by MAPKKKs. Selective activation of MAPKKK isoforms by stress
stimuli creates parallel signaling cascades involving sequential
phosphorylation of MAPKKs and MAPKs (Fig. 4). These MAPKKKs
therefore function to trigger MAPK pathway activation in response to
specific stimuli.
The mixed-lineage protein kinase (MLK) group of MAPKKKs are
activated by members of the Rho GTPase family, including Rac1 and Cdc42
(Gallo and Johnson 2002). MLK protein kinases are autoinhibited by an
interaction between an amino-terminal SH3 domain and a proline motif in the
carboxy-terminal region. Binding of GTP-bound Rac1 or Cdc42 to MLK
protein kinases, mediated by a CRIB motif, causes displacement of SH3-
mediated autoinhibition and subsequent protein kinase activation that leads to
activation of the MAPKK and, subsequently, the MAPK. This mechanism
contributes to stress signaling mediated by inflammatory cytokines (Kant et
al. 2011). The response to activated Rho proteins may also be mediated by
members of the MEKK group of MAPKKKs, including MEKK1 and
MEKK4 (Fanger et al. 1997).
The MAPKKK isoform MEKK2 is activated by receptor tyrosine kinases
that bind epidermal growth factor, fibroblast growth factor 2 (FGF2), and
stem cell factor (SCF, also known as Kit ligand) by a mechanism that has not
been defined (Garrington et al. 2000; Kesavan et al. 2004). Activated
MEKK2 causes coordinate activation of the ERK5 and JNK pathways
(Kesavan et al. 2004).
The stress-induced immediate early gene response can also cause
activation of the JNK and p38 MAPK pathways. GADD45 α, β, and γ
proteins are expressed in cells exposed to inflammatory cytokines or
environmental stress. These are small (18 kDa), acidic, mainly nuclear
proteins belonging to the L7Ae/L30e/S12e RNA-binding protein
superfamily. They lack obvious enzymatic activity and exert their pleiotropic
function by interaction with multiple effectors that influence processes as
diverse as cell cycle progression, apoptosis, and DNA repair and
demethylation (Niehrs and Schäfer 2012). All GADD45 isoforms can bind to
and activate the MAPKKK MEKK4 (Takekawa and Saito 1998). Indeed,
mice lacking GADD45 or MEKK4 show similar defects in MAPK signaling
(Chi et al. 2004). The binding of GADD45 to MEKK4 disrupts autoinhibition
by the MEKK4 amino-terminal domain and causes dimerization,
transphosphorylation, and activation of MEKK4 (Miyake et al. 2007).
 Redox stress can activate the MAPKKK isoform ASK1 by causing the
release of the inhibitor thioredoxin (Matsukawa et al. 2004). Consequently,
knockout mice lacking ASK1 expression show severe defects in MAPK
signaling during the response to oxidative stress (Matsuzawa et al. 2002).
Stress-activated MAPKs are activated during the DNA damage response
by the ATM protein kinase (Ch. 6 [Rhind and Russell 2012]), which
phosphorylates and activates members of the TAO group of atypical
MAPKKKs (Raman et al. 2007). This mechanism appears to be selective for
the p38 branch of stress-activated MAPK signaling.
Exposure of cells to the inflammatory cytokines TNF and IL1 causes
activation of stress-activated MAPKs (p. 121 [Lim and Staudt 2013]; Ch. 15
[Newton and Dixit 2013]). This pathway requires the MAPKKK isoform
TAK1 and the ubiquitin-binding accessory proteins TAB2 and TAB3. These
TAB proteins activate TAK1 when bound to K63-linked polyubiquitin chains
formed by the inflammatory receptor-signaling complex (Chen 2012). The
TNF receptor causes K63-linked ubiquitylation of RIP1 and TRAF2/5 by the
E3 ligases cIAP1/2, whereas the IL1 receptor causes K63-linked
autoubiquitylation of the E3 ligase TRAF6. A similar ubiquitin-mediated
TAK1 activation mechanism is engaged when TLRs bind pathogen-
associated molecular patterns to stimulate stress-activated MAPK-signaling
pathways (p. 121 [Lim and Staudt 2013]; Ch. 15 [Newton and Dixit 2013]).
These roles of TAK1 in MAPK activation are coordinated with TAK1-
mediated activation of the NF-κB-signaling pathway (p. 121 [Lim and Staudt
2013; Ch. 15 [Newton and Dixit 2013]).

3.2 Inactivation of Stress-Activated MAPK-Signaling

Pathways
The major mechanism for inactivation of MAPK-signaling pathways is to
reverse phosphorylation-mediated activation. Dual-specificity phosphatases
(DUSPs, also known as MAPK phosphatases) play a key role in MAPK
inactivation (Owens and Keyse 2007; Caunt and Keyse 2013). In addition,
MAPKs can be inactivated by the serine/threonine protein phosphatases
PP2A and PP2C, tyrosine phosphatases, and the death-effector-domain
protein PEA15. The PP2C family member WIP1 plays a major role in
switching off the response of p38 to DNA damage (Le Guezennec and
Bulavin 2010) and the tyrosine phosphatase HePTP regulates p38 MAPK in
B cells following stimulation by adrenalin (McAlees and Sanders 2009). This
regulation can display complex dynamics because of phosphorylation-
induced regulation of DUSP activity and signal-induced DUSP expression
(Keyse 2008). Note that stress-activated MAPK pathways can also be
inactivated by bacterial pathogens (Ch. 20 [Alto and Orth 2012]).

3.3 The Role of Scaffold Proteins

The protein kinase cascade that is initiated by an activated MAPKKK and
includes subsequent MAPKK and MAPK activation represents a defined
signaling module. The interactions among the constituent proteins can
involve a series of binary docking interactions. For example, as outlined
above, a D domain located in the amino-terminal region of a MAPKK can
interact with a docking site on MAPKs (Bardwell and Thorner 1996; Enslen
and Davis 2001). Cleavage by anthrax lethal factor protease that removes this
D domain accounts for the prevention of MAPKK-mediated MAPK
activation by this pathogenic bacterium (Duesbery et al. 1998). Similarly,
docking interactions between a MAPKKK and MAPKK have been described,
including the Phox/Bem1p domain-mediated interactions between MEKK2/3
and MKK5 (Nakamura and Johnson 2007).
MAPK-signaling modules can be assembled by scaffolding proteins
(Morrison and Davis 2003; Good et al. 2011; Witzel et al. 2012). The best-
characterized scaffold is Ste5, a component of the yeast-mating response
MAPK pathway (Good et al. 2011). Other proteins that regulate mammalian
MAPK signaling include scaffolds for the ERK pathway (KSR1), the p38
pathway (OSM, JIP2, and JIP4), and the JNK pathway (JIP1 and JIP3)
(Morrison and Davis 2003). Gene disruption studies have confirmed that JIP1
plays a key role in JNK activation caused by the neurotransmitter glutamate
(Whitmarsh et al. 2001) and metabolic stress caused by a high-fat diet
(Jaeschke et al. 2004; Morel et al. 2010). JIP1 binds to JNK, the MAPKK
isoform MKK7, and members of the MLK group of MAPKKKs to form a
functional JNK-signaling module. The formation of this signaling complex is
regulated by phosphorylation (Morel et al. 2010). Moreover, JIP1 interacts
with kinesin light chain and is dynamically localized within the cell by
microtubule-mediated protein trafficking. This scaffold protein allows
regulatory control of localization, module activation, and substrate access to
direct stress-activated MAPK signaling to specific targets within the cell
(Morrison and Davis 2003).

3.4 Physiological Role of Stress-Activated MAPKs

Like the UPR, stress-activated MAPKs contribute to many aspects of normal
cellular physiology and pathology (Davis 2000; Cuadrado and Nebreda
2010). Below, we discuss their roles in the context of three specific
examples: cell death versus survival signaling, inflammatory stress, and
metabolic stress.

3.4.1 Stress-Activated MAPKs and Cell Death

The initial response of cells to stress exposure is to mount a survival
response, but prolonged exposure to stress may lead to cell death (Ch. 19
[Green and Llambi 2014]). MAPKs are implicated in these processes (Xia et
al. 1995). Studies of TNF-stimulated cell death show that JNK initially
promotes a survival response and that prolonged JNK activation is required
for cell death (Lamb et al. 2003; Ventura et al. 2006). Sustained activation of
JNK and p38 can contribute to both necrotic and apoptotic cell death by
regulating the expression of cytotoxic ligands (e.g., FasL and TNF) (Das et
al. 2009) and synthesis of ROS (Ventura et al. 2004). In addition, stress-
activated MAPKs can regulate the intrinsic apoptosis pathway, mediated by
mitochondria, by regulating members of the Bcl2 family (Tournier et al.
2000). Thus, JNK can phosphorylate and inhibit prosurvival signaling by
Mcl1 by inducing ubiquitin-dependent proteasomal degradation (Morel et al.
2009) and JNK can cause induction of the proapoptotic BH3-only protein
Bim (Wong et al. 2005; Perier et al. 2007). Bim is also subject to
posttranslational regulation by MAPKs (Puthalakath and Strasser 2002). ERK
phosphorylation on multiple serine residues sites triggers ubiquitin-
independent proteasomal degradation of Bim (Wiggins et al. 2011). In
contrast, JNK phosphorylation of Bim on a threonine residue disrupts the
interaction of Bim with the dynein microtubule motor complex that can
sequester Bim on the microtubule cytoskeleton (Lei and Davis 2003; Hubner
et al. 2008). The Bim-related protein Bmf is similarly regulated by JNK-
mediated phosphorylation, which disrupts the interaction of Bmf with the
myosin V motor complex that sequesters Bmf on the actin cytoskeleton (Lei
and Davis 2003; Hubner et al. 2010). JNK therefore promotes the intrinsic
apoptotic pathway by degrading the antiapoptotic protein Mcl1 and up-
regulating the proapoptotic protein Bim by multiple mechanisms, including
increased transcription and sequestration of Bim by the cytoskeleton. The
loss of the antiapoptotic protein Mcl1 combined with the increase in Bim
activity causes a change in the balance of pro- and antiapoptotic pathways
that leads to cell death. This balance of cell death and survival regulated by
stress-activated MAPKs is also influenced by the activation state of survival
signaling pathways, including those involving Akt, ERK, and NF-κB.
In vivo studies have shown that stress-activated MAPKs play a critical
role in neurodegenerative disorders, including stroke (Kuan et al. 2003),
seizure (Yang et al. 1997), Alzheimer’s disease (Mazzitelli et al. 2011), and
Parkinson’s disease (Perier et al. 2007). Cell death regulated by stress-
activated MAPKs may also play an important role during tumor development
(Davis 2000).

3.4.2 Stress-Activated MAPKs and Inflammation

MAPKs contribute to both innate and adaptive immune responses (Dong et
al. 2002). In particular, stress-activated MAPKs play an important role in the
regulation of inflammatory cytokine expression. The p38 and JNK pathways
target transcription factors (e.g., ATF, Jun, and MEF2 family members) and
chromatin-remodeling enzymes that can regulate expression of chemokines
(e.g., CCL2 and CCL5) and cytokines (e.g., IL1, IL6, IL12p40, and TNF)
during inflammatory responses (Davis 2000; Cuadrado and Nebreda 2010).
In addition, p38α increases cytokine mRNA stability controlled by the AU-
rich element-binding proteins HuR (phosphorylated by p38) and TTP
(phosphorylated by p38α-MAPK-activated MK2) (Clark et al. 2009). p38α
can also stimulate cytokine mRNA translation by phosphorylation and
activation of eIF4E/MAPK-interacting kinases (MNK1 and MNK2)
(Noubade et al. 2011). Moreover, p38γ and p38δ increase cytokine
translation by phosphorylation and activation of eEF2K, which increases
protein synthesis (Gonzalez-Teran et al. 2013). Thus, JNK and p38
coordinately regulate transcription, mRNA stability, and mRNA translation
to promote cytokine/chemokine expression.
The immune response elicited during inflammation depends on the
involvement of specific innate and adaptive immune cells. These types of
cells are influenced by stress-activated MAPK-signaling pathways.
Macrophages represent an important part of the innate immune response.
Classical activation of macrophages by interferon γ or endotoxin causes
polarization to the M1 phenotype that is associated with expression of
inflammatory cytokines (e.g., TNF) and inflammation. In contrast,
alternatively activated macrophages polarized to the M2 phenotype can
express anti-inflammatory cytokines (e.g., IL10) and are implicated in
resolution of the immune response and tissue remodeling. The M2 phenotype
is complex and represents a mixture of different cell types, including cells
exposed to IL4 or IL13 (M2a), immune complexes (M2b), and IL10 or TGFβ
(M2c). Stress-activated MAPKs can control macrophage polarization. For
example, JNK is required for M1, but not M2, macrophage development
(Han et al. 2013). Consequently, JNK can promote inflammation by
polarizing macrophages to the M1 inflammatory phenotype. In contrast, JNK
inhibition can suppress inflammation by increasing polarization to the M2
anti-inflammatory phenotype.
Stress-activated MAPKs can also regulate the formation of specific T-cell
subsets. For example, JNK is required for CD8+ cell proliferation and IL2
secretion (Conze et al. 2002). In contrast, it is not required for CD4+ T-cell
activation or IL2 secretion, but JNK is required for naïve CD4+ T-cell
differentiation into Th1 or Th2 effector cells (Dong et al. 2000). Moreover,
p38α is required for differentiation of CD4+ T cells into Th17 cells (Noubade
et al. 2011). A consequence of these roles in T cells is that JNK is required
for efficient viral clearance (Arbour et al. 2002) and p38 is required for
suppression of autoimmunity (Noubade et al. 2011).
Collectively, these mechanisms lead to inflammation in tissues with
increased stress-activated MAPK activity, which can promote metabolic
dysfunction (Sabio and Davis 2010) and carcinogenesis (Das et al. 2011).

3.4.3 Stress-Activated MAPKs and Metabolism

Feeding a high-fat diet causes metabolic stress and activates stress-activated
MAPK-signaling pathways. This is mediated by increased amounts of
saturated free fatty acids that activate the MLK group of MAPKKKs
(Jaeschke and Davis 2007; Kant et al. 2013). The mechanism of fatty acid
signaling may require G-protein-coupled receptors (Talukdar et al. 2011) or
nonreceptor mechanisms (Holzer et al. 2011) that activate the tyrosine kinase
Src in lipid raft domains of the plasma membrane (Holzer et al. 2011). p38α
and p38δ appear to have roles in insulin resistance, oxidative stress-induced
β-cell failure, and hepatic gluconeogenesis (Sumara et al. 2009; Lee et al.
2011b). Moreover, the JNK pathway is required for high-fat-diet-induced
obesity and insulin resistance (Hirosumi et al. 2002).
The effects of JNK on obesity are due to a requirement for JNK in
negative-feedback regulation of energy expenditure by the hypothalamus-
pituitary-thyroid axis (Sabio and Davis 2010). The activation of JNK inhibits
the expression of hypothalamic thyrotropin-releasing hormone and pituitary
gland expression of thyroid-stimulating hormone. The consequence of JNK-
mediated suppression of the hypothalamic-pituitary-thyroid axis is that levels
of circulating thyroid hormone, thyroid-hormone-dependent target gene
expression, and oxidative metabolism are all decreased, which reduces
energy expenditure, and obesity is increased.
The effects of JNK on insulin resistance depend on roles of JNK in
peripheral tissues that are independent of obesity (Sabio and Davis 2010).
Activated JNK causes inhibition of signaling by the insulin receptor. Initial
studies indicated that this might be mediated by inhibitory phosphorylation of
IRS1 (Aguirre et al. 2000), but this conclusion was not supported by later
studies (Copps and White 2012). The mechanism of inhibition of insulin
receptor signaling by JNK remains to be elucidated. However, one potential
mechanism that may contribute to insulin resistance is the requirement of
JNK for macrophage polarization to the M1 phenotype because tissue
infiltration by inflammatory macrophages is a key determinant of the
development of insulin resistance (Han et al. 2013). It is clear that loss of
JNK function in murine models protects mice against the development of
insulin resistance when fed a high-fat diet (Sabio and Davis 2010). This
observation is important because compensation of insulin resistance by
hyperinsulinemia can lead to β-cell failure and diabetes. Because stress-
activated MAPKs play a key role in energy homeostasis and normal
glycemia, they represent potential targets for therapies aimed at treatment of
metabolic syndrome and prediabetes (Sabio and Davis 2010).

4 CONCLUSIONS
Stress-signaling pathways are evolutionarily conserved and play an important
role in the maintenance of homeostasis and adaptation to new cellular
microenvironments (Fig. 5). Key areas for future research include structural
studies of stress-response signaling proteins and integrated analysis of the
signaling network’s response to stress.
Figure 5. Integrated response to metabolic stress. A high-fat diet causes metabolic stress associated
with increased amounts of saturated free fatty acids, which engage the UPR and stress-activated
MAPKs. The UPR includes three different signaling pathways that are initiated by IRE1, PERK, and
ATF6. The stress-activated MAPK response is initiated by the MLK group of MAPKKKs and leads to
the activation of the JNK and p38 MAPKs. Cross talk between the UPR and stress-activated MAPK
signaling leads to an integrated adaptive response.

Finally, studies of stress-signaling pathways that translate to the clinic are

needed if we are to realize their potential for therapeutic intervention in
disease processes. Effective and specific means to target both MAPK
signaling and the UPR are required. An important area of research will be the
development of molecular tools that allow us to specifically modulate the
function of the key components of MAPK pathways. Similarly, we must
define new targets within the UPR and develop new tools to modulate its
components.

ACKNOWLEDGMENTS
Studies in the Hotamisligil laboratory are currently supported by grants from
the National Institutes of Health (NIH), the Juvenile Diabetes Research
Foundation, the American Diabetes Association, Servier and UCB
Pharmaceuticals, and the Simmons Fund. Special thanks to Scott
Widenmaier, Ling Yang, and Takahisa Nakamura for critical discussions,
thoughtful comments, and help in preparing the manuscript, Ana Paula
Arruda and Suneng Fu for help in illustrations, and Megan Washack and
Claudia Garcia Wagner for editorial assistance. Studies in the Davis
laboratory are currently supported by grants from the NIH, the American
Diabetes Association, and the Howard Hughes Medical Institute. Kathy
Gemme provided expert editorial assistance. We thank the students and
fellows who contributed to the studies in our groups over the years and to our
collaborators. We regret the inadvertent omission of references to important
work by our colleagues because of space limitations.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006072
CHAPTER 19

Cell Death Signaling

Douglas R. Green and Fabien Llambi

Department of Immunology, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105
Correspondence: douglas.green@stjude.org

SUMMARY

In multicellular organisms, cell death is a critical and active process that

maintains tissue homeostasis and eliminates potentially harmful cells.
There are three major types of morphologically distinct cell death:
apoptosis (type I cell death), autophagic cell death (type II), and necrosis
(type III). All three can be executed through distinct, and sometimes
overlapping, signaling pathways that are engaged in response to specific
stimuli. Apoptosis is triggered when cell-surface death receptors such as
Fas are bound by their ligands (the extrinsic pathway) or when Bcl2-
family proapoptotic proteins cause the permeabilization of the
mitochondrial outer membrane (the intrinsic pathway). Both pathways
converge on the activation of the caspase protease family, which is
ultimately responsible for the dismantling of the cell. Autophagy defines
a catabolic process in which parts of the cytosol and specific organelles
are engulfed by a double-membrane structure, known as the
autophagosome, and eventually degraded. Autophagy is mostly a
survival mechanism; nevertheless, there are a few examples of
autophagic cell death in which components of the autophagic signaling
 pathway actively promote cell death. Necrotic cell death is characterized
by the rapid loss of plasma membrane integrity. This form of cell death
can result from active signaling pathways, the best characterized of
which is dependent on the activity of the protein kinase RIP3.

Outline
1 Introduction
2 Type I cell death: Apoptosis
3 Type II cell death and autophagy
4 Type III cell death: Necrosis
5 Beyond type III—Other forms of cell death
6 Conclusion
References

1 INTRODUCTION
Although cell death can happen as a result of overwhelming damage, most
cell deaths in animals occur in an active manner, as a consequence of specific
signaling events. In general, there are three types of cell death, defined in
large part by the appearance of the dying cell: apoptosis (also known as type I
cell death), autophagic cell death (type II), and necrosis (type III) (Galluzzi et
al. 2007).
Apoptosis is characterized by cell shrinkage, membrane blebbing, and
condensation of the chromatin (pyknosis) (Kerr et al. 1972). It can be further
defined as cell death accompanied by the activation of caspase proteases
(Galluzzi et al. 2012). Two major signaling pathways trigger apoptotic cell
death: the mitochondrial (the intrinsic) pathway and the death receptor (the
extrinsic) pathway. The latter involves a classical ligand–cell-surface-
receptor interaction. For example, cytotoxic lymphocytes can kill infected or
transformed cells by expressing ligands for death receptors (DRs), a subset of
the tumor necrosis factor (TNF) receptor (TNFR) family. These ligands
induce apoptotic cell death of the targeted cells provided they express such
DRs. DR-induced cell death in general is critical for immune system function
and homeostasis. In contrast, the mitochondrial apoptotic pathway is usually
initiated in a cell-autonomous manner. Most cellular stresses, such as DNA
damage (induced by genotoxic agents or defects in DNA repair) or
endoplasmic reticulum (ER) stress (induced by the accumulation of unfolded
proteins), actively engage apoptosis when cells are damaged beyond repair.
Conversely, the lack of a signal, such as those activated by growth factors
(e.g., cytokines and neurotrophic factors), can lead to cell death. This
mechanism is critical for the development of the nervous system in
vertebrates and it is estimated that half of the neurons generated die during
this process (Buss et al. 2006). This cell death is due, in part, to the failure of
some neuronal precursors to properly migrate or innervate their targets and
the consequent lack of neurotrophic factor stimulation. Similarly, during an
immune response, cytokine deprivation (together with the DR pathway) is
responsible for the acute contraction of the lymphocyte population after
clearance of the pathogen. Another example of “loss-of-signal”-induced cell
death is the particular form of apoptosis called anoikis, which occurs when
epithelial or endothelial cells detach from the extracellular matrix (ECM). In
this scenario, unligated ECM receptors of the integrin family cease to induce
prosurvival signaling pathways, eventually leading to apoptosis. This
mechanism prevents cells shedding from their original location from
colonizing elsewhere (a characteristic of metastatic cancer cells). Finally,
apoptosis can be induced by oncogenes (e.g., Myc) as a safeguard mechanism
against cancer development. This process is controlled in part by a p53-
dependent apoptotic pathway, which is activated in response to aberrant
mitogenic signals resulting from oncogene overexpression or mutation. As a
consequence, evasion of apoptotic cell death is often a requisite to sustain
oncogene transformation (see Ch. 21 [Sever and Brugge 2014]).
Autophagic cell death is characterized by the appearance of large
intracellular vesicles and engagement of the autophagy machinery. Note that
although autophagy (i.e., the membrane engulfment and catabolic
degradation of parts of the cytoplasm) is a well-defined process, its function
as an active cell death mechanism remains highly controversial. Autophagy is
mainly a survival process engaged in response to a metabolic crisis (e.g., low
ATP levels and nutrient and amino acid deprivation) or to remove damaged
organelles (e.g., mitochondria with low membrane potential) and protein
aggregates. As a stress response, autophagy accompanies rather than
promotes cell death in most scenarios and merely represents a failed survival
attempt (Shen et al. 2012). Nevertheless, there are specific examples in which
the autophagy machinery is absolutely required for cell death. During
Drosophila metamorphosis, obsolete larval tissues such as the midgut and
salivary glands regress through massive autophagic cell death, a process
triggered by the steroid hormone ecdysone. In this particular case, a
deficiency in genes of the autophagic signaling pathway alters the cell death
program (Berry and Baehrecke 2007; Denton et al. 2009). Autophagic cell
death has also been reported in response to deregulated H-Ras activity and
could therefore represent a safeguard mechanism against oncogenic
transformation (Elgendy et al. 2011).
Necrosis is characterized by cell swelling and plasma membrane rupture,
and a loss of organellar structure without chromatin condensation. Although
necrosis can occur as a consequence of irreparable cell damage, at least one
pathway of active necrosis exists. This form of cell death, sometimes called
necroptosis, is engaged by several signaling pathways that all converge on
the activation of receptor-interacting protein kinase 3 (RIP3). RIP3 is
activated upon recruitment to macromolecular complexes downstream from
various cell-surface receptors: DRs, Toll-like receptors (TLRs), and the T-
cell receptor (TCR). Additionally, DNA damage can directly induce the
formation of a RIP3-activation platform, independently of cell-surface
receptor ligation. Finally, RIP3-dependent necrosis is also triggered by the
cytosolic DNA sensor, DNA-dependent activator of interferon (DAI)
regulatory factors, following virus infection and the presence, in the cytosol,
of double-stranded viral DNA.
Here we are concerned with signaling leading to cell death in vertebrates,
and focus on processes that are at least partially understood at the molecular
level. We do not discuss the physiology and pathology of cell death in detail
or processes involved in the clearance of dying cells. Readers will find a
more complete overview of these topics and other aspects of cell death
elsewhere (Green 2011b).

2 TYPE I CELL DEATH: APOPTOSIS

Apoptotic cell death is predominantly initiated either by the DR or
mitochondrial pathway, although additional pathways exist. DRs—for
example, Fas (also known as CD95), Trail receptor (TRAIL-R), or TNFR1—
induce apoptosis by directly recruiting a caspase-activation platform upon
binding to their respective ligand. The mitochondrial pathway of apoptosis,
on the other hand, is triggered upon loss of integrity of the mitochondrial
outer membrane, which allows the release of proapoptotic factors (e.g.,
cytochrome c) from the mitochondria into the cytosol. This process is
controlled by the Bcl2 protein family. Once in the cytosol, cytochrome c
induces the assembly of a caspase-activation complex: the apoptosome. Both
pathways culminate in the activation of caspase proteases and the cleavage of
intracellular proteins, ultimately leading to the dismantling of the cell. Below,
we explore these processes in detail, starting with the end point: caspase
activation.

2.1 Caspase Activation, Function, and Regulation

Apoptosis involves the activation of caspases, which orchestrate all of the
morphological changes that characterize this form of cell death. Caspases are
cysteine proteases with specificity for aspartic acid residues in their
substrates. Although at least 17 different caspases exist in mammals, our
focus is on only a subset of these for which the activation is at least partially
understood and roles in cell death have been established.
The executioner caspases (caspase-3, caspase-6, and caspase-7) effect the
destruction and are produced as inactive dimers that lack protein-interaction
domains (Fig. 1). Activation is due to proteolytic cleavage between what will
be the large and small subunits of the mature enzyme (Salvesen and Riedl
2008). Upon cleavage, the new ends fold into the dimer interface and
promote conformational changes to create two active sites in the mature
protease. Cleavage of caspase-6 is mediated by caspase-3 and caspase-7 (Slee
et al. 1999), whereas activation of the latter two caspases is generally the
function of “initiator” caspases. It is these initiator caspases, and their
activation, that define the apoptotic signaling pathways (see below).

Figure 1. The caspase protein family. Initiator caspases (caspase-2, caspase-8, and caspase-9) are the
apical caspases of the apoptotic-signaling cascade. Initiator caspases are produced as inactive zymogens
composed of a prodomain (containing a CARD or a death effector domain [DED]) and a large and
small subunit. They are recruited through their prodomains into large activation platforms and activated
by dimerization. In contrast, executioner caspases (caspase-3, caspase-6, and caspase-7) are activated
by cleavage of the zymogen between the large and small subunits and are therefore dependent on
initiator caspases for their activation. Catalytically active caspases are composed of a heterotetramer of
two small and two large subunits.

Following activation, the executioner caspases, particularly caspase-3 and

caspase-7, can process at least 1000 proteins (Crawford and Wells 2011). The
cleavage of these caspase substrates can result in either a gain or a loss of
function of these proteins and eventually leads to the cellular changes
associated with apoptosis. Notably, caspase proteolysis inactivates
components of essential physiological processes. For example, caspase
cleavage of the p75 subunit of complex I of the electron transport chain
disrupts the mitochondrial transmembrane potential, electron transport, and
ATP production during apoptosis (Ricci et al. 2004). Conversely, caspase
cleavage can also activate specific pathways. This is the case for caspase-
activated nuclease (CAD), a DNAse that cuts chromatin between
nucleosomes when its inhibitor, iCAD, is cleaved by caspase-3 (Enari et al.
1998; Sakahira et al. 1998). Additionally, caspases can hijack signaling
pathways through constitutive activation of some of their components. For
example, the characteristic morphology of apoptotic cells is caused by
caspase-mediated activation of several actin cytoskeleton modulators:
gelsolin, p21-activated kinase 2 (PAK2), and Rho-associated kinase I
(ROCKI). Gelsolin, a calcium-regulated actin-severing protein, becomes
constitutively active following caspase processing (Kothakota et al. 1997).
PAK2 and ROCK I are serine–threonine kinase effectors of the Rho GTPase
family (Rac1, Cdc42, and Rho) that regulate actin polymerization and actin–
myosin contractility. Upon caspase cleavage, their kinase activity becomes
independent of the GTPases, thus inducing an aberrant reorganization of the
actin cytoskeleton and the characteristic membrane blebbing observed in
apoptotic cells (Rudel and Bokoch 1997; Coleman et al. 2001; Sebbagh et al.
2001) (for more details on cytoskeleton-modulating proteins, see Ch. 8
[Devreotes and Horwitz 2014]).
Unlike the executioner caspases, initiator caspases (Fig. 1) exist as
inactive monomers in cells and are not activated by cleavage. Instead, adaptor
molecules that assemble into caspase-activation platforms recruit these
initiator caspases, forcing monomers into close proximity and causing
conformational changes that result in the formation of active sites. In some
(but not all) cases, subsequent autocleavage of the caspase is necessary to
stabilize the mature, active enzyme (Pop et al. 2007; Oberst et al. 2010). Note
that although cleavage of an executioner caspase is indicative of its
activation, that of initiator caspases is not necessarily an indication of
activation (McStay et al. 2008).
The interactions between caspase-activation platforms and caspases
involve “death fold” domains of the proteins (Kersse et al. 2011). Such death
fold elements are present in adaptor proteins and caspases: the caspase-
recruitment domain (CARD), and the death effector domain (DED). Other
death folds, the death domain (DD) and the pyrin (PYR) domain, are
involved in the assembly of some caspase-activation platforms but not
present on caspases (Kersse et al. 2011). Death fold domains typically
mediate protein–protein interaction through homotypic interaction. They do
not share sequence similarity but have similar structures composed of six
amphipathic α helices.

2.2 Caspase-9 Activation: The Mitochondrial Pathway of

Apoptosis
The mitochondrial pathway of apoptosis, also called the intrinsic pathway, is
the most common mechanism of apoptosis in vertebrates. It is activated in
response to a variety of cellular stresses, including DNA damage, growth
factor deprivation, ER stress, and developmental cues. In this pathway, the
executioner caspases are cleaved and activated by caspase-9, which is itself
activated by a caspase-activation platform called the apoptosome (Fig. 2)
(Bratton et al. 2001; Bratton and Salvesen 2010).
Figure 2. The mitochondrial apoptotic pathway. In response to various cellular stresses, proapoptotic
members of the Bcl2 family induce mitochondrial outer membrane permeabilization (MOMP),
allowing the release into the cytosol of proapoptotic factors that are normally sequestered in the
intermembrane space of the mitochondria (including cytochrome c, Smac, and Omi). In the cytosol,
cytochrome c binds to APAF1 and triggers its oligomerization. Caspase-9 is then recruited and
activated by this platform, known as the apoptosome. Catalytically active caspase-9 cleaves and
activates the executioner caspases-3 and -7. Upon release in the cytosol, Smac and Omi bind to and
inhibit the caspase inhibitor X-linked inhibitor of apoptosis (XIAP). In doing so, they relieve XIAP
inhibition of caspase-9, caspase-3, and caspase-7, and potentiate overall caspase activation by the
apoptosome.

Apoptotic protease-activating factor 1 (APAF1) constitutes the scaffold

around which the apoptosome is assembled. During intrinsic apoptosis,
cytochrome c is released from mitochondria into the cytosol (see below) and
binds to Apaf1 (Zou et al. 1997). This interaction triggers hydrolysis of the
Apaf1 cofactor dATP to dADP (Kim et al. 2005). The subsequent exchange
of dADP with exogenous dATP allows the oligomerization of seven APAF1–
dATP–cytochrome-c units into an active apoptosome. At the center of the
apoptosome, exposed CARDs on APAF1 bind to the CARD of caspase-9,
thus bringing the inactive caspase-9 monomers into close proximity for
activation and autoprocessing (Yu et al. 2005; Yuan et al. 2010). Owing to its
higher affinity for the apoptosome, full-length caspase-9 displaces the
processed form, creating a continuous cycle of caspase-9 recruitment,
activation, processing, and release (Malladi et al. 2009). Because caspase-9
only sustains catalytic activity in this bound state (Rodriguez and Lazebnik
1999; Stennicke et al. 1999; Bratton et al. 2001), the apoptosome functions as
a molecular timer in which its lifetime is directly proportional to the amount
of unprocessed caspase-9 present (Malladi et al. 2009).
In healthy cells, cytochrome c is found only in the mitochondrial
intermembrane space. For it to interact with APAF1, mitochondrial outer
membrane permeabilization (MOMP) triggered by apoptotic stimuli must
occur (Tait and Green 2010). MOMP induces the release of all soluble
proteins of the mitochondrial intermembrane space into the cytosol. In
addition to cytochrome c, two other proapoptotic proteins are released during
that process: Smac (also known as Diablo) and Omi (also known as HtrA2).
Smac and Omi potentiate the apoptosome activity by antagonizing the
caspase inhibitor X-linked inhibitor of apoptosis (XIAP) (Fig. 2). In the
absence of Smac and Omi, XIAP binds to, and inhibits the catalytic activity
of, the initiator caspase-9 and the executioner caspases-3 and -7. XIAP
further dampens intrinsic apoptosis induction through direct ubiquitylation
and proteasomal degradation of active caspases (Eckelman et al. 2006).
MOMP is a tightly regulated event controlled by members of the Bcl2
family. These share one or more Bcl2 homology (BH) regions defined by
sequence, structure, and function (Fig. 3A). There are three broad classes of
Bcl2 proteins: the proapoptotic effector proteins (Bax and Bak), which are
necessary and sufficient for MOMP; the antiapoptotic Bcl2 proteins (e.g.,
Bcl2, Bcl-xL, and Mcl1), which block MOMP; and the BH3-only proteins
(e.g., Bid, Bim, Bad, and Noxa), which activate the proapoptotic effectors
and/or neutralize the antiapoptotic Bcl2 proteins. Bcl2 proteins also control
several other cellular processes, including mitochondrial fusion, autophagy,
and calcium efflux from the ER (Chipuk et al. 2010).

Figure 3. Regulation of mitochondrial outer membrane integrity by the Bcl2 protein family. (A)
Members of the Bcl2 protein family are characterized by the presence of one or more Bcl2 homology
(BH) region. The antiapoptotic Bcl2 proteins (e.g., Bcl2, Bcl-xL, and Mcl1) and the proapoptotic
effectors (e.g., Bax and Bak) share four BH regions and a similar globular structure. BH3-only proteins
(e.g., Bid, Bim, Bad, and Noxa) are characterized by a single BH region (BH3). (B) The proapoptotic
effectors reside in cells in inactive forms tethered to the outer mitochondrial membrane (Bak) or soluble
in the cytosol (Bax). Upon activation, Bax and Bak oligomerize and further insert into the membrane,
causing mitochondrial outer membrane permeabilization (MOMP) and thus apoptosis. (C) Bax/Bak
activation is triggered following the transient binding of a subset of direct activator BH3-only proteins
(e.g., Bid and Bim). Antiapoptotic Bcl2 proteins inhibit MOMP by sequestering the direct activator
proteins and/or the effectors. Another group of BH3-only proteins, called sensitizers or derepressors
(e.g., Bad and Noxa) promote MOMP by antagonizing antiapoptotic Bcl2 proteins, thereby releasing
both direct activator BH3-only proteins and Bax/Bak.

Bax and Bak are directly responsible for the loss of mitochondrial outer
membrane integrity (Fig. 3B). Upon activation, they form large oligomers
that insert into the mitochondrial outer membrane, disrupting it (Eskes et al.
2000; Korsmeyer et al. 2000; Dewson et al. 2008, 2009). The precise nature
of the disruption remains unclear, but it allows the near simultaneous release
of all intermembrane space proteins (Goldstein et al. 2000; Munoz-Pinedo et
al. 2006).
Bax and Bak act redundantly in MOMP and at least one of them is
required to permeabilize mitochondria. In living cells these proteins are
generally inactive, but become activated in response to upstream events. At
least two of the BH3-only proteins (Bim and active Bid) activate Bax and
Bak through transient interaction (Fig. 3C), although other conditions (such
as heat, changes in pH, and changes in the lipid milieu) may activate the
effectors independently of BH3-only proteins (Wei et al. 2000; Kuwana et al.
2002, 2005; Letai et al. 2002).
MOMP is antagonized by the antiapoptotic Bcl2 proteins, which bind to
and inhibit both Bax/Bak and the BH3-only proteins by interacting with their
BH3 domains (Llambi et al. 2011) (Fig. 3C). Antiapoptotic Bcl2 proteins are
also regulated at both the transcriptional and posttranslational levels. In
particular, Mcl1 degradation by the ubiquitin-proteasome system participates
in apoptosis induction following several cellular stresses. Upon DNA
damage, the BH3-domain-containing protein Mcl1 ubiquitin ligase E3
(MULE) directly binds to Mcl1 and catalyzes its ubiquitylation and
degradation (Warr et al. 2005). During antitubulin chemotherapeutic-induced
mitotic arrest, Mcl1 is phosphorylated by stress-activated and mitotic kinases
such as the MAP kinase (MAPK) Jun amino-terminal kinase (JNK), casein
kinase II (CKII), and p38 MAPK. These phosphorylation events unveil a
degron on Mcl1 that recruits the SCF-Fbw7 ubiquitin ligase complex, thus
targeting Mcl1 for ubiquitylation and degradation (Wertz et al. 2011).
Similarly, Mcl1 is degraded following growth factor withdrawal (e.g., IL3)
and the subsequent loss of phosphoinositide 3-kinase (PI3K)-Akt signaling.
This process relieves glycogen synthase kinase 3 (GSK3) from Akt
inhibition. GSK3 then phosphorylates Mcl1, allowing its ubiquitylation by
the E3 ligase β-transductin-repeat-containing protein (β-TrCP) and its
subsequent degradation (Maurer et al. 2006; Ding et al. 2007). Conversely,
cancer cells can increase Mcl1 stability and overall resistance to cellular
stress by expressing USP9X, a deubiquitylase that removes polyubiquitin
chains from Mcl1 (Schwickart et al. 2010).
Proteins of the Bcl2 family integrate pro- and antiapoptotic signals in
healthy and stressed cells and therefore constitute one of the main signaling
nodes in the life or death decision. Within this family, BH3-only proteins
constitute the main upstream sensors of the mitochondrial apoptotic pathway.
A wide variety of signaling pathways converge on the BH3-only family of
proteins and regulate their expression level and activity both transcriptionally
and posttranscriptionally (see Table 1). For example, Bid is activated upon
cleavage by caspase-8 following DR ligation (Li et al. 1998, Luo et al. 1998).
In doing so, Bid coordinates the cross-regulation between the extrinsic and
intrinsic apoptotic pathways. Additionally, proteolytic activation of Bid can
be achieved by granzyme B during the cytotoxic lymphocyte killing process
(Heibein et al. 2000; Sutton et al. 2000). The apoptotic response to genotoxic
stress is performed, in part, by Puma and Noxa (Jeffers et al. 2003; Villunger
et al. 2003). Both are direct transcriptional targets of the tumor suppressor
p53 (Oda et al. 2000; Nakano and Vousden 2001; Yu et al. 2003), although
other stimuli regulate their expression levels (see Table 1). Similarly, Bim is
transcriptionally up-regulated by the forkhead transcription factor FOXO3A
upon cytokine deprivation (Dijkers et al. 2000; Gilley et al. 2003; Urbich et
al. 2005). Bim is also a major apoptotic factor of the ER stress pathway
engaged in response to accumulation of unfolded proteins (Puthalakath et al.
2007). Finally, Bad activity is negatively regulated through phosphorylation
by several kinases, such as Akt (Datta et al. 1997; del Peso et al. 1997),
which induces its sequestration by 14-3-3 proteins. Upon growth factor
deprivation and loss of Akt signaling, Bad is released and antagonizes
antiapoptotic Bcl2 proteins (Zha et al. 1996; Datta et al. 1997).

Table 1. Signaling to BH3-only proteins

BH3-
only Signaling
protein Stimulus/input Type of regulation pathway Output References
Bad Growth factors and Phosphorylation Akt Cell survival Datta et al. 1997, 2002; del Peso
cytokines et al. 1997
 Phosphorylation p70 S6K Cell survival Harada et al. 2001
Phosphorylation PKA Cell survival Harada et al. 1999
Phosphorylation PIM kinases Cell survival Fox et al. 2003; Yan et al. 2003
Phosphorylation PAK Cell survival Schürmann et al. 2000; Cotteret
et al. 2003
Sequestration 14-3-3 Cell survival Zha et al. 1996; Datta et al. 2000
TNF Phosphorylation IKK Cell survival Yan et al. 2013
Neuronal activity Phosphorylation Cdc2 Apoptosis Konishi et al. 2002
deprivation
Growth factor or Phosphorylation JNK Apoptosis Donovan et al. 2002
cytokine deprivation Dephosphorylation PP1, PP2A, Apoptosis Ayllón et al. 2000; Chiang et al.
and PP2C 2001; Klumpp et al. 2003
Calcium Dephosphorylation Calcineurin Apoptosis Wang et al. 1999
Bid Cytotoxic T cell Cleavage Granzyme B Apoptosis Heibein et al. 2000; Sutton et al.
2000
Fas/TNF/TRAIL Cleavage Caspase-8 Apoptosis Li et al. 1998; Luo et al. 1998
Heat shock or ER Cleavage Caspase-2 Apoptosis Bonzon et al. 2006; Upton et al.
stress 2008
Ischemia or cisplatin Cleavage Calpain Apoptosis Chen et al. 2001; Mandic et al.
2002
Lysosome Cleavage Cathepsin Apoptosis Stoka et al. 2001; Reiners et al.
permeabilization 2002
Bim Growth factors or Phosphorylation ERK1/2 and Cell survival Ley et al. 2003; Hubner et al.
cytokines RSK1/2 2008; Dehan et al. 2009
Ubiquitylation βTrCP Cell survival Akiyama et al. 2003; Ley et al.
2003; Dehan et al. 2009
mRNA stability Hsc70 Cell survival Matsui et al. 2007
? mRNA stability miR-17-92 Cell survival Xiao et al. 2008
ER stress Transcription CHOP- Apoptosis Puthalakath et al. 2007
C/EBPa
Dephosphorylation PP2A Apoptosis Puthalakath et al. 2007
Glucocorticoid Transcription GR Apoptosis Erlacher et al. 2005; Ploner et al.
2008
Growth factor Phosphorylation JNK Apoptosis Lei and Davis 2003; Putcha et al.
deprivation or DNA 2003
damage
Growth factor or Transcription FOXO3a Apoptosis Dijkers et al. 2000; Gilley et al.
cytokine deprivation 2003
Noxa DNA damage Transcription p53 Apoptosis Oda et al. 2000; Villunger et al.
2003; Naik et al. 2007
DNA damage Transcription p63 Apoptosis Kerr et al. 2012
DNA damage Deubiquitylation UCH-L1 Apoptosis Brinkmann et al. 2013
Glucose deprivation Transcription ? Apoptosis Alves et al. 2006
Hypoxia Transcription HIF1α Apoptosis Kim et al. 2004
Proteasome inhibition Transcription/protein Myc, others Apoptosis Fernandez et al. 2005; Qin et al.
stabilization 2005; Nikiforov et al. 2007
Puma Cytokine deprivation Transcription FOXO3a Apoptosis Jeffers et al. 2003; Villunger et al.
2003; You et al. 2006; Ekoff et
al. 2007
DNA damage Transcription p53 Apoptosis Nakano and Vousden 2001; Yu et
al. 2001; Jeffers et al. 2003;
Villunger et al. 2003
DNA damage or ER Transcription p63 Apoptosis Pyati et al. 2011; Kerr et al. 2012
 stress
Glucocorticoid Transcription GR Apoptosis Villunger et al. 2003; Erlacher et
al. 2005
BIK DNA damage Transcription Smad Proapoptotic Spender 2009
TGFβ Transcription E2F1 Proapoptotic Real 2006
Ubiquitylation? Proapoptotic Nikrad 2005; Zhu 2005
Proteasome inhibition Cleavage RHBDD1 Antiapoptotic Wang et al. 2008a
Phosphorylation Casein Proapoptotic Verma 2001; Li 2003
kinase
Transcription Smad Proapoptotic Ramjaun 2007
BMF TGFβ Sequestration DLC2 Antiapoptotic Puthalakath 2001
Transcription HIFα Proapoptotic Bruick 2000; Guo 2001
BNIP3 Hypoxia Transcription E2F1 Proapoptotic Yurkova 2008
Transcription NF-κB Antiapoptotic Shaw 2006, 2008
inhibition
Transcription Jun Proapoptotic Ma 2007
HRK Potassium deprivation Transcription E2F1 Proapoptotic Hershko 2004
Transcription DREAM Antiapoptotic Sanz 2001
inhibition
Cytokines Transcription HIFα Proapoptotic Sowter 2001
NIX Hypoxia

MOMP often condemns a cell to death even if caspase activation is

blocked or disrupted (Tait and Green 2010). This is probably the
consequence of a catastrophic loss of mitochondrial function that results in
bioenergetic failure (Lartigue et al. 2009). Under these conditions, some cells
can recover from MOMP, however, and survive (Tait et al. 2010). Therefore,
the regulation of caspase activation downstream from MOMP may be
important in some settings. For example following cytochrome c binding to
APAF1, the failure to replace hydrolyzed dADP by exogenous dATP triggers
nonfunctional aggregation and irreversible inactivation of APAF1 (Kim et al.
2005). Nucleotide exchange, and therefore functional apoptosome assembly,
is facilitated by a complex composed of heat shock protein 70 (Hsp70),
cellular apoptosis susceptibility (CAS) protein, and putative HLA-DR-
associated protein I (PHAPI) (Kim et al. 2008). Accordingly, cellular levels
of these proteins modulate caspase-activation efficiency during intrinsic
apoptosis. tRNA levels also regulate apoptosome activity by binding to
cytochrome c, blocking its interaction with APAF1 (Mei et al. 2010). Finally,
intrinsic apoptosis can be perturbed downstream from MOMP through
phosphorylation and inhibition of caspase-9 (see below) (Allan and Clarke
2009).
2.3 Caspase-8 Activation: The Death Receptor Pathway
Caspase-8 is activated predominantly by the DR pathway of vertebrate
apoptosis. DRs are a subset of the TNFR superfamily and include TNFR1,
Fas, and TRAIL-R1/2 (Dickens et al. 2012). They contain a DD in their
intracellular regions. Through a series of homotypic interactions, these
initiate the assembly of large macromolecular complexes that recruit and
activate caspase-8 for apoptotic signaling, as well as other signaling
molecules that control processes such as inflammation and cell adhesion (Ch.
15 [Newton and Dixit 2012]).
When Fas or a TRAIL-R is bound by its ligand, clustering of the receptors
recruits a DD-containing adaptor molecule, FADD, through DD-DD
interactions (Fig. 4A). This exposes another death fold domain in FADD, its
DED. The complex represents a caspase-activation platform called a death-
inducing signaling complex (DISC), as the FADD DEDs bind to DEDs in the
prodomain of caspase-8. This brings caspase-8 monomers into close
proximity, triggering their protease activity. Caspase-8 then undergoes
autocatalytic cleavage both between the large and small subunits and between
the large subunit and the prodomain. The first cleavage stabilizes the active
dimer (and is required for homodimer activity in this pathway); the second
cleavage releases it from the DISC (Dickens et al. 2012).
Figure 4. Death receptor signaling pathway. The death receptor signaling pathway is triggered by
ligation of a death receptor (TNFR1, Fas, or TRAIL-R1/2) and potentially leads to three different
outcomes: survival, apoptosis, or necrosis. (A) Upon ligation (by FasL and TRAIL, respectively),
Fas/CD95 and TRAIL-Rs assemble a caspase-activation platform called the DISC. This platform
recruits and activates caspase-8 via the adaptor protein FADD and thus engages the extrinsic apoptotic
pathway. (B) Upon ligation to TNF, TNFR1 recruits the adaptor protein TRADD and the kinase RIP1,
which in turn mobilizes additional partners, such as the ubiquitin ligases TRAF2 and cIAP1/2. These
catalyze the nondegradative ubiquitylation of RIP1 as well as other components of the protein complex,
resulting in the stabilization of a prosurvival and proinflammatory signaling platform called complex I.
(C) The removal of ubiquitin chains of RIP1 by inhibition of cIAP1/2 or through the action of
deubiquitylases destabilizes complex I, thus allowing the release of TRADD and RIP1. TRADD and
RIP1 then promote the formation of a series of cytosolic complexes (complex II) that can initiate either
apoptotic or necrotic cell death. In the proapoptotic configuration of the complex, TRADD and RIP1
recruit FADD and caspase-8, resulting in caspase activation and apoptosis. When FADD or caspase-8
are absent, or when caspase activity is blocked, RIP1 binds to and activates RIP3, thus triggering
necrotic cell death. Expression of FLICE-like inhibitory protein (FLIP) inhibits the activity of both
complexes. FLIP forms a heterodimer with caspase-8 that cannot induce apoptosis but still displays
catalytic activity, and this activity antagonizes the activation of RIP3. Therefore, complexes containing
FLIP–caspase-8 heterodimers simultaneously block apoptosis and RIP-dependent necrosis.

In some cells (called type I cells), active caspase-8 then promotes

apoptosis by cleaving and activating caspase-3 and caspase-7. However, in
many cell types (type II cells), the active executioner caspases are inhibited
by XIAP, and thus apoptosis is blocked (Jost et al. 2009). In these cases,
another caspase-8 substrate comes into play, the BH3-only protein Bid (see
above). Caspase-8-mediated cleavage activates Bid, which in turn activates
Bax and Bak to promote MOMP. The IAP antagonists Smac and Omi are
then released, and these neutralize XIAP to allow apoptosis to proceed.
The signaling pathways engaged downstream from TNFR1 are more
complex and potentially lead to three different outcomes: survival, apoptosis
or necrosis (Fig. 4B). Upon TNFR1 ligation, a different DD-containing
adaptor, TNFR-associated death domain protein (TRADD), is first recruited.
TRADD does not directly bind or activate caspase-8 but instead acts as a
membrane-bond scaffold for the recruitment of additional signaling
molecules, including a kinase, receptor-interacting protein 1 (RIP1), and the
ubiquitin ligases TRAF2 and cIAP1/2. Components of this complex,
including RIP1, are modified by a complex series of nondegradative
ubiquitylation events performed by both the cIAPs and the linear ubiquitin
assembly complex (LUBAC). The assembly and ubiquitylation of this
signaling platform (referred to as complex I) culminates in the activation of
the NF-κB signaling pathway and an inflammatory response rather than cell
death (Ch. 15 [Newton and Dixit 2012]).
TNFR1 ligation leads to cell death when ubiquitylation of RIP1 is
compromised either by inhibition of ubiquitin ligases contained in complex I
(e.g., cIAP1/2) or through direct de-ubiquitylation of RIP1 by enzymes such
as CYLD (and probably other signaling events). RIP1 and TRADD are then
released from TNFR1 to form a series of dynamic cytosolic signaling
platforms (collectively referred to as complex II) that have the potential to
cause both apoptotic and necrotic cell death (Fig. 4C). Cytosolic TRADD
recruits FADD (via DD-DD interaction), which in turn can bind to and
activate caspase-8 similarly to the DISC (Fig. 4C, left panel) (Christofferson
and Yuan 2010; Declercq et al. 2009). In this configuration, complex II
formation leads to apoptotic cell death. In many cases, however, apoptosis
does not proceed upon TNFR1 ligation, at least in part because the activation
of NF-κB induces the expression of the caspase-8-like protein FLICE-like
inhibitory protein (FLIP) (Micheau et al. 2001). FLIP has a domain structure
similar to that of caspase-8, but it lacks a catalytic cysteine. It preferentially
binds to FADD (via DED-DED interactions) and a monomer of caspase-8.
The caspase-8–FLIP heterodimer is catalytically active (Micheau et al. 2002;
Pop et al. 2011), but does not promote apoptosis. Why this is remains
obscure, but may relate to more rapid degradation of the complex (Geserick
et al. 2009). Consequently, the assembly of complex II under conditions of
active NF-κB signaling, and therefore FLIP expression, leads to a survival
outcome (Fig. 4C, middle panel). Finally, complex II formation can engage a
necrotic form of cell death (see section below). As we will see below, the
activity of the caspase-8–FLIP heterodimer is also important for preventing
this necrotic cell death signaling engaged by ligated TNFR1.

2.4 Caspase -1 and Caspase-5/-11 Activation: The

Inflammasome Pathway
Caspase-1 and human caspase-5 (caspase-11 in rodents) have large
prodomains containing CARDs. These are engaged by caspase-activation
platforms called inflammasomes. Generally these platforms form in response
to infectious agents (e.g., viruses, bacteria, and fungi) and inert substances
that induce inflammation (e.g., uric acid crystals, calcium phosphate crystals,
alum, asbestos) (Franchi et al. 2012).
The platforms that engage caspase-1 are established by activated NOD-
like receptors (NLRs), which bear either a CARD or, more often, a PYR
domain (Fig. 5). In most cases, the NLR engages the adaptor molecule ASC
via PYR–PYR interactions. ASC also contains a CARD that binds to and
activates caspase-1 (Martinon et al. 2002; Faustin et al. 2007; Franchi et al.
2009).
Figure 5. The inflammasomes. The inflammasomes are caspase-activation platforms that assemble in
response to infectious agents (e.g., viruses, bacteria, and fungi) and inert substances that induce
inflammation (e.g., uric acid crystals, calcium phosphate crystals, alum, asbestos). They recruit and
activate the inflammatory caspase-1 and -11. The caspase-1 activation platforms are supported by the
NOD-like receptors (NLRs; e.g., NLRP3 and NLRC4) and AIM. Upon activation by inflammatory
agents, these proteins recruit the adaptor molecule ASC and caspase-1. The subsequent activation of
caspase-1 induces cleavage and secretion of two inflammatory cytokines, interleukin (IL) 1β and IL18.

Another caspase-1 inflammasome involves the sensor AIM2, which binds

to cytosolic DNA (e.g., from viruses) and also engages ASC (Fig. 5)
(Hornung et al. 2009). Caspase-1 cleaves and promotes the secretion of two
inflammatory cytokines, interleukin (IL) 1β and IL18. It can also cleave and
activate Bid, as well as caspase-3 and caspase-7 to promote apoptosis. This
form of caspase-1-mediated cell death is sometimes called pyroptosis
(Brennan and Cookson 2000; Cookson and Brennan 2001; Fink and Cookson
2006; Bergsbaken and Cookson 2007).
Little is known about the activation platform for caspase -5/-11, except
that it does not involve ASC or the NLRs that function in caspase-1
activation (Kayagaki et al. 2011). Nevertheless, caspase-11 activation can
result in apoptosis (Kayagaki et al. 2011). Caspase-11 can probably also bind
to and activate caspase-1 (Green 2011a). In addition, caspase-11, but not
caspase-1, is involved in the lethal effects of bacterial lipolysaccharides in
mice in vivo (endotoxemia) (Kayagaki et al. 2011), which may also involve
one of the executioner caspases, caspase-7 (Lamkanfi et al. 2009).

2.5 Caspase-2 Activation: The PIDDosome Pathway

The functions of caspase-2 in mammalian apoptosis remain somewhat
obscure (Krumschnabel et al. 2009). Caspase-2 is activated in response to
heat shock, microtubule disruption and DNA damage (Bouchier-Hayes et al.
2009), and it has been implicated in apoptosis in oocytes (Nutt et al. 2005)
and degenerating neurons (Troy et al. 1997, 2000, 2001).
The activation platform for caspase-2 includes the adaptor molecule
RAIDD (Duan and Dixit 1997; Hofmann et al. 1997; Chou et al. 1998),
which bears a CARD that binds to the CARD in the prodomain of the
caspase. Like caspase-8, caspase-2 is activated by induced proximity,
following which intrachain autocleavage stabilizes the mature enzyme
(Baliga et al. 2004).
In addition to a CARD, RAIDD also has a DD. This binds to the DD-
containing protein PIDD (Lin et al. 2000; Telliez et al. 2000) to form what
has been called the PIDDosome (Tinel and Tschopp 2004). PIDD contains
two intein regions that promote its autocleavage. The first cleavage produces
PIDD-C, a molecule that functions in NF-κB activation (Janssens et al. 2005;
Tinel et al. 2007). The second generates PIDD-CC, which binds to RAIDD
(Tinel and Tschopp 2004; Tinel et al. 2007). However, caspase-2 activation
occurs in the absence of PIDD (Manzl et al. 2009; Ribe et al. 2012), and it is
not clear when or if PIDD is required (Manzl et al. 2012). The interaction of
caspase-2 with RAIDD is regulated by phosphorylation of the caspase (see
below).

2.6 Regulation of Caspase Activation by Kinases

Much of the regulation of apoptosis inevitably occurs upstream of caspase-
activation platforms. But other signaling events can modify caspases such
that, even when a suitable activation platform forms, their activation is
inhibited. XIAP and FLIP are two examples. Below we consider some others.
Regulation of caspase-2 activation is particularly well described. When
NADPH is plentiful in the cell (e.g., owing to the activity of the pentose
phosphate pathway) calcium/calmodulin-dependent protein kinase II
(CaMKII) phosphorylates its CARD (Nutt et al. 2005). This allows the
binding of 14-3-3ζ, which prevents association with the PIDDosome (Nutt et
al. 2009). If NADPH levels decrease, protein phosphatase 1 (PP1)
dephosphorylates caspase-2, permitting its activation by the PIDDosome
(Nutt et al. 2009). SIRT1-mediated acetylation of 14-3-3ζ inhibits its binding
to phospho-caspase-2, reinforcing this signal (Andersen et al. 2011).
Intriguingly, the Drosophila initiator caspase Dronc (see Box 1) is also
regulated by NADPH and phosphorylation by CaMKII (Yang et al. 2010; Ch.
6 [Rhind and Russel 2012]).

BOX 1. CASPASE ACTIVATION IN INVERTEBRATE

ORGANISMS

Homologs of mammalian components of the mitochondrial pathway

(e.g., caspases, APAF1 and Bcl2 proteins) are found throughout the
animal kingdom, including invertebrates. However, in neither of the two
invertebrates studied extensively—Drosophila and Caenorhabditis
elegans—has a role for the mitochondrial pathway involving MOMP
and cytochrome-c-mediated activation of the apoptosome been
unambiguously shown. Recent studies indicate that mitochondrial
pathway may exist in helminths (Lee et al. 2011; Bender et al. 2012),
and cytochrome c (Cyt c) triggers caspase activation in cytosolic extracts
of a helminth and several echinoderms (Bender et al. 2012). Therefore,
it is possible that the insect and nematode pathways outlined below are
derived from an ancestral mitochondrial pathway resembling that of
mammals (see the figure below, part A).
 In C. elegans, the single caspase involved in apoptosis, CED3,
contains a CARD and is activated by a platform composed of the
APAF1 ortholog CED4 (Yuan et al. 1993; Chinnaiyan et al. 1997a).
Monomeric CED4 is held inactive in living cells by the Bcl2 ortholog
CED9 (Chinnaiyan et al. 1997b; Seshagiri and Miller 1997; Spector et
al. 1997; Wu et al. 1997a,b). (Recall that this is not the case in
mammals, in which Bcl2 proteins do not sequester APAF1, but instead
restrict access to cytochrome c [Newmeyer et al. 2000].) In cells that are
triggered to undergo apoptosis, the BH3-only protein Egl1 binds to
CED9, releasing CED4, which then assembles in the caspase-activation
platform for CED3 (Conradt and Horvitz 1998; Yan et al. 2004). The
control of expression of Egl1 is the major way in which apoptosis in this
organism is regulated (see the figure above, part B).
In Drosophila, there are several initiator and executioner caspases.
The initiator caspase Dronc contains a CARD and is activated on a
platform composed of the APAF1 ortholog, Dark (Kanuka et al. 1999;
Rodriguez et al. 1999; Zhou et al. 1999). The Dark apoptosome appears
to be constitutively active (Yu et al. 2006) but the activity of Dronc is
 inhibited by an IAP, DIAP1 (Rodriguez et al. 2002). The expression of
one of several DIAP1 inhibitors, Reaper, Hid, Grim, or Sickle (Goyal et
al. 2000; Lisi et al. 2000; Yoo et al. 2002), leads to displacement of
DIAP1, allowing Dronc to cleave and thereby activate the executioner
caspases DCP1, drICE, and Decay. Therefore, the expression of the
DIAP1 inhibitors appears to directly control apoptosis in flies (see the
figure above, part C). The roles of MOMP and cytochrome c release in
Drosophila apoptosis are controversial. Although MOMP occurs, it
appears to be predominantly caspase dependent and therefore is
probably an effect rather than a cause of caspase activation.

The cell-cycle regulator cyclin-dependent kinase 1 (CDK1) bound to

cyclin B1 also phosphorylates caspase-2 (Andersen et al. 2009). In this case,
the phosphorylation is in the region between what will be the large and small
subunits of the active caspases, and appears to directly inhibit the generation
of the mature, stable enzyme.
Caspase-9 is phosphorylated on a threonine residue in the region between
the prodomain and the large subunit by several kinases such as the ERK2
MAPK and CDK1 (Allan and Clarke 2009). Inhibition of caspase-9 activity
by ERK2 occurs downstream from Ras in response to prosurvival stimuli
such as growth factor stimulation (Allan et al. 2003). CDK1–cyclin-B1
controls caspase-9 phosophorylation and activity during mitosis. This process
is thought to dampen the threshold for intrinsic apoptosis signals during the
cell cycle, especially upon prolonged mitotic arrest (Allan and Clarke 2007).
The mechanism by which this phosphorylation event inhibits caspase-9
activity remains obscure because it does not affect binding to APAF1.

3 TYPE II CELL DEATH AND AUTOPHAGY

3.1 The Autophagy Pathway
Macroautophagy, referred to here simply as autophagy, is a cellular process
that is highly conserved in eukaryotes.1 It is a catabolic process engaged
under metabolic stress (such as nutrient starvation and bioenergetics failure),
to ensure availability of critical metabolic intermediates. It is also important
for the removal of damaged organelles (including mitochondria), protein
aggregates, and infecting organisms (Levine and Kroemer 2008; Kroemer et
al. 2010).
Autophagy, in general, is a survival process. Its presence during so-called
autophagic (type II) cell death usually represents a failed attempt to overcome
lethal stress, and disruption of this process promotes rather than inhibits cell
death in many cases (but see below). This form of cell death is therefore often
referred to as caspase-independent cell death accompanied by autophagy
(Kroemer and Levine 2008; Shen et al. 2012).
The autophagy pathway culminates in the formation of a double
membrane structure, the autophagosome. This envelops intracellular material
and ultimately fuses with lysosomes allowing degradation of the enveloped
material (Fig. 6). Autophagosome formation and maturation is regulated by
the sequential function of multiple autophagy-related (ATG) proteins. The
process starts with the activation of a preinitiation complex composed of the
kinase Unc-51-like kinase 1 (ULK1), FIP200, and ATG13. The activity of
this kinase complex is directly regulated by two major metabolic
checkpoints: mammalian target of rapamycin complex 1 (mTORC1) and
AMP-activated protein kinase (AMPK) (see Ch. 14 [Hardie 2012]; p. 91
[Laplante and Sabatini 2012]). In healthy cells, mTORC1 inhibits the
preinitiation complex through phosphorylation of ULK1 and ATG13. Upon
metabolic stress (e.g., amino acid deprivation), mTORC1-mediated inhibition
of ULK1 and ATG13 is abolished (Ganley et al. 2009; Hosokawa et al. 2009;
Jung et al. 2009). In contrast, AMPK activates the preinitiation complex
(Egan et al. 2011; Kim et al. 2011). When ATP synthesis is unable to meet
the demands of ATP consumption in a cell, AMP and ADP accumulate and
activate AMPK. Active AMPK promotes autophagy indirectly through
suppression of mTORC1 activity and directly by phosphorylating and
activating ULK1. Therefore, conditions under which mTORC1 is inhibited
and/or AMPK is activated engage the activity of the preinitiation complex.
Figure 6. The autophagic signaling pathway. Under metabolic stress, AMPK activation and/or
mTORC1 inhibition lead to the activation of the preinitiation complex (ULK1, FIP200, and ATG13).
The latter activates the initiation complex (beclin 1, VPS15, and VPS34) that generates PI3P and
recruits ATG7 to the phagophore. ATG7 functions similarly to an E1-ubiquitin ligase and initiates two
conjugation pathways necessary for membrane elongation and closure of the autophagosome. In the
ATG5-ATG12 conjugation pathway, ATG12 is sequentially transferred to ATG7, ATG10, and ATG12.
The ATG5-12 conjugate recruits ATG16L and forms a complex necessary to stabilize the phagophore
and to complete the second conjugation pathway. In the LC3-PE pathway, LC3 is cleaved by ATG4
and sequentially conjugated to ATG7 and ATG3. The ATG5-ATG12-ATG16L complex carries out the
final step by transferring LC3 to PE to form an LC3-PE conjugate (also called LC3-II). LC3-II
associates with the autophagosomal membrane and is crucial for the targeting of autophagosomes to
lysosomes, as well as for the selective autophagy of organelles and protein aggregates.

The preinitiation complex recruits and activates an initiation complex

composed of beclin 1, a class III PI3K (Vps34), and the protein kinase
Vps15, which results in the generation of the lipid phosphatidylinositol 3-
phosphate (PI3P) (He and Levine 2010). The activity of the initiation
complex is negatively regulated by several independent signaling pathways.
Akt directly phosphorylates beclin 1, thus allowing it to bind to 14-3-3
(Wang et al. 2012). Beclin-1–14-3-3 complexes are sequestered by vimentin,
a component of intermediate filaments. This Akt-mediated cytoskeletal
sequestration dampens the lipid kinase activity of the initiation complex.
Therefore, growth factors and the PI3K-AKT pathway can inhibit autophagy
both directly through beclin 1 and indirectly through the mTOR pathway.
Beclin 1 is a scaffold protein that recruits several additional partners (in
addition to Vps34 and Vps15) that regulate the lipid kinases activity of the
preinitiation complex (for a more comprehensive review of beclin-1-
interacting proteins, see Kroemer et al. 2010). For example, beclin-1 binds to
autophagy/beclin-1 regulator 1 (AMBRA1), a protein that tethers the beclin-
1–Vps34 complex to microtubules through the dynein motor complex (Di
Bartolomeo et al. 2010; Fimia et al. 2012). Upon autophagy induction,
AMBRA1 is phosphorylated by ULK1 and released from the cytoskeleton to
allow autophagosome formation.
Interestingly, members of the Bcl2 family can regulate the initiation
complex. Beclin 1 contains a bona fide BH3 domain, which allows its
sequestration by antiapoptotic proteins such as Bcl2 and Bcl-xL (Pattingre et
al. 2005; Maiuri et al. 2007b; Oberstein et al. 2007). This dampens the
activity of the initiation complex. Under stress conditions (e.g., starvation or
hypoxia), the BH3-only proteins Bad and Bnip3 release beclin 1 to permit its
participation in the autophagic process (Maiuri et al. 2007a,b; Bellot et al.
2009). Alternatively, dissociation of beclin 1 from Bcl2 and Bcl-xL can be
achieved through phosphorylation of the beclin–1 BH3 domain by the death-
associated protein kinase (DAPK) (Zalckvar et al. 2009a,b). Finally, kinases
of the JNK family disrupt this complex by directly phosphorylating Bcl2,
thus decreasing its affinity for beclin 1 (Wei et al. 2008). This process is
essential for regulation of exercise-induced autophagy and glucose
metabolism in muscles (He et al. 2012).
The elongation and ultimate closure of the autophagosome is regulated by
two distinct but complementary ubiquitin-like protein conjugation systems:
the ATG5-12 and LC3-PE conjugation pathways (Fig. 6). Both pathways are
initiated by a single E1-ligase-like activating enzyme, ATG7, recruited to the
phagophore through PI3P generated by the initiation complex. First, a small
protein, ATG12, is covalently attached to the active site cysteine residue of
ATG7 by a thioester linkage. ATG12 is then transferred via thioester
exchange to an E2-like conjugating enzyme, ATG10, which further transfers
ATG12 to ATG5. Finally, the ATG5-ATG12 conjugate noncovalently
recruits ATG16L to form a large multimeric complex necessary to stabilize
the forming phagophore and to complete the second conjugation pathway
(Hanada et al. 2007). The LC3-PE conjugation pathway is initiated following
cleavage of LC3 by the cysteine protease ATG4. Cleaved LC3 binds to
ATG7 and is subsequently transferred to the E2 enzyme ATG3. The ATG5-
ATG12-ATG16L complex then acts as an E3 ligase, conjugating LC3 to
phosphatidylethanolamine (PE) to form an LC3-PE conjugate (also referred
to as LC3-II) (Hanada et al. 2007).
This lipidated, membrane-associated form, LC3-II, is crucial for the
targeting of autophagosomes to lysosomes. Once completed, the
autophagosome fuses with lysosomes to form an autolysosome, thus
degrading the engulfed material. This fusion event is achieved when
membranes from both organelles are brought into close proximity through
interaction between the autophagosomal protein syntaxin 17 (Stx17),
SNAP29, and VAMP8 on the lysosome (Itakura et al. 2012). Additionally,
two lysosomal proteins, LAMP1 and LAMP2, are required for the fusion
process, and in their absence the autophagosome fails to fuse to lysosomes
(Tanaka et al. 2000; Eskelinen et al. 2002, 2004).
During starvation (e.g., amino acid or serum deprivation), large portions
of the bulk cytosol are randomly engulfed and degraded by autophagy.
Additionally, autophagy can be a selective process and remove specific
subcellular structures (e.g., protein aggregates and damaged or excess
organelles) in the absence of metabolic stress. During selective autophagy,
substrate specificity is conferred by adaptor proteins that tether the targeted
structure to the nascent phagophore. Aggregates formed by unfolded proteins
are ubiquitylated and linked to the phagophore by proteins such as p62 and
neighbor of breast cancer 1 (NBR1) that bind both LC3 and ubiquitin (Shaid
et al. 2013). Similarly, autophagic removal of mitochondria (a process called
mitophagy) is controlled by mitochondrion-anchored LC3-binding proteins
such as NIP-like protein X (NIX, also called Bnip3L) and FUN14-domain-
containing 1 (FUNDC1). NIX, a Bcl2-family-related protein, promotes
removal of the entire mitochondrial pool during terminal erythrocyte
differentiation (Schweers et al. 2007; Sandoval et al. 2008), and FUNDC1
participates in hypoxia-induced mitochondrial clearance (Liu et al. 2012).
Additionally, damaged mitochondria that display low membrane potential are
also removed by mitophagy following ubiquitylation of mitochondrial
proteins by the ubiquitin ligase parkin (Narendra and Youle 2011), although
the exact mechanism by which the phagophore is recruited in that scenario is
not clear.
It is relatively easy to see how autophagy is engaged under conditions of
cellular stress and can ameliorate the stress to protect the cells. Two questions
persist: What kills the cells in type II cell death, and can autophagy itself ever
be a mechanism that promotes cell death? Currently, we do not have a
satisfactory answer to the first question but we are closer to answering the
second.

3.2 Can Autophagy Kill Cells?

Autophagy often accompanies type II cell death without participating in it.
There are cases, however, in which it appears to actively promote cell death.
This is best exemplified during Drosophila metamorphosis, in which
autophagy-dependent cell death drives the involution of the salivary gland
(Berry and Baehrecke 2007) and of the midgut (Denton et al. 2009). This
physiological form of cell death is triggered by the steroid hormone ecdysone
and occurs (in the salivary gland) following growth arrest and inhibition of
the PI3K signaling pathway. In both cases, mutation of any of several
essential ATG genes reduces these developmental cell deaths. Reciprocally,
forced expression of the ULK1 ortholog ATG1 is sufficient to drive this form
of cell death.
Autophagy-dependent cell death also occurs in mammalian cells
transformed with a constitutively active H-Ras (Elgendy et al. 2011). In this
setting, Ras-dependent cell death is inhibited if ATG5, ATG7, or beclin 1 are
silenced. Intriguingly, this form of autophagic cell death appears to be
regulated by Bcl2 proteins. In particular, displacement of beclin 1 from Mcl1
is thought to be triggered by the proapoptotic BH3-only protein Noxa. Again,
how autophagy components promote the death of the cell remains to be
elucidated.
Untransformed breast epithelial cells that lose attachment to a substrate
can engulf each other, resulting in the death of the engulfed cell by entosis
(Overholtzer et al. 2007). The engulfed cell undergoes apoptosis, which can
be inhibited by expression of Bcl2, but this does not rescue the cell.
However, if the engulfing cell lacks components of the autophagy pathway,
the engulfed cell can survive (Krajcovic et al. 2011). Recent studies have
shown that components of the autophagy pathway can be recruited to
phagosomes that have engulfed organisms or cells, facilitating the
degradation of the phagocytosed cargo (Sanjuan et al. 2007; Martinez et al.
2011). The preinitiation complex does not appear to be required. In the case
of entosis for example, FIP200 is dispensable (Krajcovic et al. 2011). Thus,
in these cases, autophagy may act as a “murder weapon” allowing one cell to
efficiently kill another.

4 TYPE III CELL DEATH: NECROSIS

4.1 Types of Necrotic Cell Death
Cellular necrosis or necrotic cell death encompasses a wide variety of cell
death processes with one common denominator: the loss of plasma
membrane integrity followed by cytoplasmic leakage (Yuan and Kroemer
2010). Necrosis can occur simply as a consequence of such extensive damage
that cell integrity is disrupted—for example, at high temperature, following
freeze-thaw, or upon mechanical stress. In such cases, cell death is passive
and does not require the activation of any particular signaling pathway. Note
that a necrotic morphology (i.e., rupture of the plasma membrane) can also be
observed at late stages of an apoptotic or autophagic cell death program,
when dead cells fail to be cleared from the system by phagocytosis. This
process is referred to as secondary necrosis and is independent of any other
signaling event than those initially engaged (apoptotic or autophagic).
However, necrotic cell death is not always an accidental or passive process
and can also be the result of a directed signaling cascade.
The best-characterized form of programmed necrosis is RIP-kinase-
dependent necrosis (also referred to as “necroptosis”) (Degterev et al. 2005).
This requires the kinase activity of RIP3 and can result in a rapid cell death
with the features of necrosis (Cho et al. 2009; He et al. 2009; Zhang et al.
2009). Necroptosis can be engaged downstream from TNFR1 (Schulze-
Osthoff et al. 1994; Vercammen et al. 1998; Holler et al. 2000; Matsumura et
al. 2000). Following TNFR1 ligation, RIP3 is activated through recruitment
to complex II by RIP1 (Fig. 4C). The interaction between RIP1 and RIP3 is
mediated by their respective RIP homotypic interaction motifs (RHIMs). In
this configuration, complex II induces necrosis rather than apoptosis and is
referred to as the RIPoptosome (Feoktistova et al. 2011a; Tenev et al. 2011).
The assembly and activation of the necrotic signaling platform are controlled
by a series of posttranslational modifications of components of this complex.
RIPoptosome formation is negatively regulated by ubiquitylation of RIP1.
Consequently, treatments with cIAP1/2 inhibitors or expression of the
deubiquitylase CYLD promote TNFR1-induced necrosis (Wang et al. 2008b;
O’Donnell et al. 2011; Moulin et al. 2012). Additionally, RIP3 activation
depends on the kinase activity of RIP1 (Cho et al. 2009; He et al. 2009;
Zhang et al. 2009), which can be inhibited by the drug necrostatin (Degterev
et al. 2008). Accordingly, RIP-dependent necrosis is blocked by necrostatin
(Degterev et al. 2005). In addition, necrosis induction by the RIPoptosome is
negatively regulated by the catalytic activity of the caspase-8–FLIP
heterodimers (see below).
RIP-dependent necrosis can be triggered by TLR3 and TLR4 (for more
details on TLR signaling pathways, see p. 121 [Lim and Staudt 2013]). TLR3
and TLR4 promote the assembly of the RIPoptosome by recruiting the
RHIM-containing protein TIR-domain-containing adaptor-inducing
interferon-β (TRIF) (Imtiyaz et al. 2006; Feoktistova et al. 2011b). TRIF
recruits RIP1 and RIP3 to induce RIP-dependent necrosis (Feoktistova et al.
2011a). TRIF might, however, recruit RIP3 independently of RIP1 through
direct RHIM–RHIM interaction. RIP-dependent necrosis can also be engaged
during viral infection (Cho et al. 2009; Upton et al. 2010) via the RHIM-
containing cytosolic DNA sensor, DNA-dependent activator of interferon
regulatory factors (DAI). DAI is activated by double-stranded viral DNA and
recruits RIP3 through its RHIM domain (Upton et al. 2012). Finally, DNA
damage signaling can also cause RIPoptosome formation independently of
any membrane receptor signaling (Tenev et al. 2011).
The mechanism by which RIP3 causes cell death remains obscure.
Several mechanisms have been proposed, including the generation of reactive
oxygen species (either mitochondrial or via NADPH oxidase) and elevation
of metabolic processes to deplete ATP and/or NADH, but the evidence for
them is not compelling (Hitomi et al. 2008). Recent studies have implicated
the pseudokinase mixed lineage kinase-like (MLKL) (Sun et al. 2012; Zhao
et al. 2012) as a critical player in the execution of RIP-dependent necrosis.
Phosphorylation by RIP3 triggers MLKL oligomerization and translocation
to the plasma membrane (Cai et al. 2014; Chen et al. 2014). The exact
mechanism by which MLKL impairs the plasma membrane integrity is not
known but is thought to involve a disruption of ionic homeostasis.

4.2 Control of RIPK-Dependent Necrosis by Caspases

Stimuli that activate RIP1 and the RIPoptosome (e.g., ligation of TNFR1,
ligation of TLR3/4, and possibly DNA damage) engage caspase-8 and FLIP.
The catalytic activity of caspase-8–FLIP heterodimers antagonizes activation
of RIP3 and the ensuing necrosis (Pop et al. 2011). As a result, cells that are
treated with TNF or TLR3/4 ligands do not generally undergo cell death.
However, if caspase-8–FLIP activity is blocked or disrupted, this can engage
necrosis in a manner that is dependent on both RIP1 and RIP3 (Cho et al.
2009; He et al. 2009; Zhang et al. 2009; Oberst et al. 2010). RIP1, RIP3, and
CYLD are substrates of caspase-8 (Lin et al. 1999; Feng et al. 2007; Lu et al.
2011; O’Donnell et al. 2011). However, in the absence of FLIP, activation of
caspase-8 is not sufficient to prevent RIPK-dependent necrosis, even if
apoptosis downstream from caspase-8 activation is blocked (Oberst et al.
2011).

4.3 Other Necrosis Signaling Pathways

Several other forms of necrosis signaling can occur in cells, although, like
RIP-dependent necrosis, none of these is fully characterized.
Ischemia/reperfusion injury (I/R), for example, causes cell death when a
tissue is transiently deprived of blood supply and is then restored. High levels
of calcium influx and generation of reactive oxygen species (ROS) have been
associated with this form of cell death (Halestrap and Pasdois 2009). These
trigger cell death, in part, by causing a mitochondrial permeability transition
(mPT), in which the mitochondrial inner membrane becomes permeable to
small solutes (Ricchelli et al. 2011). This results in a rapid dissipation of the
transmembrane potential, followed by matrix swelling and often rupture of
the mitochondrial outer membrane. Cyclophilin D (CypD), a peptidyl-proline
trans-isomerase, plays a major role in mPT. Mice lacking CypD display
defective mPTs (Baines et al. 2005; Basso et al. 2005; Nakagawa et al. 2005;
Schinzel et al. 2005) and display diminished tissue damage under conditions
of I/R (Baines et al. 2005; Nakagawa et al. 2005; Schinzel et al. 2005).
Importantly, mice lacking CypD have normal apoptosis (Baines et al. 2005;
Nakagawa et al. 2005; Schinzel et al. 2005) and RIP-dependent necrosis
(Ch’en et al. 2011).
Another form of active necrosis involves NADPH oxidase1 (Nox1) and
can be triggered by TNF (Kim et al. 2007). Nox1 generates a ROS burst that
may be important. There may be a relationship between Nox1 and RIP-
dependent necrosis (Vanden Berghe et al. 2007), but further evidence
supporting this connection is needed.
In neurons, engagement of glutamate receptors can trigger a form of
necrotic cell death called excitotoxicity (Wang and Qin 2010). This appears
to involve a calcium influx that triggers an ROS burst and also activates
calpain (Higuchi et al. 2005; Vosler et al. 2008). Excitotoxicity is associated
with I/R and may occur in other pathological conditions (Szydlowska and
Tymianski 2010).

5 BEYOND TYPE III—OTHER FORMS OF CELL DEATH

There are additional forms of cell death that appear to require the
participation of the cell in its demise. These include mitotic catastrophe
(Castedo et al. 2004), ferroptosis (Dixon et al. 2012), and others. Although
outside the scope of our discussion, a useful distinction might be made
between cellular suicide and cellular sabotage. Suicide mechanisms
(including apoptosis, necroptosis, and perhaps autophagic cell death, as
outlined above) represent signaling pathways that appear to have evolved to
execute the cell. In contrast, sabotage can be thought of as the consequence of
disruption of normal cellular processes that have not evolved for the purpose
of cell death. Much as a train must be moving if removal of a railroad tie (a
sabot) is to damage it, these forms of cell death occur when cellular
machinery confronts a disruption that results in cellular destruction. For
example, if chromosomes are cross-linked, mitosis may sufficiently damage
them that the cell dies, even if mechanisms of active cell suicide cannot be
engaged (mitotic catastrophe, although it is not clear that mitotic catastrophe
proceeds in this way [Castedo et al. 2004]).

6 CONCLUSION
Active or programmed cell death is essential to maintain homeostasis in
multicellular organisms as well as for the selective elimination of potentially
harmful or infected cells. Accordingly, deregulation of the signaling
pathways that trigger cell death can lead to the development of catastrophic
diseases such as cancer and autoimmunity (too little cell death) as well as
degenerative diseases (too much cell death). Therefore, the existence of
tightly controlled and efficient means to induce cell death can be interpreted
as the logical consequence of the evolution of multicellular organisms.
However, the necessity for so many different death-signaling pathways might
appear counterintuitive. Taken together, cell death induction could be viewed
as a simple signaling process with multiple inputs/stimuli and one outcome:
the death of the cell. However, the way by which a cell dies has important
consequences for neighboring cells and sometimes the entire organism. For
example, apoptotic and necrotic cells display divergent inflammatory
properties and trigger different immune responses. Additionally, particular
death programs include the release of proliferative signals that trigger
compensatory proliferation in surrounding tissues. These signals might differ
from one type of cell death to the next. Finally, death-signaling pathways are
clearly interconnected. For example, autophagic cell death is often
potentiated by caspase activation, whereas RIP-dependent necrosis is
antagonized by a caspase-dependent activity. Cross talk between these
pathways potentially provides numerous backup mechanisms for cell death
programs and could explain why inhibition of a single program often has
minor consequences for the organism. A better understanding of the impact
of each type of cell death on surrounding tissues and of the interplay between
these cell death programs might help to answer some of these questions.

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1Two other processes, microautophagy and chaperone-mediated autophagy, also exist, but are not
 further discussed here.

Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006080
CHAPTER 20

Subversion of Cell Signaling by

Pathogens

Neal M. Alto and Kim Orth

UT Southwestern Medical Center, Dallas, Texas 75390
Correspondence: kim.orth@utsouthwestern.edu

SUMMARY

Pathogens exploit several eukaryotic signaling pathways during an

infection. They have evolved specific effectors and toxins to hijack host
cell machinery for their own benefit. Signaling molecules are
preferentially targeted by pathogens because they globally regulate
many cellular processes. Both viruses and bacteria manipulate and
control pathways that regulate host cell survival and shape, including
MAPK signaling, G-protein signaling, signals controlling cytoskeletal
dynamics, and innate immune responses.

Outline
1 Introduction
2 Corruption of MAPK signaling
3 Manipulation of G-protein signaling
 4 Highjacking lipid signaling
5 Other actin regulators targeted by pathogens
6 Targeting ubiquitin-mediated signal transduction
7 Conclusion
References

1 INTRODUCTION
Viruses and bacteria both produce proteins that hijack cellular signaling
machinery to ensure their survival despite the constant negative pressure of
their hosts’ innate and adaptive immune systems. Studies of these have not
only revealed methods by which microbial pathogens cause infectious
disease, but also provided critical insights into mechanisms of cellular
regulation, particularly in the case of viral oncoproteins (see Box 1). They
also yielded valuable tools for dissecting mechanisms involved in regulating
signaling.

BOX 1. VIRAL ONCOPROTEINS

Studies of viruses have provided critical biological insights into the

signal transduction mechanisms of host cells. More than a century ago,
Peyton Rous described Rous sarcoma virus (RSV) as a transforming
agent that induces solid tumors in chickens. Although this discovery was
met with some skepticism at the time, researchers in the 1970s and
1980s isolated and sequenced the viral non-receptor tyrosine kinase-
encoding v-src gene as the genetic element that causes tumor formation.
In fact, v-src was the first retroviral oncogene discovered, and this
discovery helped define the first proto-oncogene in the vertebrate
genome (c-Src, now known simply as Src). Further investigations into
oncoviruses also revealed v-Abl, the retroviral oncoprotein from the
Abelson murine leukemia virus (A-MuLV). Like v-src, v-Abl encodes a
nonreceptor tyrosine kinase that is genetically and functionally related to
the ABL gene in mammals. In humans, the translocation involving
chromosomes 22 and 9 (known as the Philadelphia chromosome) causes
the first exon in ABL1 to be replaced by sequences from the BCR gene,
resulting in expression of the BCR–ABL fusion protein. This genetic
translocation results in abnormally high levels of BCR–ABL kinase
activity, leading to chronic myeloid leukemia (CML) and a subset of
acute lymphocytic leukemia (ALL). Biochemical and structural studies
led to one of the successful medicinal treatments for cancer: a specific,
small molecule kinase inhibitor referred to as imatinib or GLEEVAC.
Studies of the transforming activity of murine sarcoma viruses were
also particularly influential in stimulating work that elucidated small G-
protein signaling in human cancer. In the 1960s, the first Ras (for Rat
sarcoma) genes were identified as transduced oncogenes expressed by
the Harvey and Kirsten strains of acutely transforming murine
retroviruses. More than a decade later, researchers discovered that these
viral Ras proteins interface with guanine nucleotide signaling
mechanisms through an intrinsic GTPase activity. Several human genes
were subsequently found to display sequence homology to viral Ras.
The identification of RAS as the first human transforming gene set off a
global research initiative aimed at discovering the molecular
mechanisms of small G proteins.
Interestingly, the major downstream substrate of Ras signal
transduction is Raf kinase, encoded by the cellular homolog of the v-raf
oncogene expressed by the murine retrovirus 3611-MSV. Under normal
conditions, binding of extracellular ligands such as growth factors,
cytokines, and hormones to cell-surface receptors activates Ras, and this
initiates Raf activation. Indeed, both lie downstream from the EGF
receptor, which also has a viral oncogene homolog, v-erbB. Thus, not
only has research on viral oncogenes been an entry point into the
molecular genetics of cancer, but it has also revealed critical links
between infectious disease mechanisms and eukaryotic signal
transduction.

These proteins help a pathogen achieve its goals of survival, replication,

and virulence. For example, they may induce a slow cell death to allow time
for replication or cause a rapid cell death so that the pathogen avoids
engulfment. Alternatively, they may manipulate the actin cytoskeleton to
prevent or accelerate phagocytosis by the host. In the case of viral
oncoproteins, they may hijack the cell cycle machinery to promote virus
replication.
Here, we do not attempt to be comprehensive, but provide representative
examples of how pathogens manipulate host cell signaling, emphasizing the
virulence factors produced by bacterial pathogens (Table 1). Targets for these
virulence factors include GTPases and their regulators that control the
cytoskeleton and vesicular trafficking, kinase cascades involved in intra- and
extracellular signaling, and ubiquitin-dependent pathways that regulate signal
stability or dictate other outputs.

Table 1. Bacterial toxins and effectors and their targets

Pathogen Toxin Effector Target Activity
Vibrio cholerae Cholera toxin Gαs ADP ribosylation
Vibrio cholerae EF edema Calmodulin Adenylate cyclase
factor
Vibrio cholerae LF lethal factor MKK1,2 Metalloprotease
Bordetella pertussis Pertussus toxin Gαi ADP ribosylation
Clostridium botulinum C3 botulin Rho GTPases ADP ribosylation
toxin
Escherichia coli CNF1 Rho GTPases Deamination
EPEC/EHEC O157:H7 Tir Actin Recruits NCK adaptor
EPEC/EHEC O157:H7 Map Rho GTPases GEF
EPEC/EHEC O157:H7 EspFu N-WASP Activator of N-WASP
EPEC/EHEC O157:H7 EspG p21-activated kinase (PAK) Activator of PAK
EPEC/Burkholderia Cif/CBHP Ubiquitin, Nedd8 Ubiquitylation inhibitor
spp.
Yersinia spp. YopH p130Cas Tyrosine phosphatase
Yersinia spp. YopE Rho-like GTPases GAP
Yersinia spp. YopT Rho GTPase Cysteine protease
Yersinia spp. YpkA Gαq, Rho GTPases Ser/Thr kinase, GDI
Yersinia spp. YopJ MAPKKs, IKK-β Ser/Thr acetyltransferase
Vibrio VopA/p MAPKKs Ser/Thr/Lys acetyltransferase
parahaemolyticus
Vibrio VopS Rho-GTPases AMPylation
parahaemolyticus
Vibrio VPA0450 Phosphatidylinositol 4,5- Lipid phosphatase
parahaemolyticus bisphosphate
Vibrio VopL Actin Actin nucleator
parahaemolyticus
Histophilus somni IbpA Rho GTPases Ampylation
Legionella pneumophila DrrA/SidM Rab1b AMPylation, GEF
Legionella pneumophila SidD Rab1b DeAMPylation
Legionella pneumophila AnkX Rab1b Phosphocholination
Shigella spp. OspF MAPK Phosphothreonine lyase
Shigella spp. IpgD Phosphatidylinositol 4,5- Lipid phosphatase
bisphophate
Shigella spp. IpaH9.8 Ste7 MAPK E3 ubiquitin ligase
Shigella spp. IpgB2 Rac1 and RhoA GEF
Salmonella spp. SopB Phosphatidylinositol 4,5- Lipid phosphatase
bisphophate
Salmonella spp. SopE Cdc42 and Rac GTPases GEF
Salmonella spp. SptP Small GTPase GAP, tyrosine phosphatase
Pseudomonas ExoS Small GTPases ADP ribosylation, GAP
aeruginosa
Listeria monocytogenes ActA Arp2/3, actin Activator of Arp2/3
Viral effector
Vaccinia virus A36R Actin Adaptor recruits Nck and
Grb2
Adenovirus E1B-55K p53, Mre11, BLM helicase Ubiquitin ligase adaptor
Adenovirus E4orf6 p53, Mre11, BLM helicase Ubiquitin ligase adaptor
Papillomavirus E6 E6-AP, p53 E3 ubiquitin ligase
KSHV RTA IRF-7 E3 ubiquitin ligase
Gammaherpesviruses K3 and K5 MHC class 1 E3 ubiquitin ligase
Herpes simplex virus 1 ICP0 PML E3 ubiquitin ligase

Bacteria produce different types of virulence factors. Note that these are
not always proteins—they may also be peptides or small molecules—but here
we confine our discussion to proteins. The first type, called a toxin, is
secreted by the pathogen at high concentration and delivered into the host
cytoplasm by a variety of mechanisms, including endocytosis or via protein
pores formed by the bacterial toxin itself (Fig. 1) (Henkel et al. 2010).
Cholera toxin from Vibrio cholerae, for example, is an enzyme that enters the
cell by endocytosis and ADP-ribosylates the αs subunit of the heterotrimeric
Gs protein. This modification prevents αs from hydrolyzing GTP, locking it
into an active state that constitutively stimulates its downstream effector,
adenylyl cyclase (see p. 99 [Sassone-Corsi 2012]). In the intestinal
epithelium, the elevated levels of cyclic AMP (cAMP) generated cause an
efflux of chloride ions and water, resulting in severe diarrhea and
dehydration. Pertussis toxin produced by Bordetella pertussis, which causes
whooping cough, by contrast, ADP ribosylates the αi subunit of the
heterotrimeric Gi protein so that it remains in the GDP-bound state and
cannot be activated by upstream signals. The modified GDP-bound αi is no
longer able to bind to adenylyl cyclase and inhibit production of cAMP.
Pertussis toxin causes major trauma in airways during infection because
signaling pathways are constitutively activated and results in abnormally high
levels of insulin and histamine sensitivity. These toxins have proven very
useful for the elucidation of molecular signaling by G-protein-coupled
receptors (GPCRs) and their downstream signaling partners.

Figure 1. Bacterial secretion systems. Bacteria use several different mechanisms to secrete molecules
into the extracellular space and to translocate molecules into a host cell. Toxins, including peptides and
proteins, are typically secreted through a type I or II secretion system. Effectors are translocated
through a type III or IV secretion system, whereas DNA is only transferred through the type IV
secretion system.

Another type of virulence factor, called an effector, is a protein that is

directly translocated from bacteria into the host cell by specialized needle-
like delivery systems, including the type III and type IV secretion systems
(T3SS and T4SS, respectively) (Fig. 1) (Hayes et al. 2010). The effectors are
made in the bacterium but appear to be inactive because of association with a
chaperone, lack of appropriate substrate, and/or absence of eukaryotic
activators. After delivery into the eukaryotic host, the effectors display very
potent activities that manipulate signal transduction pathways. Many of these
activities mimic an endogenous activity of the host.
The first such effector analyzed at the molecular level was YopH
(Yersinia outer protein H) from Yersinia, the causal agent of the plague (also
known as the Black Death) (Guan and Dixon 1990). This protein contains an
unregulated, highly active tyrosine phosphatase domain linked to a leader
sequence that both guides its translocation from the bacterium into the host
cell and determines its localization after delivery. YopH is translocated into
eukaryotic cells through the Yersinia T3SS and proceeds to focal adhesions,
where it dephosphorylates critical phosphorylated tyrosine residues on
protein substrates including p130Cas and Fyb (see Ch. 8 [Devreotes and
Horwitz 2014]). The resulting dephosphorylated focal adhesion complex
disassembles and, therefore, is unable to promote phagocytosis of the
bacterial pathogen. YopH is a typical bacterial effector for the following
reasons.
1. Although an extremely active enzyme, YopH has no effect on the
pathogen itself, owing to the lack of a substrate.
2. The YopH protein contains information in its amino-terminal domain for
both secretion by the T3SS apparatus and localization in the infected host.
3. YopH contains a potent activity that efficiently targets and destroys the
Achilles heel of a process, in this case, phagocytosis, by targeting focal
adhesions that are regulated by phosphorylated tyrosine residues.
4. YopH plays a critical role in virulence.
Below, we describe several other pathogenic effectors that show these
general characteristics, breaking them down by the signaling pathways they
target.

2 CORRUPTION OF MAPK SIGNALING

Mitogen-activated protein kinase (MAPK) signaling pathways are cascades
of kinases that sequentially activate each other by phosphorylation (p. 81
[Morrison 2012]). A MAPK kinase kinase (MAPKKK) activates a MAPK
kinase (MAPKK), which, in turn, activates a MAPK, and there are multiple
family members at each stage. Pathogens target these signaling pathways
because they regulate many types of cellular behaviors, including cell
proliferation, innate immune responses, cell migration, apoptosis, and
autophagy.

2.1 Bacillus anthracis Lethal Factor Hydrolyzes the MAPKK

MKK1/2
Bacillus anthracis, the causal agent of anthrax, releases a multi-subunit
complex called anthrax toxin, composed of protective antigen (PA), edema
factor (EF), and lethal factor (LF) (Collier 2009). The toxin binds via PA to
either anthrax toxin receptor 1 or 2 on the surface of the host cell. After
uptake by receptor-mediated endocytosis and acidification of the resulting
endosome, EF and LF are released into the cytoplasm. The calcium-binding
protein calmodulin binds to cytoplasmic EF, causing a change in
conformation that generates an active enzyme that produces cAMP from
cellular ATP. The excess cAMP globally disrupts signaling by binding and
activating downstream effectors, such as cAMP-dependent kinase (PKA).
Cytoplasmic LF is an active metalloprotease that cleaves the amino-terminal
extensions from MAPKKs MKK1 and MKK2, producing kinases that can no
longer interact with their substrates to activate a proliferative response (Fig.
2). Both of the toxins have an irreversible toxic effect on the infected cell.
Figure 2. Corruption of MAPK and NFκB signaling pathways by bacterial effectors. Yersinia YopJ is
an acetyltransferase that acetylates and inhibits MAPKK and IKKβ activation by blocking
phosphorylation. Similarly, Vibrio VopA/P blocks activation of MAPKK. Lethal factor (LF) from B.
anthracis, the causal agent of anthrax, is a metalloprotease that inhibits MAPKK by proteolysis. OspF
is a phosphothreonine lyase that irreversibly dephosphorylates MAPK by elimination of a phosphate
group from activated MAPK.

2.2 Yersinia YopJ Acetylates MAPKK/IKKβ Activation Loop

Yersinia ssp. have a very efficient strategy for disrupting the innate immune
response and promoting apoptosis in infected cells, using one molecule, YopJ
(also termed YopP). This effector is injected directly into the host’s
cytoplasm through a T3SS. YopJ blocks all of the MAPK pathways and the
NF-κB pathway by preventing the activation of all MAPKKs and IKKβ (but
not IKKα) (Fig. 2) (Orth et al. 1999; Hao et al. 2008; p. 81 [Morrison 2012]
and p. 121 [Lim and Staudt 2013]). The activity of this 32-kDa effector
remained elusive for many years because it contains a catalytic triad similar
to that in some cysteine proteases, specifically clan CE proteases, which
include adenoviral proteases and ubiquitin-like protein proteases (Orth et al.
2000). However, a classical biochemical approach finally revealed that YopJ
does not cleave MAPKKs or any other substrate; instead, it modifies
MAPKKs with a small acetyl moiety. This acetyltransferase activity requires
an intact catalytic triad and uses acetyl-CoA to modify serine and/or
threonine residues in the activation loops of MAPKKs and IKKβ, generating
a novel posttranslational modification that competes with phosphorylation
(Mittal et al. 2006; Mukherjee et al. 2006).
These findings revealed a new paradigm for signaling, in which serine
and threonine residues could be substrates for acetylation (Mukherjee et al.
2007). So how is the catalytic triad of a presumed protease used for
acetylation? In fact, YopJ acetyltransferases and clan CE cysteine proteases
are both proposed to use the same catalytic “ping-pong” mechanism (Fig. 3).
Acetyltransferases containing a catalytic triad react with acetyl-CoA to form
a covalent acetyl-enzyme intermediate and release CoA. They then bind their
substrate and transfer the acetyl group to the substrate’s attacking
nucleophile. Cysteine proteases use the same mechanism to form a covalent
acyl-enzyme intermediate, release the carboxy-terminal peptide, and then use
a second substrate, water, to attack the acyl-enzyme covalent intermediate to
complete the hydrolase reaction. Acetyl transferases do not allow water in the
catalytic site; otherwise, these enzymes would simply hydrolyze the essential
metabolite acetyl-CoA.

Figure 3. Catalytic triads: same chemistry, different substrates. Yersinia YopJ is proposed to use a
ping-pong mechanism whereby its catalytic cysteine attacks acetyl-CoA to form a covalent acetyl-
enzyme intermediate, followed by a subsequent attack by its second substrate, a hydroxyl on MAPKK,
to transfer the acetyl group to MAPKK. A cysteine protease uses the same mechanism with a peptide to
form a covalent acetyl-enzyme intermediate. The second substrate in this reaction is water and leads to
cleavage of a peptide bond.
 During an infection, signals for host survival and apoptosis are induced.
In the presence of YopJ, the default pathway will always be death because
YopJ blocks the NF-κB survival pathway. By inhibiting signaling pathways
that alert the immune system and induce survival signals, YopJ attenuates the
immune response to Yersinia during infection. In contrast, VopA/P, a YopJ
relative from the seafood-borne pathogen Vibrio parahaemolyticus that
causes food poisoning, inhibits MAPK signaling pathways but not the NF-κB
pathway (Fig. 2) (Trosky et al. 2004). Additionally, VopA/P acetylates not
only the activation loop of MAPKKs but also a conserved lysine residue in
the catalytic loop of MAPKKs that is required for coordination of the γ-
phosphate of ATP (Trosky et al. 2007). This inhibits the binding of ATP but
not ADP, resulting in an inactive kinase. During infection by V.
parahaemolyticus, VopA/P efficiently blocks proliferative pathways while
allowing activation of survival pathways. Our understanding of this
infectious process is in its infancy; thus the activities of the other secreted
effectors will need to be uncovered to shed light on the importance of
VopA/P inhibition. Note that this type of serine/threonine acetylation has not
yet been observed as an endogenous protein modification in eukaryotes.

2.3 Shigella OspF Irreversibly Eliminates a Phosphate

Shigella, the causal agent of bloody dysentery, contains the T3SS effector
OspF, which is a phosphothreonine lyase that translocates to the host nucleus
upon infection. This enzyme eliminates a phosphate group from a
phosphothreonine residue in the activation loop of the MAPKs ERK1 and
ERK2 by β-elimination of the hydroxyl moiety, an unprecedented reaction
mechanism for removing a phosphate from a protein, which generates
dehydroalanine (Fig. 2) (Li et al. 2007). This irreversible dephosphorylation
inhibits ERK-mediated activation of downstream mitogen- and stress-
activated kinase 1 (MSK1) and MSK2, thereby preventing phosphorylation
of histone H3 on S10. This modification is a prerequisite for chromatin
reorganization and priming of transcription-factor-binding sites in NF-κB-
regulated promoters (Ch. 15 [Newton and Dixit 2012]). Initial studies
supported the hypothesis that OspF works to diminish a proinflammatory
response during a Shigella infection, but other studies implicate an OspF-
mediated inhibition of a negative-feedback loop to partially activate immune
signaling, which may create an advantageous environment for this
intracellular pathogen.

3 MANIPULATION OF G-PROTEIN SIGNALING

Among the Ras superfamily of small G proteins, the Ras, Rho, and Ran
subfamilies primarily regulate cell division/differentiation, cytoskeleton
remodeling, and nuclear import, respectively (Takai et al. 2001; Ch. 2 [Lee
and Yaffe 2014]). Members of the Arf and Rab subfamilies facilitate many
aspects of intracellular trafficking (Takai et al. 2001). As in the case of
heterotrimeric G proteins, nucleotide binding regulates small G proteins:
GTP-bound small G proteins are in an active conformation, and GDP-bound
small G proteins are in an inactive conformation. Unsurprisingly, they are
important targets of viral and bacterial virulence factors, as are the guanine-
nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs)
that regulate them (Ch. 2 [Lee and Yaffe 2014]). Indeed, the diverse
regulatory processes they control make them prime targets, allowing
pathogens to subvert host machinery in order to mediate cellular attachment
and entry and promote growth, replication, and dissemination of the pathogen
within the harsh environment of the host. There is an impressive list of
secreted virulence factors that mimic or modify the behavior of host small G
proteins (Table 1).

3.1 Bacterial Guanine-Nucleotide Exchange Factor (GEF)

Mimics
The pathogenic strategy underlying the subversion of small G proteins during
infection depends on the bacterial life cycle. For example, Salmonella, the
causal agent of typhoid fever, is intracellular and deploys bacterial GEF
proteins such as SopE and SopE2 to activate actin polymerization, which
facilitates internalization of the bacterium into host cells. SopE directly
activates the Cdc42 and Rac small G proteins to induce membrane ruffling at
the site of Salmonella invasion (Fig. 4) (Hardt et al. 1998). SopE2
(Salmonella spp.), BopE (Burkholderia pseudomallei), and CopE
(Chromobacterium violaceum) are similar to SopE. A second class of
bacterial GEFs shares very low sequence similarity (<15%) with one another,
but all contain an invariant WxxxE motif (Alto et al. 2006; Huang et al.
2009). This extends the group of pathogens that directly activate host small G
proteins to the facultative intracellular pathogens Shigella spp. and the
extracellular attaching/effacing (A/E) Escherichia coli pathogens, including
entrohemorrhagic E. coli O157:H7 (EHEC O157:H7) and enteropathogenic
E. coli (EPEC).

Figure 4. Multilevel regulation of small G proteins by bacterial effectors. Small G proteins cycle on
and off membranes, exchange guanine nucleotides (GDP to GTP) for activation, hydrolyze GTP for
inactivation, and stimulate downstream signaling pathways. Bacterial pathogens have evolved toxins
and effector proteins to usurp nearly every aspect of small G-protein function, using molecular mimicry
as well as novel stimulatory and inhibitory mechanisms.

Bioinformatics analyses reveal that SopE-type and WxxxE-type bacterial

GEFs share no overall sequence similarity, yet both classes adopt a conserved
V-shape structure and promote the exchange of guanine nucleotides by
presenting conserved acidic and amide residues necessary for stabilization of
switch I and switch II regions on the target small G proteins (Buchwald et al.
2002; Upadhyay et al. 2008; Huang et al. 2009). However, unlike many
forms of pathogenic mimicry, the bacterial GEFs are functional mimics that
have structures completely different from those of their eukaryotic
counterparts. Indeed, they resemble neither the eukaryotic Dbl-homology
(DH) domain nor the dock homology region 2 (DHR2) domain proteins, the
two major classes of eukaryotic Rho-family GEFs. Nevertheless, both
bacterial and eukaryotic GEFs induce similar nucleotide-free transition states
essential for guanine-nucleotide exchange. The catalytic loops of bacterial
GEFs are important for making contacts with the switch I and switch II
regions of the host small G proteins. Recent studies have shown that these
loops are flexible and that proper orientation is important for small G-protein
recognition and activation (Klink et al. 2010). Reorientation of the catalytic
loop may therefore be a mechanism for bacterial GEFs to control host small
G-protein activity in response to bacterial or host stimuli.

3.2 Bacterial GTPase-Activating Protein (GAP) Mimics

Many pathogens have evolved proteins that directly engage small G proteins
and stimulate GTP hydrolysis, thus turning the molecular switch off. A key
example of this activity is the YopE protein from Yersinia
pseudotuberculosis (Fig. 4). YopE has in vitro GAP activity toward RhoA,
Rac1, and Cdc42 (Black and Bliska 2000; Von Pawel-Rammingen et al.
2000; Andor et al. 2001). It engages the small G proteins directly and
presents a critical arginine residue (R144) that stabilizes the β phosphate of
the GTP moiety, thus increasing the hydrolytic rate. In fact, mutant bacteria
expressing YopE (R144A) that lacks GAP activity are incapable of inducing
cytotoxicity. During a Yersinia infection, YopE works in concert with YopH
to prevent phagocytosis by the host cell. The former effector induces collapse
of the actin cytoskeleton and rounding of the host cell, whereas the latter
effector disassembles focal adhesions (see above).
Salmonella spp. subvert host small G proteins to gain entry into normally
nonphagocytic cells, such as intestinal epithelial cells. It induces its own
engulfment by injecting proteins like SopE that reorganize the actin
cytoskeleton (discussed above). The type III secreted SptP protein uses a
tyrosine phosphatase activity and Rho-GTPase GAP activity in order to
return the cytoskeleton back to a preinvasion state (Fu and Galan 1999;
Stebbins and Galan 2000). Much like YopE from Yersinia, SptP from
Salmonella has an “arginine finger” that induces efficient GTP hydrolysis by
small G proteins. Interestingly, the temporal shift in activity between actin-
polymerization-promoting factors such as SopE and actin inhibitors such as
SptP is dictated by ubiquitin-dependent degradation (Kubori and Galan
2003). Degradation of SopE allows SptP to rapidly inhibit nonspecific Rho-
family GTPase signaling, thus preventing spurious actin assembly so that
immune cells no longer detect intracellular Salmonella.
Finally, Pseudomonas, an opportunistic, extracellular pathogen associated
with severe infection in cystic fibrosis patients and burn patients, expresses
the effector protein ExoS, which contains two catalytic domains that inhibit
small G proteins. The first is an ADP-ribosyltransferase domain that transfers
ADP-ribose from NAD+ to R41 of several Ras-like small G proteins (Coburn
et al. 1989; Coburn and Gill 1991). This activity uncouples signaling by
preventing GEF-mediated activation of small G proteins. The second is a
GAP domain that potently inactivates several host small G proteins
(Goehring et al. 1999). Whereas many bacterial effector proteins have
evolved to specifically target a particular small G protein, ExoS functions to
perturb the activation of multiple GTP-binding proteins and thus the signal
transduction pathways they control (Henriksson et al. 2002). Together, the
down-regulation of small G-protein signaling by bacterial GAPs dampens
actin cytoskeleton dynamics at the bacterium–host interface.

3.3 A Yersinia Protease Destroys the Small G-Protein

Membrane Anchor
The Yersinia effector protein YopT can disrupt the actin cytoskeleton,
causing rounding up of host cells, and thereby inhibit phagocytosis (Fig. 4)
(Iriarte and Cornelis 1998). YopT is a cysteine protease that cleaves amino-
terminally to the prenylated cysteine residue in RhoA, Rac, and Cdc42 (Shao
et al. 2002, 2003). This liberates Rho proteins from the membrane and
irreversibly inhibits signaling to the actin cytoskeleton. YopT is similar to
papain proteases and recognizes basic amino acid residues upstream of the
cleavage site as well as both the GDP-bound and GTP-bound forms of RhoA.
Thus, cleavage does not depend on the conformational state of the small G
protein. When RhoA is no longer tethered to the membrane, actin-mediated
cytoskeletal responses during infection are effectively thwarted.

3.4 Yersinia YpkA: A Kinase and a GDI

Another Yersinia effector protein that regulates host membrane remodeling is
YpkA. This three-domain effector contains an amino-terminal kinase domain,
central guanine nucleotide dissociation inhibitor (GDI) domain, and a
carboxy-terminal actin-binding domain (Juris et al. 2000; Prehna et al. 2006).
YpkA preferentially phosphorylates the active GTP-bound form of the αq
heterotrimeric G-protein subunit at position S47 (Navarro et al. 2007). This
modification prevents αq from binding GTP, thereby inhibiting its activity
and the subsequent membrane remodeling that would otherwise enhance
uptake of bacteria. Interestingly, mice with deficiencies in αq function have
increased bleeding times and defective platelet activation, which is a
hallmark of the plague (Offermanns et al. 1997).
The central GDI domain in YpkA from Yersinia directly binds Rac1 and
mimics the host GDI for Rho small G proteins (Prehna et al. 2006).
Consequently, Rac1 cannot undergo GDP for GTP exchange and localization
to the membrane, where it would normally function in cell adhesion,
migration, and regulation of epithelial cell differentiation.

3.5 Bacterial Effectors Can Modify Small G Proteins

As indicated above, Rho family small G proteins are high-value targets for
bacterial effector proteins because they control the eukaryotic cytoskeleton.
Therefore, unsurprisingly, some bacterial effector proteins not only mimic the
endogenous regulators but can posttranslationally modify eukaryotic small G
proteins (Etienne-Manneville and Hall 2002). C3 toxin from Clostridium
botulinum, more generically known as botulinum toxin, has evolved the
ability to ADP-ribosylate and inactivate Rho-family small G proteins,
specifically RhoA, RhoB, and RhoC, on an invariant N41 residue (Mohr et
al. 1992). Treatment of adherent cells with C3 toxin results in the
disassembly of actin microfilaments and rounding up of cells (Chardin et al.
1989). N41 is located at the border of the switch I region of Rho-family
proteins, and ADP ribosylation induces close association of the Rho small G
protein with its cognate GDI, thereby inhibiting cytosol-to-membrane cycling
and preventing activation by GEFs (Sehr et al. 1998). Bacteria with
extracellular life cycles, like E. coli, must avoid phagocytosis. This can be
accomplished through covalent modification of Rho, Rac, and Cdc42.
Specifically, cytotoxic necrotizing factor 1 (CNF1) deamidates Q63 of Rho
or Q61 of Rac and Cdc42 (Flatau et al. 1997; Schmidt et al. 1997). By
removing the functional amine group from the critical catalytic glutamine
residue, this renders the small G protein constitutively active. Deamination-
induced activation of these small G proteins results in cell ruffling and the
formation of stress fibers, focal adhesions, and lamellipodia, as well as
extension of filopodia. These effects lead to unregulated membrane
protrusions that appear to inhibit normal phagocytic events. An additional
interesting feature of CNF1 is its ability to rescue epithelial cells from
apoptosis, presumably prolonging the infectivity of the pathogenic bacterium
(Miraglia et al. 2007).
Another posttranslational modification used by bacterial effectors is
AMPylation, covalent attachment of AMP to a hydroxyl side chain on a
protein substrate (Woolery et al. 2010). In the 1960s, Earl Stadtman and
colleagues found that E. coli glutamine synthetase is regulated by glutamine
synthetase adenylyl transferase (GS-ATase), a bifunctional enzyme that
catalyzes addition and removal of AMP from glutamine synthetase. This
results in inactivation and activation of the enzyme, respectively, which
permits cellular regulation of nitrogen metabolism (Brown et al. 1971).
VopS, a T3SS effector protein from V. parahaemolyticus, which causes
gastroenteritis due to consumption of contaminated raw seafood, has been
found to AMPylate the conserved threonine residue in the switch I region of
Rho. The AMPylated small G protein can no longer bind to downstream
substrates such as PAK and rhotekin, which results in disorganization of the
actin cytoskeleton (Yarbrough et al. 2009). This benefits the pathogen
because the host cell’s actin assembly machinery is compromised, and it can
no longer induce phagocytosis.
Bacterial pathogens use AMPylators to disrupt host signaling pathways to
promote bacterial survival and replication (for review, see Woolery et al.
2010). IbpA secreted from Histophilus somni, like VopS, also modifies RhoA
with AMP but on a tyrosine residue instead of a threonine in the switch I
region (Worby et al. 2009). Both VopS and IpbA proteins contain a Fic
domain (Filamentation induced by cAMP) that mediates this enzymatic
activity. The Fic and doc domains share a conserved HPFx[D/E]GN[G/K]R
motif, in which the invariant histidine residue is essential for AMPylation
activity in Fic proteins (Luong et al. 2010) and cytotoxicity in doc (Garcia-
Pino et al. 2008). Indeed, the two domains are grouped in the same family
and classified as FIDO domains based on structural similarities (Kinch et al.
2009). Interestingly, Fic domains, found in bacteria, archaea, and metazoans,
are thought to function as endogenous signaling elements and, therefore, are
regulated so as not to cause harm to the host (Kinch et al. 2009; Engel et al.
2012). Indeed, 90% of Fic domains are regulated by an inhibitory α-helix that
prevents constitutive binding of ATP, and binding of a specific substrate is
predicted to relieve this inhibition to allow AMPylation (Engel et al. 2012).
AMPylators are comparable to kinases in that they both hydrolyze ATP and
reversibly transfer a part of this metabolite onto a hydroxyl side chain of the
protein substrate.
A more specialized Fic domain can use the substrate CDP-choline, instead
of ATP, in a phosphotransfer reaction, producing small G proteins modified
by a phosphocholine moiety (Mukherjee et al. 2011). Legionella, causal agent
of Legionnaires’ disease, produces the effector AnkX, which contains an Fic
domain that modifies Rab1 (a host small G protein involved in membrane
transport) at its switch II region with phosphocholine. The modified Rab1 is
no longer recognized by the host GEF, connecdenn, but can bind to and be
activated by the Legionella GEF DrrA. The inactivation and activation of
Rab1 on Legionella-containing vacuoles (LCVs) thus become dictated by the
pathogen by posttranslational modifications of Rab GTPases.
The adenylyl transferase domain is part of the larger nucleotidyl
transferase domain family and, like Fic domains, can catalyze AMPylation. It
is characterized by a conserved Gx11DxD motif in which the aspartate
residues are essential for the AMPylation activity (Jiang et al. 2007; Muller et
al. 2010). This domain has been identified in more than 1400 bacterial
proteins among 685 bacterial species, of which the large majority are
proteobacteria (Finn et al. 2009).
DrrA (also known as SidM) is a virulence factor secreted from
Legionella, an intracellular pathogen that survives in LCVs in the cell (Muller
et al. 2010). Legionella uses a T4SS to secrete proteins from the LCV into the
host cell. DrrA is composed of three domains: an amino-terminal adenylyl
transferase domain, a GEF domain, and a carboxy-terminal
phosphatidylinositol-4-phosphate-binding (P4M) domain (Murata et al. 2006;
Brombacher et al. 2009). The first domain of DrrA very closely resembles the
carboxy-terminal adenylyl transferase domain of E. coli GS-ATase and
contains the conserved Gx11DxD motif (Muller et al. 2010). The GEF
domain is capable of catalyzing the exchange of GDP for GTP on Rab1,
which plays a role in the regulation of vesicular transport from the
endoplasmic reticulum (Murata et al. 2006). The P4M domain anchors the
effector to the cytoplasmic side of the LCV membrane (Brombacher et al.
2009). DrrA hijacks Rab1 by locking it into its GTP-bound active state, using
both the DrrA GEF and adenylyl transferase domains. The GEF domain
exchanges GDP for GTP, and the adenylyl transferase domain AMPylates
Y77 in the switch II region of Rab1, which blocks its interaction with host
GAPs, preventing the hydrolysis of GTP. AMPylated and prenylated GTP-
Rab1 localizes with DrrA and targets ER vacuoles to the LCV (Muller et al.
2010). Another virulence factor from Legionella, SidD, contains a
deAMPylating activity, allowing its recycling (Neunuebel et al. 2011; Tan
and Luo 2011). This protein is a phosphodiesterase that, on the basis of
structural analysis, resembles a protein phosphatase (Rigden 2011).

3.6 Bacterial Effectors Mimic Host Small G Proteins

Another strategy used by bacterial pathogens is to produce proteins that
mimic small G proteins themselves. EHEC O157:H7 secretes the effector
protein EspFu (also known as TccP) in order to create actin-based pedestals,
allowing attachment of the bacterium to the intestinal epithelium
(Campellone et al. 2004; Garmendia et al. 2004). The Wiskott-Aldrich
syndrome protein (WASP) family of actin nucleators is normally regulated
by Rac and Cdc42, which bind to the Cdc42/Rac1-interaction-binding
domain (CRIB) motif within the G-protein-binding domain (GBD) of N-
WASP (Ch. 8 [Devreotes and Horwitz 2014]). This releases N-WASP from
an autoinhibited conformation that depends on interaction of the GBD and
the verprolin-homology, cofilin-homology, acidic (VCA) domain.
Remarkably, EspFu mimics a 17-residue stretch of the VCA motif, thus
competing for binding with the GBD, and thereby constitutively activates N-
WASP and activates downstream actin polymerization (Cheng et al. 2008;
Sallee et al. 2008).
EspFu is not the only bacterial effector to directly activate small G-protein
substrates. A second EHEC O157:H7 effector, EspG, is an activator of class I
PAK family serine/threonine kinases (Selyunin et al. 2011). Under normal
circumstances, PAKs are responsible for regulating cytoskeletal dynamics
and are activated by Rac1 or Cdc42 small G proteins in a manner similar to
the activation of N-WASP discussed above. The PAKs contain an amino-
terminal autoinhibitory domain and a carboxy-terminal kinase domain.
Binding of activated Cdc42 or Rac1 to the CRIB domain within the
autoinhibitory domain of PAK potently induces kinase activation. In contrast,
EHEC EspG binds to and activates PAK but does not recognize the CRIB.
Rather, EspG specifically interacts with the Iα3 helix, which sits upstream of
the CRIB domain and serves two primary functions: (1) it occludes the
substrate-binding site of the kinase domain, and (2) it positions an inhibitory
loop into the kinase catalytic cleft. Binding of EspG to Iα3 is predicted to
unfold the autoinhibitory domain and release the kinase inhibitory loop,
leading to PAK activation. Both bacterial virulence factors, EspFu and EspG,
thus mimic the basic principles of host small G-protein function to activate
their downstream targets by separating the inhibitory domain from the
activity-bearing domain, but they use distinct molecular mechanisms to
achieve this result.

4 HIGHJACKING LIPID SIGNALING

Phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PIP2),
regulate the actin cytoskeleton beneath the plasma membrane, functioning in
signaling as well as trafficking by targeting vesicles around the cell.
Disruption of phosphoinositide homeostasis at the plasma membrane by
bacterial effectors can destabilize actin dynamics and alter the morphology of
the membrane. This facilitates the entry of intracellular pathogens or, in the
case of extracellular pathogens, can disrupt membrane integrity, which leads
to rapid cell lysis in the subsequent stage of infection to facilitate pathogen
spreading (Ham et al. 2011).

4.1 The Inositol Polyphosphate 4-Phosphatase Shigella IpgD

and Salmonella SopB Promote Pathogen Entry
IpgD is an effector from the facultative intracellular pathogen Shigella that is
directly translocated into host cells through a T3SS upon contact with the cell
surface (Niebuhr et al. 2000). IpgD is a 4-phosphoinositide phosphatase that
hydrolyzes PIP2 to produce phosphatidylinositol 5-phosphate [PI(5)P]
(Niebuhr et al. 2002). Removal of PIP2 by IpgD decreases the tethering of the
plasma membrane to PIP2-binding cytoskeleton-anchoring proteins, causing
extension of membrane filopodia and massive cellular blebbing (observed as
bubble-like protrusions) (Charras and Paluch 2008). This reorganization of
the actin cytoskeleton at the bacterial entry site promotes the uptake of the
pathogen by the host cells.
Like IpgD, the Salmonella effector protein SopB hydrolyzes PIP2 to
promote bacterial invasion and establish a niche for its vacuolar life cycle
inside the host (Norris et al. 1998; Terebiznik et al. 2002; Hernandez et al.
2004). Thus, modulation of phosphoinositide metabolism appears to be a
common strategy for bacterial pathogens to usurp signaling at plasma and
vesicular membranes.

4.2 Inositol Polyphosphate 5-Phosphatase V.

parahaemolyticus VPA0450 Promotes Blebbing
A similar molecular mechanism is used by the T3SS effector VPA0450 from
the extracellular pathogen V. parahaemolyticus (Broberg et al. 2010).
VPA0450 contains catalytic motifs that mimic the activity of the eukaryotic
inositol polyphosphate 5-phosphatases (IPP5Cs), which hydrolyze PIP2 at the
membrane surface. In contrast to IpgD, VPA0450 hydrolyzes the D5
phosphate, producing PI(4)P. The removal of PIP2 disrupts actin dynamics,
causing the local detachment of the cortical cytoskeleton from the plasma
membrane, which leads to extensive membrane blebbing. Whereas IpgD uses
the same molecular mechanism to facilitate internalization of the bacteria,
blebbing induced by VPA0450 instead accelerates lysis of the infected host
cell (Broberg et al. 2011).

5 OTHER ACTIN REGULATORS TARGETED BY

PATHOGENS
The actin cytoskeleton supports focal adhesions and controls cell contraction,
cell motility, endocytosis, phagocytosis, and cell division. A characteristic
feature of all of these processes is the dynamic transition of cellular actin
between its monomeric (G) and polymeric (F) actin states, which is
controlled by a myriad of regulatory proteins that act on distinct states of the
actin polymer network. For example, the Arp2/3 complex nucleates filaments
that grow from the side of existing filaments, creating branched networks,
whereas formins and SPIRE nucleate unbranched filaments (Campellone and
Welch 2010). A common mechanistic feature of all three systems is the
ability to assemble actin or actin-like proteins into an arrangement that can
serve as a template for growth of a new filament. The Arp2/3 complex
contains two actin-related subunits, which form a pseudo-actin trimer with an
actin monomer provided by activators of the WASP family, such as N-WASP
(see above). Formins bind two actin monomers and are thought to position
them appropriately for filament growth. SPIRE proteins have multiple repeats
of Wiskott-Aldrich homology 2 (WH2) domains that bind to actin and appear
to create a three-actin template for filament extension. Bacteria and viruses
can hijack these mechanisms to directly regulate actin nucleation, producing
the characteristic pathogen motility observed in Listeria-, Shigella-,
Rickettsia-, and Vaccinia-virus-infected cells. In addition, several bacterial
species from the Vibrio genus translocate actin-elongation factors into host
cells.

5.1 Pathogenic Actin Nucleation Factors

A few bacterial pathogens and viruses use actin polymerization to move
around within and between cells. Bacteria from at least three genera (Listeria,
Shigella, and Rickettsia) and Vaccinia virus all use membrane-anchored
proteins to produce actin comet tails that propel the microorganism through
the cytoplasm, along the surface of the cell, or through the plasma membrane
into a neighboring cell. Although the proteins used by each of these
pathogens are unique in structure and function, they all share the common
feature of nucleating actin filaments de novo at the membrane surface. For
example, ActA directly recruits and activates the Arp2/3 complex at the
surface of Listeria monocytogenes (Welch et al. 1998). It has, in fact, been an
essential tool for studies of various biological processes including cell
motility and provided the first physiological evidence for the nucleating
activity of the Arp2/3 complex (Welch et al. 1998). Like ActA, Shigella
VirG/IcsA induces formation of actin comet tails, but this pathogen uses a
distinct mechanism. Whereas ActA directly activates Arp2/3, VirG/IcsA on
the bacterial surface can recruit N-WASP and induce actin nucleation via
Arp2/3 (Egile et al. 1999). Finally, Vaccinia virus uses the membrane-
anchored protein A36R to facilitate intracellular movement that is strikingly
similar. A large domain of A36R on the viral surface is phosphorylated by
Src-family tyrosine kinases and then directly interacts with the adaptor
protein Nck and subsequently recruits N-WASP. These processes are all
essential for the bacteria and viruses to invade systemic tissues of their host
organism and therefore represent key virulence factors in a wide range of
infectious diseases.
Extracellular pathogens including EPEC also hijack Arp2/3, albeit by a
mechanism distinct from that described above. EPEC secretes a cell surface
receptor, Tir, which embeds in the host plasma membrane and forms a
complex with the bacterial adhesion molecule intimin (Kenny et al. 1997).
This causes Tir to cluster at the cell surface, resulting in tyrosine
phosphorylation of its cytoplasmic tail and subsequent recruitment of Nck via
its SH2 domain (Gruenheid et al. 2001). This recruits N-WASP and the
Arp2/3 complex to nucleate branched actin filaments at the EPEC–host
interface, resulting in the formation of pedestals. These molecular events are
instrumental in the tight attachment of EPEC to the intestinal epithelial cell
wall and also induce the characteristic attaching and effacing (A/E) lesion
that defines EPEC infections.

5.2 Pathogenic Elongation Factors: Vibrio VopL/F

In addition to inducing branched actin networks through activation of Arp2/3,
V. parahaemolyticus produces VopL, which has three closely spaced WH2
domains that bind actin (Fig. 4) (Liverman et al. 2007). VopL directly
induces the nucleation of actin independently of any other eukaryotic factor
and is more efficient than its eukaryotic counterparts (Namgoong et al. 2011;
Yu et al. 2011). Interspersed with the WH2 domains are three proline-rich
motifs (PRMs). PRMs have many potential interacting partners, including
WW domains, SH3 domains, and the actin-binding protein profilin. The
PRMs in VopL closely resemble those found in the FH1 domains of formins,
which are known to bind profilin and profilin–actin complexes (Holt and
Koffer 2001). V. parahaemolyticus uses VopL to induce unregulated
production of stress fibers and thereby disrupts actin homeostasis in the
epithelial cells of the gut during infection, resulting in an enterotoxic effect in
the intestine. Another T3SS virulence factor from V. cholera, VopF, contains
a similar WH2/PRM domain architecture and also promotes actin assembly
independently of host proteins. VopF induces the formation of small actin
protrusions, rather than stress fibers, and may help efficient colonization
during infection.

6 TARGETING UBIQUITIN-MEDIATED SIGNAL

TRANSDUCTION
Evolutionarily conserved ubiquitylation machinery regulates a diverse set of
cellular processes, including development, transcription, replication, cell
signaling, and immune function (Pickart 2004; Mukhopadhyay and Riezman
2007; Ch. 2 [Lee and Yaffe 2014]). The versatility of this system to
reversibly modify protein function makes it an attractive target for a wide
range of pathogens, including viruses and bacteria. These microbes are
particularly adept at coopting the ubiquitylation machinery. Indeed, ubiquitin
itself is encoded by a large number of viral genomes, and many viruses and
bacteria encode the ubiquitin ligases or adaptor proteins required for
ubiquitin posttranslational modification (Randow and Lehner 2009; Collins
and Brown 2010).
Ubiquitin can be covalently linked to protein substrates as either a single
molecule (monoubiquitylation) or a polypeptide chain (polyubiquitylation)
(Ch. 2 [Lee and Yaffe 2014]). It is first activated by a ubiquitin-activating
enzyme, E1, which involves an ATP-dependent transfer of ubiquitin to the
enzyme’s catalytic cysteine residue. It is then transferred to the active-site
cysteine of an E2 ubiquitin-conjugating enzyme. The ubiquitin residue on the
charged E2 enzyme is then targeted to substrates via an E3 ligase. K48-linked
chains of ubiquitin mark the substrate for proteasomal degradation. In
contrast, monoubiquitylation or K63-linked chains serve as regulatory signals
in signal transduction, membrane trafficking, DNA repair, and chromatin
remodeling. Below, we highlight a few specific examples of pathogens
exploiting the ubiquitin system. These interactions have not only informed us
regarding microbial pathogenesis, but also continue to reveal novel
mechanisms of ubiquitin regulation in cell signaling.
Many viruses, including baculoviruses, poxviruses, and herpes simplex
virus, encode their own ubiquitin molecules but have significantly altered the
ubiquitin gene. Human ubiquitin shares only 75% similarity with baculovirus
ubiquitin, compared with 96% similarity with yeast ubiquitin (Haas et al.
1996). The viral ubiquitylation machinery may therefore function differently
from the host ubiquitylation machinery. Many bacteria also secrete enzymes
that modify host ubiquitin or ubiquitin-like molecules (UBLs). Recent studies
of EPEC revealed that host ubiquitin is deamidated on Q40 by the bacterial
type III effector Cif (Cui et al. 2010). Similarly, the Cif homolog CHBP
encoded by Burkholderia pseudomallei deamidates Q40 both on ubiquitin
and the UBL Nedd8 (Cui et al. 2010; Jubelin et al. 2010; Morikawa et al.
2010). These posttranslational modifications potently inhibit polyubiquitin
chain synthesis, resulting in accumulation of host substrates and severe
cytopathic effects.
Viruses and bacteria can also encode their own E3 ubiquitin ligases or
adaptor proteins that link host E3 enzymes to specific host substrates. Most
known eukaryotic E3 ligases belong to one of three types: RING, HECT, and
U-box. There are currently no known viral HECT family E3 ubiquitin ligases.
Instead, viruses encode RING family or unconventional E3 ligases. Two
examples of RING type E3 ligases are the RING-CH family and the Infected
Cell Protein 0 (ICP0) family. Initially identified in the murine and human
gammaherpes viruses, respectively, these virulence factors down-regulate
immune cell surface receptors (Coscoy and Ganem 2000; Ishido et al. 2000;
Stevenson et al. 2000; Haque et al. 2001). For example, the K3 and K5 gene
products of Kaposi’s sarcoma–associated herpesvirus (KSHV) provide
immune protection by ubiquitylating MHC class I molecules that present
antigen at the cell surface, targeting them for endocytosis and lysosomal
degradation. In contrast, ICP0 of herpes simplex virus type I (HSV-1) is
required for reactivation of latency and suppression of innate immunity
(Everett 2000). The RING domain of ICP0 promotes the accumulation of
ubiquitylated proteins and their subsequent proteasomal degradation. In
particular, it causes the degradation of RNF8 and RNF168, host cell E3
ligases that are essential for the cellular response to DNA damage. By
degrading these proteins, ICP0 blocks the cellular DNA damage response that
HSV infection activates, which would otherwise shut off viral transcription.
Another well-characterized example of a virally encoded E3 ligase is
adenovirus E4orf6, which, together with adenovirus E1B-55K, substitutes for
the substrate recognition subunits of the cullin-EloB-C core complex
(Querido et al. 2001a). This host–pathogen complex forms a novel ubiquitin
ligase that targets the tumor suppressor p53, Mre11, and the BLM helicase to
abrogate the cellular DNA damage response during viral infection (Dobner et
al. 1996; Querido et al. 2001b).
The KSHV immediate-early transcription factor RTA shows
unconventional E3 ubiquitin ligase activity that targets host immune protein
IRF7 for proteasomal degradation (Yu et al. 2005). Similarly, the ubiquitin
ligase domain of the IpaH family of bacterial type III effectors is structurally
distinct from both the HECT and RING families (Rohde et al. 2007; Singer et
al. 2008; Zhu et al. 2008). However, like the HECT-type E3 ligases, IpaH
transfers ubiquitin from UbcH5 E2 to substrates by forming a ubiquitin
thioester intermediate at a conserved cysteine residue. A series of leucine-rich
repeats (LRRs) in IpaH and its family members is responsible for recognizing
a diverse array of host substrates and targeting these substrates for
ubiquitylation.
Pathogens may also encode adaptor proteins that link E3 ligases to target
substrates. In a classic example, the E6 oncoprotein encoded by human
papillomavirus (HPC) facilitates ubiquitylation and proteasomal degradation
of p53 (Scheffner et al. 1993; Huibregtse et al. 1995). The dimeric E6 forms a
complex with human E6-AP, the founding member of the HECT-type E3
ubiquitin ligase family (Huibregtse et al. 1995). The E6–E6-AP complex
binds to and targets p53 for ubiquitin-dependent proteolysis, thus interfering
with the growth-regulating activities of this tumor suppressor. These
discoveries have provided essential insights into cancer caused by high-risk
HPV, and have defined an entire class of E3 ubiquitin ligases involved in a
myriad of biological processes.

7 CONCLUSION
Virulence factors produced by pathogens have evolved to efficiently
manipulate host signaling pathways (Table 1). Mechanisms range from
constitutive activation of a pathway, to irreversible inactivation of a critical
signaling molecule, to subversion of a whole signaling system to favor the
invading pathogen. A major challenge in the future is to determine the
enzymatic activities and host substrates for the bacterial and viral virulence
factors that show no obvious homology to eukaryotic proteins. Another, even
more complex challenge is to understand how these factors work together to
orchestrate a successful infection. Temporal and spatial considerations are
extremely important for regulating a host cell during infection. Likewise,
within the pathogen, determining the regulatory mechanisms that control the
activation patterns and spatial dynamics of virulence factors will help reveal
how microbial pathogens coopt signal transduction systems during infection.
Finally, the use of model organisms to complement studies in mammalian
cells will provide valuable insights into the physiological roles of bacterial
effector proteins. Such information is essential to gain a system-level view of
the infectious disease process and to ultimately design therapeutics that target
host–pathogen interactions. Inevitably, by discovering the mechanisms of
pathogenic effectors, we have gained a greater understanding into critical
steps in eukaryotic signaling. Although a great deal has been learned, given
the number and diversity of the yet-to-be-studied bacterial and viral
pathogens, much more is left to be discovered.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a006114
CHAPTER 21

Signal Transduction in Cancer

Richard Sever1 and Joan S. Brugge2

1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
2Harvard Medical School, Department of Cell Biology, Boston, Massachusetts 02115

Correspondence: joan_brugge@hms.harvard.edu

SUMMARY

Cancer is driven by genetic and epigenetic alterations that allow cells to

overproliferate and escape mechanisms that normally control their
survival and migration. Many of these alterations map to signaling
pathways that control cell growth and division, cell death, cell fate, and
cell motility, and can be placed in the context of distortions of wider
signaling networks that fuel cancer progression, such as changes in the
tumor microenvironment, angiogenesis, and inflammation. Mutations
that convert cellular proto-oncogenes to oncogenes can cause
hyperactivation of these signaling pathways, whereas inactivation of
tumor suppressors eliminates critical negative regulators of signaling.
An examination of the PI3K-Akt and Ras-ERK pathways illustrates how
such alterations dysregulate signaling in cancer and produce many of the
characteristic features of tumor cells.

Outline
 1 Introduction
2 Mutations as the cause of cancer
3 Dysregulation of cellular processes by oncogenic signaling
4 Cell proliferation
5 Cell survival
6 Cell metabolism
7 Cell polarity and migration
8 Cell fate and differentiation
9 Genomic instability
10 The tumor microenvironment
11 Concluding remarks
References

1 INTRODUCTION
The development of cancer involves successive genetic and epigenetic
alterations that allow cells to escape homeostatic controls that ordinarily
suppress inappropriate proliferation and inhibit the survival of aberrantly
proliferating cells outside their normal niches. Most cancers arise in epithelial
cells, manifesting as carcinomas in organs such as the lung, skin, breast, liver,
and pancreas. Sarcomas, in contrast, arise from mesenchymal tissues,
occurring in fibroblasts, myocytes, adipocytes, and osteoblasts. Nonepithelial
tumors can also develop in cells of the nervous system (e.g., gliomas,
neuroblastomas, and medulloblastomas) and hematopoietic tissues (leukemia
and lymphoma).
In solid tumors, these alterations typically promote progression from a
relatively benign group of proliferating cells (hyperplasias) to a mass of cells
with abnormal morphology, cytological appearance, and cellular
organization. After a tumor expands, the tumor core loses access to oxygen
and nutrients, often leading to the growth of new blood vessels
(angiogenesis), which restores access to nutrients and oxygen. Subsequently,
tumor cells can develop the ability to invade the tissue beyond their normal
boundaries, enter the circulation, and seed new tumors at other locations
(metastasis), the defining feature of malignancy (Fig. 1). This linear sequence
of events is clearly an oversimplification of complex cancer-associated events
that proceed in distinct ways in individual tumors and between tumor sites;
however, it provides a useful framework in which to highlight the critical role
of dysregulated signaling in processes associated with the initiation and
progression of cancer.

Figure 1. Cancer progression.

 The root cause of cancer is usually genetic or epigenetic alterations in the
tumor cells (see below). Progression of the cancer, however, is associated
with a complex interplay between the tumor cells and surrounding non-
neoplastic cells and the extracellular matrix (ECM). Moreover, the tumor
cells develop several well-defined features (Hanahan and Weinberg 2000;
Solimini et al. 2007). In addition to increased cell proliferation, these include
resistance to apoptosis and other forms of cell death, metabolic changes,
genetic instability, induction of angiogenesis, and increased migratory
capacity. Dysregulation of cellular signal transduction pathways underlies
most of these characteristics.
Here, we describe how tumor cells co-opt signaling pathways to allow
them to proliferate, survive, and invade other tissues. To cover all of the
signaling molecules involved and their myriad contributions to cancer would
require an entire textbook (Weinberg 2013). We therefore focus primarily on
two pathways—Ras-ERK (p. 81 [Morrison 2012]) and PI3K-Akt signaling
(p. 87 [Hemmings and Restuccia 2012])—that play central roles in multiple
processes associated with cancer, while highlighting the involvement of some
other key signaling molecules.

2 MUTATIONS AS THE CAUSE OF CANCER

Most tumors arise as a consequence of genetic alterations to cellular genes,
which may be inherited or arise spontaneously, for example, as a result of
DNA damage induced by environmental carcinogens or mutations arising
from replication errors. These alterations confer a selective advantage to the
cells, which together with changes in the microenvironment, promote tumor
growth and progression. Some are gain-of-function mutations, producing so-
called oncogenes that drive tumor formation. Others inactivate tumor
suppressor genes that normally ensure that cells do not proliferate
inappropriately or survive outside their normal niche.
Tumors can possess tens to hundreds or even thousands of mutations, but
many of these are merely so-called “passengers.” Typically only two to eight
are the “driver mutations” that cause progression of the cancer (Vogelstein et
al. 2013). These may be point mutations (such as G12V Ras), deletions (as
seen with PTEN), inversions, or amplifications (as seen with Myc). Large-
scale rearrangements also occur, for example, the BCR-ABL fusions
involving chromosomes 9 and 22, which are associated with several
leukemias and generate an oncogenic version of the tyrosine kinase Abl. Loss
of heterozygosity due to gene conversion or mitotic recombination between
normal and mutant parental alleles is another source of genetic alterations
that drive cancer. This often affects tumor suppressors such as the
retinoblastoma protein (pRB) and p53 (encoded by the TP53 gene in
humans).
Changes in the methylation state of promoters of genes that impact cancer
can also play an important role in oncogenesis (Sandoval and Esteller 2012;
Suva et al. 2013). Indeed, epigenetic silencing is more common than
mutational silencing for some genes, for example, the cyclin-dependent
kinase (CDK) inhibitor (CKI) p16 (also known as CDKN2A or INK4a) and
the mismatch repair (MMR) enzyme MLH1. Silencing of MMR enzymes can
lead to additional genetic changes because it affects proteins that prevent
errors by repairing DNA. Conversely, several mutations associated with
cancer affect epigenetic regulators that influence multiple cellular programs,
for example, DNMT1 and TET1, which control DNA methylation, and the
histone-modifying enzymes EZH2, SETD2, and KDM6A are deleted or
mutated in cancer (Delhommeau et al. 2009; Ley et al. 2010; Wu et al. 2012).
Interestingly, mutations in the metabolic enzymes isocitrate dehydrogenase
(IDH) 1 and IDH2 may promote cancer by generating an “oncometabolite”
not present in normal cells that inhibits certain chromatin-modifying enzymes
(see below) (Ward and Thompson 2012a).
Finally, in a minority of cancers, infectious agents are the triggers. A few
human cancers are triggered by viruses that encode genes that promote
tumorigenesis through activation of oncogene pathways or inactivation of
tumor suppressors. The human papilloma virus, which is associated with
cervical and head and neck cancers, encodes a protein, E6, that promotes
degradation of p53, while another viral protein, E7, inactivates pRB and
CKIs, among other effects (Munger and Howley 2002). In hepatocellular
carcinoma caused by hepatitis B virus, in contrast, it is not clear whether viral
proteins themselves are oncogenic, viral integration promotes expression of
nearby cellular oncogenes, or cancer is simply a consequence of persistent
liver injury and inflammation (Seeger et al. 2013). Epstein–Barr virus (also
known as human herpes virus 4) produces a protein called LMP1 that acts as
a constitutively active tumor necrosis factor (TNF) receptor, engaging a
plethora of signaling pathways including NF-κB, JNK/p38, PI3K, and ERK
(Morris et al. 2009). An extreme case of a transmissible cancer is that
affecting the Tasmanian devil. All tumors are derived from a founder tumor
and are transmitted as allografts from devil to devil during intraspecies facial
biting (Murchison et al. 2012; Hamede et al. 2013).

2.1 Cancer-Causing Mutations Affect Signaling Pathways

We can connect the genetic alterations in cancer cells with signaling
pathways that control processes associated with tumorigenesis and place
these in the context of distortions of wider signaling networks that fuel cancer
progression. In each case, the result is dysregulated signaling that is not
subject to the normal control mechanisms.
Oncogenic mutations can cause the affected genes to be overexpressed
(e.g., gene amplification) or produce mutated proteins whose activity is
dysregulated (e.g., point mutations, truncations, and fusions). Examples
include proteins involved in signaling pathways that are commonly activated
in many physiological responses, such as growth factor receptor tyrosine
kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR), small
GTPases (e.g., Ras), serine/threonine kinases (e.g., Raf and Akt), cytoplasmic
tyrosine kinases (e.g., Src and Abl), lipid kinases (e.g., phosphoinositide 3-
kinases, PI3Ks), as well as nuclear receptors (e.g., the estrogen receptor, ER).
Components of developmental signaling pathways, such as Wnt, Hedgehog
(Hh), Hippo, and Notch can also be affected, as can downstream nuclear
targets of signaling pathways, for example, transcription factors (e.g., Myc
and NF-κB), chromatin remodelers (e.g., EZH2), and cell cycle effectors
(e.g., cyclins).
Alternatively, deletions and other mutations can inactivate negative
regulators that normally function as tumor suppressors. Indeed, one of the
most commonly mutated genes in cancer is the tumor suppressor p53, the so-
called “guardian of the genome.” p53 is a critical hub that controls cell
proliferation and stress signals such as apoptosis and DNA damage responses
(see below). pRB and CKIs such as p16 are other tumor suppressors whose
mutation deregulates the cell cycle. Many tumor suppressors function as
negative regulators of cytoplasmic signaling, for example, the adenomatous
polyposis protein (APC) is a negative regulator of the Wnt pathway, and the
lipid phosphatase PTEN is a negative regulator of the PI3K-Akt pathway.
It is worth noting that hyperactivated oncogene pathways can also induce
a state of irreversible cell cycle arrest termed senescence (Gorgoulis and
Halazonetis 2010; Vargas et al. 2012). This is believed to represent a fail-safe
mechanism to inhibit proliferation caused by aberrant activation of
oncoproteins in normal cells and is accompanied by changes in cellular
structure, chromatin organization, DNA damage, cytokine secretion, and
gene expression. Oncogenic transformation requires alterations that abrogate
senescence, such as loss of p53 or PTEN.

2.1.1 The PI3K-Akt and Ras-ERK Pathways as Examples of Oncogenic

Signaling Pathways
Many of the genes commonly mutated in cancer encode components or
targets of the PI3K-Akt and Ras-ERK pathways (Fig. 2). Ordinarily these
pathways are transiently activated in response to growth factor or cytokine
signaling and ligand occupancy of integrin adhesion receptors, but genetic
alterations can lead to constitutive signaling even in the absence of growth
factors. The PI3K-Akt pathway can be activated through amplification or
activating mutations affecting several PI3K-Akt-pathway proteins—the type I
PI3K isoform PIK3CA (p110a), Akt, and the adaptor protein PIK3R1—or
through deletion or inactivating mutations in the phosphatases that hydrolyze
PI3K products, such as phosphatidylinositol 3,4,5-trisphosphate, the PTEN,
and INPP4B tumor suppressors. Further downstream, mutations in the tumor
suppressors TSC1 and TSC2 hyperactivate signaling by mTORC1 (p. 91
[Laplante and Sabatini 2012]), an important target of PI3K-Akt signaling.
Similarly, the Ras-ERK pathway is activated by mutations in Ras, or its
downstream target Raf, that cause constitutive activation of these proteins or
by inactivation of GTPase-activating proteins (GAPs), such as NF1
(Cichowski and Jacks 2001), DAB2IP (Min et al. 2010), and RASAL2
(McLaughlin et al. 2013) that stimulate the hydrolysis of GTP bound to Ras
that leads to its inactivation. The transcription factor Myc is an important
downstream target of Ras-ERK signaling and many other pathways. It is
frequently amplified or overexpressed in cancer; interestingly, Myc can not
only bind to promoter regions of genes, but also enhance transcriptional
elongation of polymerase II, thus extending its effects beyond genes with
Myc-binding sites in their promoters. Myc can thus serve as a universal
amplifier of expressed genes rather than merely binding to promoters and
initiating transcription de novo (Rahl et al. 2010; Lin et al. 2012; Nie et al.
2012).
 Figure 2. The Ras-ERK and PI3K pathways.

Oncogenic mutations, amplification, or gene fusions involving upstream

tyrosine kinases lead to constitutive signaling through both the Ras-ERK and
PI3K-Akt pathways. RTKs including EGFR, ErbB2, fibroblast growth factor
receptor (FGFR), and platelet-derived growth factor receptor (PDGFR) are
mutated or amplified in a variety of cancers. Similarly, oncogenic mutations
in G-protein-coupled receptors (GPCRs) can also activate these pathways.
Finally, it is important to recognize that deregulated synthesis of growth
factors themselves plays an important role in many cancers. Inappropriate
synthesis of growth factors by cells expressing the appropriate receptor can
generate an autocrine loop driving signaling. This can also be achieved
through cleavage and release of anchored soluble growth factors by surface
ADAM proteases, which are activated downstream from oncogenic signaling
pathways (Turner et al. 2009). Alternatively, the growth factor may be
synthesized by a neighboring cell (paracrine stimulation). In both cases,
signaling via the Ras-ERK and PI3K-Akt pathways may be increased.

3 DYSREGULATION OF CELLULAR PROCESSES BY

ONCOGENIC SIGNALING
How, then, does dysregulation of cellular signaling drive cancer progression
and produce the characteristic features of tumor cells mentioned above?
Below we discuss the role of signal transduction in cancer-associated
processes, surveying the major signals involved and focusing on Ras-ERK
and PI3K-Akt signaling to illustrate how their targets influence the behavior
of the tumor cells.

4 CELL PROLIFERATION
Excessive cell proliferation is a feature of most cancers. Limited availability
of growth factors or nutrients, contact inhibition, and other feedback
mechanisms ensure that the pathways that regulate proliferation (see Fig. 3)
are normally tightly controlled. As outlined above, however, mutations in
proto-oncogenes and tumor suppressors or inappropriate synthesis of
ligands/receptors can hyperactivate these pathways, leading to activation of
the cell cycle machinery. Note that signaling targets that represent critical
components of cell cycle control mechanism can also undergo genetic
alterations in cancer; for example, the genes encoding cyclin D, cyclin E, and
CDK4 are amplified in certain cancers and the G1 restriction point inhibitor
pRB and p16 can be deleted or mutated as well.
 Figure 3. Regulation of cell proliferation by the Ras-ERK and PI3K-Akt pathways.

The Ras-ERK and PI3K-Akt pathways are important regulators of normal

cell proliferation and thus their constitutive hyperactivation can lead to
excessive proliferation. One important target of the Ras-ERK pathway is
Myc, which is phosphorylated by ERK; this leads to its stabilization by
suppression of ubiquitylation (Sears et al. 2000). Myc stimulates cell
proliferation by inducing numerous genes that promote cell proliferation,
including those encoding G1/S cyclins, CDKs, and the E2F-family
transcription factors that drive the cell cycle (Ch. 5 [Duronio and Xiong
2013]). In addition, it represses expression of various cell cycle inhibitors
(e.g., CKIs), blocks the activity of transcription factors that promote
differentiation (see below), induces genes that enhance translation, and shifts
cells to anabolic metabolism. ERK also phosphorylates numerous other
transcription factors important for cell proliferation. Elk1, for example, in
combination with the SRF transcription factor, induces the immediate early
gene FOS, whose product is also stabilized by ERK phosphorylation
(Murphy et al. 2002). FOS, also an oncogene, encodes a component of the
transcription factor AP1, which regulates many genes involved in cell
proliferation.
Multiple kinases in the ribosomal S6 kinase (RSK), mitogen- and stress-
activated kinase (MSK), and mitogen-activated protein kinase (MAPK)-
interacting kinase (MNK) families are also phosphorylated by ERK, and
these kinases, in turn, phosphorylate transcription factors that regulate cell
cycle progression, for example, FOS and CREB (Roux and Blenis 2004).
MSKs represent the predominant kinases responsible for the nucleosomal
response involving phosphorylation of histone H3 at S10, which is commonly
induced by mitotic stimuli (Soloaga et al. 2003). MNKs play an important
role regulating translation following mitogenic stimulation by
phosphorylating the translation initiation factor eIF4E, and loss of the MNK
phosphorylation site completely abrogates its ability to transform cell lines or
promote tumors in animal models (Soloaga et al. 2003). Activation of RSK
family members by ERK also leads to activation of the mammalian target of
the rapamycin (mTOR) pathway through TSC2 phosphorylation and relief of
mTOR inhibition. In addition, RSK regulates translation by phosphorylating
eIF4B, which increases its interaction with the translation initiation factor
eIF3. The promotion of translation by these mechanisms is important for cell
growth and, consequently, cell proliferation.
PI3K-Akt signaling controls cell proliferation at various levels. Akt
regulates cell growth during cell cycle progression by controlling mTORC1.
It inhibits the GAP activity of the TSC1–TSC2 complex toward Rheb, thus
allowing GTP-bound Rheb to activate mTORC1. This then phosphorylates
eIF4-binding protein, releasing the eIF4E cap-binding factor and allowing it
to bind mRNAs, and p70 RSK. This promotes increased protein synthesis,
which is critical for enhanced cell growth during cell cycle progression
(Richardson et al. 2004). Akt also phosphorylates the kinase GSK3,
inhibiting its catalytic activity. Phosphorylation of cyclin D and Myc by
GSK3 targets them for degradation; thus, inhibition of this kinase by Akt
causes stabilization of these important cell cycle regulators (Diehl et al. 1997;
Sears et al. 2000).
In addition, Akt inhibits several cell cycle inhibitors, such as the CKIs
p27 (also known as KIP1) and p21 (also known as CIP1); phosphorylation
leads to their sequestration in the cytoplasm by 14-3-3 proteins. In the case of
p27, phosphorylation also targets it for degradation. Akt-mediated
phosphorylation of p21 prevents it from forming a complex with proliferating
cell nuclear antigen (PCNA) to inhibit DNA replication, reduces its binding
to CDK2/CDK4, and attenuates its inhibitory activity toward CDK2 (Rossig
et al. 2001). Furthermore, Akt blocks FoxO-dependent transcription of cell
cycle inhibitors such as p27 and RBL2 (retinoblastoma-like protein 2)
(Burgering and Medema 2003). It also phosphorylates and activates MDM2
(Ogawara et al. 2002), a ubiquitin ligase that promotes degradation of p53,
thereby releasing a key brake on the cell cycle. Later on in the cell cycle, Akt
can regulate several enzymes involved in the G2/M transition (Xu et al.
2012b).
Phosphorylation and consequent inhibition of GSK3 by Akt may, in
certain contexts, lead to stabilization and nuclear translocation of the Wnt
target β-catenin (Haq et al. 2003; Korkaya et al. 2009; Ma et al. 2013), a
transcriptional regulator whose degradation would otherwise be promoted by
GSK3 (Polakis 2001; Korkaya et al. 2009). This leads to induction of β-
catenin target genes that regulate proliferation, including those encoding Myc
and cyclin D. Akt can also phosphorylate β-catenin directly, causing its
dissociation from cadherin cell–cell adhesion complexes (see below), thus
increasing the pool of β-catenin available and its transcriptional activity
(Fang et al. 2007).
Numerous other signaling pathways can, of course, drive cell proliferation
in cancer. Cytokine and RTK signaling, for example, activate STAT3, which
stimulates synthesis of Myc and cyclin D (p. 117 [Harrison 2012]). Notch,
Wnt/β-catenin, and Hedgehog, all of which have been implicated in cancer,
also induce Myc and cyclin D (see below). Similarly, the transcription factor
NF-κB, which can be activated by TNF and various other signals, also targets
cyclin D expression. Cyclin E is induced by several of these signals. Estrogen
signaling (see p. 129 [Sever and Glass 2013]) stimulates cell proliferation via
activation of the ERα subtype, which induces cyclin D and Myc. Disruption
of the balance between ERα and ERβ or mutations in ERα that yield
truncated proteins or activated proteins can dysregulate this pathway
(Thomas and Gustafsson 2011; Li et al. 2013; Robinson et al. 2013; Toy et al.
2013). Note that signaling through ERs and the androgen receptor (AR) is
coupled to and enhanced by Ras-ERK and PI3K-Akt signaling (Castoria et al.
2004; Renoir et al. 2013). Growth factor stimulation (e.g., EGF and insulin-
like growth factor, IGF) and mutations that activate these pathways increase
proliferation of ER/AR-dependent tumors. In addition, these steroid receptors
form cytoplasmic complexes with Src and PI3K, which leads to activation of
their downstream effectors, and ERK can phosphorylate ERα, which causes
its activation in the absence of ligand and stimulation of cell proliferation.
The tumor suppressors that normally hold proliferative signaling in check
are obviously also critical. Furthest downstream, pRB normally directly
inhibits the transcriptional activity of the E2F proteins until it is deactivated
through phosphorylation by CDKs. p53, in contrast, normally blocks cell
proliferation in response to stress signals such as DNA damage by inhibiting
CDK activity via induction of CKIs. Consequently, mutations in this tumor
suppressor deregulate cell proliferation under potentially dangerous, cancer-
promoting conditions. The CKIs themselves directly inhibit CDKs and are
also inactivated by mutation in many cancers, p16 being the most common
example. Further upstream are pathway-specific tumor suppressors, such as
the Ras-GAP NF1 and APC, which block Wnt/β-catenin signaling (by
promoting GSK3 phosphorylation and, consequently, ubiquitin-dependent
destruction of β-catenin). In each case, mutation of the tumor suppressor
removes an important brake, allowing cells to proliferate despite signals that
would ordinarily restrain them. The Hippo pathway plays a critical role in
regulating contact inhibition of proliferation (p. 133 [Harvey and Hariharan
2012]), and disruption of this pathway, which suppresses the transcriptional
coactivator YAP, is emerging as a key tumor suppressor pathway in many
cancers (Harvey et al. 2013; Lin et al. 2013; Yu and Guan 2013). The Ras-
ERK and PI3K-Akt pathways intersect with Hippo pathway components to
inactivate its tumor suppressive activity (O’Neill and Kolch 2005; Kim et al.
2010; Collak et al. 2012).
5 CELL SURVIVAL
Cell death functions as a homeostatic mechanism that normally controls cell
number. It is also a built-in cancer-protection mechanism that is activated
during initial stages of oncogenesis because of stresses associated with
unbalanced proliferative signals, excessive cell proliferation, loss of
anchorage to natural niches, etc. Mutations that disable cell-death signaling
can thus play an important role in cancer. Overexpression of the antiapoptotic
protein Bcl2, for example, can occur as a consequence of chromosomal
rearrangements in B lymphocytes, and this contributes to follicular
lymphoma by preventing cells from undergoing apoptosis. p53 also regulates
apoptosis, both by inducing transcription of proapoptotic regulators and
binding directly to the proapoptotic protein Bax (Ch. 19 [Green and Llambi
2014]). Loss of this tumor suppressor through mutation can therefore
contribute to cancer by reducing cell death, as well as disabling normal cell
cycle control. Other cell death regulators that are mutated in cancer include
the proapo-ptotic proteins Puma and Bok (which are frequently deleted) and
the antiapoptotic proteins Mcl1 and Bcl-xL (whose genes are amplified).
Control of proapoptotic regulators (e.g., Bim and Bad) and antiapoptotic
regulators (e.g., Bcl2 and Mcl1) in normal cells ensures that cells undergo
apoptosis in the absence of appropriate signals supplied by growth factors or
the tissue microenvironment. Hyperactivation of signaling by oncogenic
mutations in the Ras-ERK and PI3K-Akt pathways, however, disrupts the
balance in favor of antiapoptotic signals, thus contributing to tumor cell
survival and abnormal expansion of the cells beyond normal tissue
boundaries.
The PI3K-Akt and Ras-ERK pathways regulate cell death in multiple
ways (Fig. 4) (review Cagnol and Chambard 2010; Zhang et al. 2011). Akt
itself intervenes at several steps in apoptotic signaling from death receptors. It
phosphorylates forkhead-family transcription factors such as FoxO3A, which
leads to their cytoplasmic sequestration by 14-3-3 proteins, thereby blocking
induction of death ligands (e.g., FasL and TRAIL) and the proapoptotic Bcl2-
family member Bim. Akt and the ERK-regulated kinase RSK also
phosphorylate the proapoptotic Bcl2-family protein Bad, another target for
sequestration by 14-3-3 proteins. In addition, Akt phosphorylates and thereby
activates the apoptosis inhibitor XIAP. Akt also activates NF-κB, which
regulates multiple survival factors, including antiapoptotic proteins (Bcl2,
BCLxl, Mcl1) and the intracellular death receptor inhibitor FLIP (Shen and
Tergaonkar 2009). Last, Akt-induced ubiquitylation and degradation of p53
suppresses p53-induced apoptosis (Ogawara et al. 2002).

Figure 4. Regulation of cell death by Ras-ERK and PI3K-Akt pathways.

ERK phosphorylates Bim and the NF-κB inhibitor IκBα (Ghoda et al.
1997), which targets them for degradation. In addition, RSK phosphorylates
the caspase-9 scaffolding protein APAF, which impedes the ability of
cytochrome c to nucleate apoptosome formation and activate the downstream
caspases that drive apoptosis (Kim et al. 2012).

6 CELL METABOLISM
Cell growth needs to be coordinated with metabolic processes involved in the
synthesis of macromolecules. Thus, growth factor pathways that regulate
both normal and tumor cells impinge on metabolic pathways to program cells
to meet the increased need for synthesis of macromolecules to produce new
daughter cells (Ch. 7 [Ward and Thompson 2012b]). Activation of oncogenes
and loss of tumor suppressors can directly regulate components of metabolic
pathways even in the absence of growth factors and, thereby, produce similar
metabolic alterations (Fig. 5).
 Figure 5. Regulation of metabolism by Ras-ERK and PI3K-Akt signaling. 1DH*, mutated 1DH.

The most common metabolic alteration in cancer cells is increased

glucose uptake and glycolysis. At first glance, this might appear a
disadvantage because glycolysis generates less ATP than oxidative
phosphorylation; however, it allows cells to redirect carbon skeletons from
glycolysis to anabolic reactions, such as the pentose phosphate pathway,
which leads to nucleotide synthesis and regulates redox homeostasis. These
also include the serine/glycine synthesis pathway, which generates several
amino acids and charges tetrahydrofolate with a methyl group that is used in
pyrimidine synthesis and leads to generation of S-adenosylmethionine, the
methyl donor for multiple cellular methyltransferase reactions and
methylation of essential molecules such as DNA, RNA, proteins,
phospholipids, creatine, and neurotransmitters. Cancer cells show increased
glutamine uptake and glutaminolysis to support oxidative phosphorylation
and biosynthesis of proteins, lipids, and nucleic acids. They also up-regulate
lipid synthesis by redirecting citrate from the Krebs cycle to fatty acid
synthesis.
The PI3K-Akt pathway targets numerous substrates to promote these
metabolic changes (Plas and Thompson 2005). Regulation of glucose
transport and hexokinase by Akt promotes glycolysis, leading to generation
of nucleotides and amino acids necessary for cell growth (Engelman et al.
2006). Akt2 regulates glucose transport through multiple mechanisms.
Regulation of the glucose transporter GLUT4 by Akt2 is critical for
circulating glucose homeostasis. The Akt substrate AS160 plays an undefined
role in insulin-stimulated GLUT4 translocation and glucose transport through
its Rab-GTPase-activating domain (Miinea et al. 2005), and phosphorylation
of the protein synip by Akt2 triggers its dissociation from the trafficking
regulator syntaxin 4 and assembly of a protein complex that mediates
translocation of GLUT4 vesicles to the plasma membrane (Yamada et al.
2005). Akt2 also regulates transcription, accumulation (Barthel et al. 1999;
Jensen et al. 2010), and trafficking of GLUT1, which is the principle glucose
transporter expressed in most cell types (Wieman et al. 2007).
Phosphorylation of TSC2 by Akt affects metabolism through mTORC1-
mediated regulation of glycolysis; however, the mechanism of regulation is
not known. mTORC1 may regulate glycolysis by increasing translation of
glycolytic enzymes or their transcriptional regulators, such as Myc (Kim et
al. 2004; Sutrias-Grau and Arnosti 2004). Other Akt targets activated by
phosphorylation are hexokinase II, whose association with mitochondria is
increased (Roberts et al. 2013), and 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphatase (Novellasdemunt et al. 2013). Both stimulate glycolysis.
mTORC1 signaling leads to increased synthesis of the transcription factor
hypoxia-inducible factor (HIF1). HIF1 induces glycolytic enzymes and
lactate dehydrogenase (LDH-A), providing another means of stimulating
glycolysis. In addition, it induces pyruvate dehydrogenase kinase (PDK),
which inhibits pyruvate dehydrogenase (PDH) in the mitochondrion and
thereby reduces flux from glycolysis into the Krebs cycle. mTORC1 can also
stimulate pyrimidine biosynthesis via S6K1 (Ben-Sahra et al. 2013;
Nakashima et al. 2013).
Akt/mTORC1 promotes lipid synthesis by activating the transcription
factor sterol-response element-binding protein 1 (SREBP), a key regulator of
lipid synthesis that is required for tumorigenicity (Bakan and Laplante 2012;
Jeon and Osborne 2012; Guo et al. 2013). Loss of SREBP uncouples fatty
acid synthase activity from stearoyl-CoA-desaturase-1-mediated desaturation.
Another direct target of Akt is ATP-citrate lyase (ACL), an enzyme that
converts citric acid to acetyl-CoA, which is required for fatty acid,
cholesterol, and isoprenoid synthesis. mTORC1 also regulates amino acid
uptake by stimulating translocation of amino acid transporters from
intracellular vesicles to the plasma membrane (Berwick et al. 2002; Edinger
and Thompson 2002).
Another family of Akt targets that affect cellular and organismal
metabolism is FoxO transcription factors. These are negatively regulated by
Akt phosphorylation, which causes their sequestration in the cytoplasm by
14-3-3 proteins. Programs regulated by FoxO transcription factors that
increase the cellular capacity for oxidative metabolism are, thus, shut off by
active Akt.
Ras-ERK signaling exerts many of its effects on metabolism via Myc.
Myc regulates glucose uptake, glycolysis, and the pentose phosphate pathway
(Ying et al. 2012) and induces synthesis of glutamine transporters and the
enzyme glutaminase (GLS), which converts glutamine into glutamate that can
be metabolized in mitochondria (Miller et al. 2012; Dang 2013). It also
induces enzymes involved in nucleotide and amino acid synthesis.
The glycolytic enzyme pyruvate kinase is of particular interest in cancer
cells. Although glycolysis rates are usually much higher than in noncancer
cells, most cancer cells produce an alternative splice form of pyruvate kinase
(PKM2) that is less active than the enzyme (PKM1) found in most terminally
differentiated cells (Vander Heiden et al. 2009). PKM1 remains active under
most physiological conditions, but PKM2 can be turned off by signaling via
tyrosine kinases, including the upstream RTKs in the Ras-ERK and PI3K-Akt
pathways (Christofk et al. 2008; Hitosugi et al. 2009) and reactive oxygen
species (ROS) (Anastasiou et al. 2011). Cancer cells can thus redirect the flux
of glycolytic intermediates into anabolic pathways for ribose, serine, and
glycine production or production of NADPH and glutathione needed to
combat oxidative stress. PKM2 can also enter the nucleus and play a role in
gene expression (Luo et al. 2011; Gao et al. 2012). However, deletion of
PKM2 accelerates rather than impairs breast tumor formation, which
indicates that it is the ability to turn off PKM2 activity that is most critical for
tumor growth (Israelsen et al. 2013).
Clearly oncogene activation or loss of tumor suppressors such as PTEN
and NF1 can drive these metabolic changes by dysregulating PI3K-Akt and
Ras-ERK signaling. Other tumor suppressors also control cell metabolism,
however. p53, for example, down-regulates glycolysis by inducing TIGAR,
an enzyme that decreases the levels of the glycolytic activator fructose 2,6-
bisphosphate. It also stimulates expression of SCO2, which is required for
assembly of cytochrome c oxidase and promotes oxidative phosphorylation.
Loss of p53 may therefore contribute to the glycolytic phenotype of cancer
cells. p53 also regulates glutaminase 2, a metabolic enzyme that controls
production of glutamate, which is converted to α-ketoglutarate for
mitochondrial respiration and, importantly, glutathione, a critical cellular
antioxidant (Hu et al. 2010; Suzuki et al. 2010). Loss of p53 leads to
increased levels of ROS and oxidative damage. p53 also regulates the
mevalonate pathway that controls cholesterol synthesis and generates
intermediates needed for protein geranylgeranylation and farnesylation.
Drugs that target this pathway to control cholesterol/ cardiovascular disease
have been shown to suppress tumor growth (Shibata et al. 2004; Kubatka et
al. 2011).
Similarly, loss of the tumor suppressor LKB1 can lead to metabolic
alterations. LKB1 activates AMP-activated protein kinase (AMPK), which
acts as a cellular energy regulator and inhibits mTORC1 (Ch. 14 [Hardie
2012]). Loss of LKB1 relieves this inhibition, allowing mTORC1 to promote
protein synthesis and lipogenesis. Activators of AMPK, such as metformin,
are currently being used in diabetes and cancer therapy.
Finally, mutations associated with cancer can lead to the elevation of
metabolites uniquely elevated in cancer cells (Kaelin and McKnight 2013).
For example, mutations in IDH1 and IDH2 result in the production of 2-
hydroxyglutarate (2HG), a metabolite not present at significant levels in
normal cells. 2HG inhibits α-ketoglutarate-dependent enzymes such as the
TET family, which regulate DNA methylation, and Jumonji C domain
histone demethylases. This leads to epigenetic dysregulation that can drive
tumorigenesis. Other oncometabolites may include succinate and fumarate,
whose levels can increase because of mutations in succinate dehydrogenase
and fumarate hydratase. Both can inhibit the activity of prolyl hydroxylases
that control HIF levels, leading to induction of PDK and the other glycolytic
enzymes mentioned above.

7 CELL POLARITY AND MIGRATION

As tumors progress toward malignancy, the cancer cells frequently become
more migratory and develop the capacity to invade surrounding tissue. This is
usually accompanied by changes in adhesion, cell polarity, cytoskeletal
dynamics, and morphology. Migration is regulated by growth factors,
chemokines, adhesion receptors, and other stimuli (Vicente-Manzanares and
Horwitz 2011; Ch. 8 [Devreotes and Horwitz 2014]), many of which are
targets for dysregulated signaling in cancer. The PI3K-Akt and Ras-ERK
pathways regulate migration and invasion through multiple downstream
effectors, including the following (Cain and Ridley 2009):
1. Rho-family GTPases (RhoA, Rac1, Cdc42, ARF6), which control
cytoskeletal regulators such as WAVE/WASP-family members, the
Arp2/3 complex, formins, the actomyosin contractile machinery, the
kinase LIMK, and cofilins (Raftopoulou and Hall 2004);
2. Integrins and associated matrix adhesion proteins (e.g., FAK, paxillin,
and calpains) (Ch. 8 [Devreotes and Horwitz 2014]);
3. Extracellular proteases, which degrade ECM proteins, facilitating tumor
cell invasion by creating space for cells to move and reducing adhesive
 contacts that may constrain them, and also release various bioactive
molecules anchored in the ECM (see below);
4. Cell–cell adhesion complexes, whose components are regulated through
modulation of their stability or protein interactions that affects the
strength of adhesion;
5. Transcription factors such as AP1 and Ets2 that regulate expression of
many proteins that control migration/polarity, including matrix
metalloproteinases (MMPs), plasminogen activator, cadherins, and actin
regulators.

As with other processes regulated by oncoprotein signaling, the outcome

of alterations in these pathways is highly context and isoform dependent. For
example, Akt1 specifically suppresses migration in many contexts through
inhibition of ERK, the transcription factor NFAT, TSC2, or phosphopalladin-
induced actin bundling, whereas Akt2 promotes migration through regulation
of integrin expression and effects on the epithelial–mesenchymal transition
(EMT) (see below; Chin and Toker 2011). Similarly, some isoforms of ERK
target RSK to promote cell motility and invasion by altering transcription and
integrin activity, whereas others impair cell motility and invasion through
effects on the actin cytoskeleton (Sulzmaier and Ramos 2013).
Polarity proteins are critical regulators of tissue architecture. Three
protein complexes play central roles in controlling polarity: Scribble, Par, and
Crumbs complexes. Through multiple interactions, components of these
pathways control signaling pathways that regulate cell polarity and tissue
organization. Dysregulation of these pathways is common in tumors and, in
some contexts, involves alterations in Ras-ERK and PI3K-Akt signaling. For
example, Scribble inhibits ERK activation by functioning as a scaffold to link
it with the protein phosphatase PP1γ (Dow et al. 2008; Nagasaka et al. 2013).
Loss of Scribble enhances invasion stimulated by H-Ras (Shaikh et al. 1996)
and tumor formation promoted by Ras and Myc (Wu et al. 2010). Similarly,
loss of the polarity protein Par3 leads to increased invasion in several tumor
models (Iden et al. 2012; McCaffrey et al. 2012; Xue et al. 2013) through
multiple pathways, including PKC-dependent activation of JAK/STAT3
signaling. This induces expression of a metalloproteinase, MMP9, with
subsequent destruction of the ECM and invasion (McCaffrey et al. 2012), and
increased Rac activation, leading to decreased cell–cell adhesion (Xue et al.
2013).
Loss of cell polarity is often coupled to cell proliferation because the loss
of cell adhesion molecules relieves contact inhibition. One example is the
cytoskeletal protein merlin (also known as neurofibromin 2), a tumor
suppressor that regulates the Hippo pathway and whose loss is well known to
cause increased cell proliferation. Polarity signaling is also coupled to
metabolism.
Some subpopulations of epithelial cells in tumors, particularly those at
tumor margins, undergo at least a partial EMT. EMTs are associated with
various normal physiological processes, for example, wound healing,
gastrulation, and branching morphogenesis (Birchmeier and Birchmeier
1995). This developmental process is orchestrated by multiple highly
coordinated pathways induced by combinations of different factors including
transforming growth factor β (TGFβ), TNF, Wnt, Notch, and some growth
factors. EMT is characterized by a loss of apical-basal polarity, down-
regulation of E-cadherin cell–cell adhesion molecules, adoption of a more
fibroblast-like appearance, and, in some contexts, acquisition of stem- or
progenitor-cell phenotypes and anchorage independence, properties that
would enhance the cell’s ability to invade other tissues and initiate tumors at
distant sites.
The Ras-ERK and PI3K-Akt pathways drive the EMT in certain contexts,
generally under conditions in which these pathways are hyperactivated
together with other pathways implicated in EMT (e.g., TGFβ, Wnt, and
Notch signaling) (Larue and Bellacosa 2005). Multiple transcription factors,
such as Snail, Slug, Twist, and ZEB, play critical roles driving EMT, and
these are regulated by ERK and Akt. For example, Akt can phosphorylate the
IκB kinases that regulate NF-κB, a transcription factor that induces Snail. Akt
also phosphorylates and inactivates GSK3, which normally promotes
ubiquitin-dependent degradation of Snail (Doble and Woodgett 2007); Akt
activation will therefore increase Snail stability, further promoting EMT. In
addition, Akt2 phosphorylates HNRNP E1, a protein that promotes
translational elongation on EMT-promoting transcripts such as those
encoding interleukin-like EMT inducer and the adaptor protein DAB2
(Hussey et al. 2011). AP1, which is regulated by the Ras-ERK pathway, can
also induce transcription factors that promote EMT as well as other gene
expression programs that control phenotypic changes associated with EMT.
These include up-regulation of specific integrin heterodimers (e.g., α5β1 and
αVβ6), vimentin, and fibronectin and down-regulation of cytokeratin, polarity
proteins (e.g., Crumbs, PATJ, LGL), and E-cadherin, all of which support
cell motility. Interestingly, the polarity protein Scribble maintains cell–cell
junctions by suppressing ERK (which stimulates ZEB1) as described above
(Elsum et al. 2013; Nagasaka et al. 2013). Dysregulation of both Ras-ERK
and PI3K-Akt signaling thus has the potential to play an important role in
cancer progression by promoting adoption of an invasive phenotype.
Finally, it is important to note that EMT is not essential for invasion and
tumor cell dissemination. Tumor cells can migrate as epithelial sheets within
tissues (as occurs during wound healing) or invade by pushing through tissue
borders (e.g., basement membrane).

8 CELL FATE AND DIFFERENTIATION

Dysregulation or co-option of developmental signaling pathways is a feature
of many cancers. This can disrupt the balance between cell proliferation and
differentiation, alter cell fate, and/or inappropriately induce morphogenetic
programs such as the EMT (see above) that promote metastasis. Although
some oncogenes can directly regulate the developmental state of cells, it is
generally believed that cancer progression requires a self-renewing
population of “stem-cell-like” cells. These may be induced into a stem-cell-
like state by an oncogene(s), or a normal stem/progenitor cell may be the cell-
of-origin that sustains the successive mutations that lead to malignancy.
The simplest examples of cancers with dysregulated development are
perhaps hematopoietic malignancies in which a differentiation program is
stalled before the cells reach their nonproliferative differentiated state. For
example, in acute promyelocytic leukemia, a form of acute myelocytic
leukemia, myeloblasts fail to differentiate into mature white blood cells
because of a translocation that leads to synthesis of a fusion protein
combining sequences from a protein called PML and the retinoic acid
receptor (RAR). The PML-RAR fusion protein represses RAR-target genes
that normally drive differentiation, thereby inactivating the RAR signaling
that normally controls this. Subsequently, additional mutations cause
overproliferation of the undifferentiated myeloblasts. Inappropriate Wnt
signaling has a similar effect in colon cancer. Ordinarily, Wnt signaling via
β-catenin (see p. 103 [Nusse 2012]) maintains enterocytes in an
undifferentiated state in colon crypts, but is inactivated by APC-induced
degradation of β-catenin as cells move up toward the luminal surface of the
intestine. Mutation of the APC tumor suppressor in colon cancer, however,
means β-catenin is not destroyed and can maintain cells in an undifferentiated
state as they move away. Further mutations can then drive neoplasia.
Developmental signals can also drive cancer progression because they
stimulate inappropriate cell proliferation (see above). Mutations that activate
Notch, for example, contribute to acute lymphocytic leukemia because Notch
signaling (p. 109 [Kopan 2012]) can stimulate the cell cycle and also inhibits
apoptosis in T cells. Importantly, Notch functions as a tumor suppressor in
some other tissues. In others, the concentration of Notch dictates its growth
suppressive or stimulatory effects (Mazzone et al. 2010), which illustrates the
importance of the signaling context. Activation of the Hedgehog signaling
pathway (see p. 107 [Ingham 2012]) by mutations in the Patched receptor
occurs in basal cell carcinomas and medulloblastomas and again drives cell
proliferation. Hedgehog signaling is also hyperactivated via autocrine loops
in many tumors that affect tissues derived from the embryonic gut.
Given that Ras-ERK and PI3K-Akt signaling pathways are activated by
growth factors such as EGF, IGF, and fibroblast growth factor (FGF), which
play major roles in control of cell fate, they can thus be considered
developmental signaling pathways that are hijacked in cancer. Signaling by
FGF4/8, for example, activates the Ras-ERK pathway to drive EMT during
gastrulation and the Ras-ERK pathway is recapitulated in several cancers
(Thiery 2002). The context is important, however; signaling by FGF has the
potential to affect cell proliferation, apoptosis, and migration (see above), as
well as angiogenesis (see below), but it can also have tumor suppressive
effects, maintaining cells in a differentiated, nonproliferative state. For
example, whereas FGFR2 is up-regulated in gastric cancers, its expression is
reduced in bladder and prostate cancer (Turner and Grose 2010).

9 GENOMIC INSTABILITY
Genomic instability is a common characteristic of cancer cells. Aneuploidy
and large-scale DNA rearrangements are frequently observed, and many
cancers display elevated mutation rates. Ordinarily, a variety of cellular
enzymes repair DNA damage, and checkpoint signaling ensures that DNA
replication and cytokinesis are arrested in dividing cells until potentially
damaging errors are corrected. Alternatively, checkpoint signaling can induce
senescence or apoptosis so that affected cells do not pass on these errors.
Whether genomic instability is a cause or a consequence of cancer is still
debated, but it clearly reflects a failure of checkpoint signaling and/or DNA
repair mechanisms.
DNA damage signals are relayed by the kinases ATM, ATR, Chk1, and
Chk2, which stimulate p53, stall the cell cycle, and activate the DNA repair
machinery (p. 109 [Kopan 2012]; Ch. 6 [Rhind and Russell 2012]).
Downstream of p53, the CKI p21 is induced, and this can halt DNA
polymerase if DNA replication has already begun. If the damage cannot be
repaired and checkpoint signaling persists, p21 and p53 will induce cells to
senesce or undergo apoptosis (see above). Clearly, mutation or epigenetic
silencing of these tumor suppressors or upstream kinases can inactivate
checkpoint signaling, allowing DNA damage to persist and potentially fuel
cancer progression. Indeed, ATM and Chk2 mutations are seen in familial
leukemias and colon/breast cancers, respectively, and proteins involved in
DNA repair itself are also often mutated, for example, MMR enzymes and
BRCA1/2.
The mitotic checkpoint (also known as the spindle assembly checkpoint)
ensures that when a cell divides each daughter receives a full complement of
chromosomes. A complex containing the proteins Bub1, Bub3, and Mad1-3
monitors attachment of chromosomes to the mitotic spindle, relaying
checkpoint signals that block chromosome segregation and subsequent
cytokinesis. Once paired, sister chromatids are all attached to microtubules
emanating from opposite poles, the signal is switched off, and cells can move
from metaphase into anaphase and, ultimately, cytokinesis can proceed (Ch. 6
[Rhind and Russell 2012]). Inactivation of this checkpoint pathway has the
potential to lead to aneuploidy, and mutations in Mad1/2 and Bub1 have been
observed in cancer (Schvartzman et al. 2010).
Akt has been implicated in multiple aspects of DNA damage responses
and genome instability (Xu et al. 2012a). It can inhibit homologous
recombinational repair through direct phosphorylation of the checkpoint
proteins Chk1 and TopBP1 or indirectly through recruitment of resection
factors such as RPA, BRCA1, and Rad51 to sites of double-stranded breaks
(DSBs) in DNA. Akt is also activated by DSBs in a DNA-dependent protein-
kinase- or ATM/ATR-dependent manner and, in some contexts, can
contribute to radioresistance by stimulating DNA repair by nonhomologous
end joining. In addition, Akt also inhibits association of BRCA1 with DNA
damage foci. As discussed above, dysregulation of the PI3K-Akt pathway
suppresses apoptosis through many effectors, thus promoting survival of cells
with DNA damage. Because Ras-ERK signaling also inhibits apoptosis, it too
could promote survival of damaged cells. Hyperactivation of Ras-ERK
signaling has been shown to lead to genomic instability, although the
molecular mechanism is unclear (Saavedra et al. 1999). Akt therefore
modifies both the response to and repair of genotoxic damage in complex
ways that are likely to have important consequences for the therapy of tumors
showing deregulation of the PI3K-Akt pathway.
The tumor suppressor PTEN can also regulate chromosome stability,
independently of its 3′-phosphatase activity. PTEN regulates the expression
of the DNA repair protein RAD51, and loss of PTEN causes extensive
centromere breakage and chromosomal translocations (Toda et al. 1993; Liu
et al. 2008; p. 109 [Kopan 2012]).
Myc overexpression can induce genomic instability. In mammalian cells
and Drosophila, overexpression of Myc increases the frequency of
chromosomal rearrangements (Prochownik and Li 2007; Greer et al. 2013).
Multiple mechanisms have been associated with such genomic
rearrangements, including ROS-induced DSBs, suppression of checkpoints
that prevent replication of damaged DNA, and telomere clustering.
10 THE TUMOR MICROENVIRONMENT
So far, we have primarily considered how signaling within cancer cells
themselves is dysregulated in cancer. However, cancer progression (at least in
solid tumors) also depends on the ECM, blood vessels, immune cells, and
noncancerous cells such as fibroblasts in the tumor microenvironment, all of
which communicate with cancer cells by subverted signaling mechanisms
(Fig. 6). Many of the changes in the tumor microenvironment during cancer
progression mimic changes that occur during wound healing and/or
developmental processes. As tumors evolve, the complexity of their
“ecosystem” increases; reciprocal paracrine and juxtacrine interactions
between populations of neoplastic cells as well as tumor cells and
nonneoplastic cells within the microenvironment control cellular signaling
pathways in both positive and negative fashions. Dissecting the roles of
individual signaling pathways in these ecosystems is complex because it is
difficult to distinguish cell-autonomous and non-cell-autonomous activities.
Figure 6. Cancer signaling networks. The figure illustrates the wide variety of intra- and intercellular
signals affected in cancer, focusing on Ras-ERK and PI3K-Akt signaling. It is by no means
comprehensive; many more pathways are involved and there are other stromal cells involved in
paracrine signaling. Oncoproteins are indicated with yellow highlighting; tumor suppressors are
indicated with dashed outlines. Arrows do not necessarily indicate direct interactions in this figure.

10.1 The ECM

The ECM is a scaffold that physically supports tissues and provides a
substrate for cell adhesion and migration, as well as a source of bioactive
molecules. Far from a static structure, it is constantly being remodeled, and
its composition plays a critical role in control of cell behavior. Fibronectin,
laminin, collagen, and various other ECM components serve as ligands that
activate integrin signaling. Integrin signaling leads to activation of canonical
pathways such as Ras-ERK, PI3K-Akt, and Src signaling, as well as other
proteins, for example, the tyrosine kinase FAK, a scaffold that links integrins
with cytoskeletal proteins, adaptors, and enzymes that transduce signals from
matrix adhesion complexes. FAK also regulates p53 and members of the
miR-200 family of microRNAs, which control apoptosis and epithelial
phenotype (Keely 2011).
Heparin sulfate proteoglycans (HSPGs) in the ECM modulate signaling
by associating with various ligands and acting as coreceptors (e.g., for FGF
and FGFR). In addition, the ECM actively sequesters a variety of growth
factors, including TGFβ, vascular endothelial growth factor (VEGF), and
platelet-derived growth factor (PDGF), which can be liberated and/or
activated by MMPs. Collagens can also be digested and remodeled by
proteinases to enhance tumor cell motility.
The ECM changes as cancer progresses (Lu et al. 2011). It stiffens as
large quantities of ECM are deposited by cancer-associated fibroblasts (see
below) and collagen fibers become more cross-linked by lysyl oxidases
secreted by stromal cells. This increases contractility of the cells, which
further fuels stiffening. The changes in stiffness of the ECM promote cell
migration and integrin signaling through regulation of Rho family GTPases
and other pathways, which synergize with oncogene-activated Ras-ERK and
PI3K-Akt pathways to promote invasive growth and cell survival (Keely
2011).
Other changes to the ECM include increased levels of molecules such as
tenascin C, a proteoglycan common around developing blood vessels that is
induced during inflammation and promotes angiogenesis. MMPs are also up-
regulated. These stimulate signaling in various ways and, by degrading the
ECM, clear a path for cell migration. Indeed, genes encoding endogenous
inhibitors of MMPs such as TIMP3 are known to be targets for
hypermethylation in some cancers. HSPGs are also overproduced in cancer
and may potentiate oncogenic signaling by FGF, Wnt, and Hedgehog.

10.2 Angiogenesis
Like all tissues, tumors require a blood supply. They acquire this by inducing
proliferation and assembly of endothelial cells to form new blood vessels
(angiogenesis), co-opting pathways that usually function in wound healing.
Central to angiogenesis are signals such as VEGF, PDGF, FGFs, interleukin
(IL) 8, and angiopoietin. The PI3K-Akt pathway regulates the induction of
angiogenesis as well as vessel integrity (Karar and Maity 2011). Synthesis
and secretion of VEGF by cancer cells is induced by HIF1. As mentioned
above, HIF1 levels are increased by PI3K-Akt signaling, and hyperactivation
of this pathway thus plays an important role in angiogenesis. HIF1 activity
can be controlled by the von Hippel–Lindau (VHL) protein, a subunit of an
E3 ligase that promotes its ubiquitin-dependent degradation under normoxic
conditions when it is proline hydroxylated by proline hydroxylases (see Ch. 7
[Ward and Thompson 2012b]), or through translational control. VHL
functions as a tumor suppressor and inactivating VHL mutations occur in a
variety of cancers. The PI3K-Akt pathway also modulates the production of
other angiogenic factors, such as nitric oxide and angiopoietins. Constitutive
endothelial activation of Akt1 has been shown to induce the formation of
structurally abnormal blood vessels.
Following its secretion, VEGF is sequestered in the ECM and cannot
exert its effects on endothelial cells until it is released by MMPs such as
MMP9. These are produced by monocytes and macrophages in the tumor
microenvironment (see below), which underscores the importance of immune
cells in angiogenesis and existence of the wider signaling network that
involves cancer cells, immune cells, and endothelial cells.
Another factor that must be overcome for angiogenesis to occur is
inhibitory signals such as thrombospondin 1 (Tsp1). Tsp1 released by various
cells normally keeps angiogenesis in check by inducing synthesis of FasL,
which causes endothelial cells to undergo apoptosis (see Ch. 19 [Green and
Llambi 2014]). Tsp1 is induced by p53, but repressed by Ras, Src, and Myc.
It thus represents another control point in angiogenesis that could be activated
by dysregulation of the Ras-ERK pathway. Moreover, the gene that encodes
Tsp1 is hypermethylated in some cancers.

10.3 Inflammation
Inflammatory cells such as macrophages and neutrophils constitute the first
defense against pathogens, but are also involved in tissue remodeling and
repair. They are recruited by chemokines secreted by tumor and stromal cells
to almost all tumors and secrete various molecules that promote cancer cell
proliferation, survival, and migration. In many respects, the contribution of
inflammatory cells to tumor progression, like that of angiogenesis,
recapitulates their role in wound healing, which also involves these
processes.
Signaling via the transcription factor NF-κB (see Ch. 15 [Newton and
Dixit 2012]) is important in both cancer cells and tumor-associated
inflammatory cells because it can promote cell survival and proliferation and
stimulates production of cytokines such as TNF. Oncogenic mutations
affecting NF-κB or upstream regulators such as MALT1 and Bcl10 occur in
some lymphoid malignancies; however, in most cancers, NF-κB activity is
simply increased by cytokine signaling. For example, in colon cancer, TNF
produced by macrophages increases NF-κB activity in intestinal epithelial
cells, which promotes cell survival; meanwhile, other cytokines such as IL6
and IL11 increase phospho-STAT3 levels (see p. 117 [Harrison 2012]),
which promote cell proliferation. A similar phenomenon occurs in
hepatocytes in hepatocellular carcinoma, the most common form of liver
cancer, and prostate cancer (Karin 2009). NF-κB activation also leads to
production of more TNF and synthesis of prostaglandin E2, which further
fuels cell proliferation and loss of cell polarity.
In addition to cytokines, inflammatory cells secrete growth factors such as
EGF and FGF. These are obviously important regulators of Ras-ERK and
PI3K-Akt signaling in cancer cells and will, therefore, dysregulate control of
cell proliferation, cell death, metabolism, and cell migration, as discussed
above. They also lead to production of colony-stimulating factor 1 (CSF1), a
key reciprocal signal that stimulates macrophages, causing them to produce
more EGF. Immune cells also produce VEGF and MMPs, which promotes
angiogenesis, ECM remodeling, and release of other bioactive molecules (see
above). Importantly, all these factors participate in paracrine loops involving
immune and cancer cells that sustain chronic inflammation and promote
tumor growth and progression.

10.4 Cancer-Associated Fibroblasts

Fibroblasts are present in most tissues, helping to shape organs and control
the composition of the ECM. They are activated by tissue injury and, in
cancer, produce various factors that stimulate proliferation and migration of
cancer cells, along with angiogenesis and inflammation. Release of signals
such as TGFβ and PDGF by macrophages and cancer cells activates
fibroblasts, which, in turn, can release EGF, HGF, IGF, and chemokines such
as CXCR12 (also known as stromal-cell-derived factor 1). These can then
further activate Ras-ERK and PI3K-Akt signaling and other pathways in the
cancer cells and drive feedback loops that amplify this. Cancer-associated
fibroblasts are also responsible for the distinct ECM associated with
advanced carcinomas, which also affects signaling within the tumor.
The interplay between cancer cells and these different components of the
tumor microenvironment is thus incredibly complex and mimics signaling
that tissues display both during development and in normal tissue
homeostasis and repair. As cancers metastasize, this extends to other organs.
Different cancers are known to seed secondary tumors in particular tissues.
Successful colonization depends on the cell surface receptors expressed by
the cancer cells and target tissue and the suitability of the microenvironment
the latter provides.

11 CONCLUDING REMARKS
Cancer cells show a number of defining characteristics. Underlying these is a
dysregulation of cellular signal transduction induced by the genetic and
epigenetic changes that drive cancer. This affects not only the cancer cells
themselves, but the wider signaling network that encompasses other cells, the
ECM, blood vessels, and the immune system. Indeed, metastatic cancer can
be considered a systemic disease that affects signaling throughout the
affected individual, and systemic effects are ultimately what kill patients in
cancer.
Pharmacologic and antibody-based inhibitors that target signaling proteins
mutated in tumors or proteins downstream from these have had significant
impact as cancer treatments. For example, inhibitors of the nonreceptor
tyrosine kinase Abl and RTK ErbB2 dramatically reduce patient mortality in
chronic myelogenous leukemia and breast cancer. Other inhibitors, such as
those that target B-Raf, EGFR, and the kinase ALK induce remarkable
reductions of tumor volume and extend survival in patients with melanoma
and nonsmall-cell lung carcinomas; however, the rate of recurrence is high
because of the development of drug resistance (Gainor and Shaw 2013;
Giroux 2013; Holohan et al. 2013; Lito et al. 2013).
The complexity of the cancer signaling network (see Fig. 6) presents a
huge challenge for efforts to develop such anticancer drugs because of the
redundancy of pathways that control cell proliferation and survival, cross talk
between pathways, and feedback inhibition mechanisms that cause pathway
reactivation. The fact that pathways such as Ras-ERK and Akt-PI3K
signaling control so many characteristics of cancer cells, and that components
of these pathways, or upstream receptors, are so commonly mutated in a
variety of cancers gives reason to be optimistic that approaches based on
targeting them will be successful. The efficacy of therapies that target these
pathways is, however, limited by multiple factors. For example, rewiring of
signaling pathways is associated with adaptive responses to inhibition of
driver mutations, and this is commonly because of either loss of feedback
inhibition or induction of stress pathways (Pratilas and Solit, 2010; Rodrik-
Outmezguine et al. 2011; Lito et al. 2013). Moreover, factors from the tumor
microenvironment may stimulate alternative pathways that maintain cell
viability despite inhibition of the targeted pathways (Castells et al. 2012;
Muranen et al. 2012). Alternatively, there can be selection for rare tumor
cells that contain drug-resistant variants of the targeted protein or mutations
in other pathways that circumvent the dependency on the targeted pathway,
and epigenetic or stochastic changes in the state of tumor cells can also
activate intrinsic resistance pathways (Holohan et al. 2013; Holzel et al.
2013).
Further complicating matters is the degree of intratumoral genetic
heterogeneity. Recent evidence emerging from sequencing of single cells and
multiple regions of tumors from individual patients has revealed this is far
greater than previously imagined (Navin et al. 2011; Ruiz et al. 2011; Ding et
al. 2012; Gerlinger et al. 2012; Xu et al. 2012b; Bashashati et al. 2013) In one
study of kidney tumors, only ∼45% of mutations were detected in all tumor
regions. This heterogeneity also contributes significantly to intratumoral
variation in sensitivity to drugs targeting mutated signaling proteins mutated
in cancer, and means that single biopsies may not be sufficient to customize
patient treatment.
Overcoming these challenges will require a deeper understanding of the
nature of resistance mechanisms and how different cellular signaling
programs mediate resistant states in heterogeneous populations of tumor
cells. Combination therapies that target these should increase the efficacy of
targeted therapies. This is a significant challenge, but one that we feel is not
insurmountable.

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Cite this chapter as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a006098
CHAPTER 22

Outlook

Jeremy Thorner1, Tony Hunter2, Lewis C. Cantley3, and

Richard Sever4
1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
2Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California
92037
3Cancer Center, Weill Cornell Medical College, New York, New York 10065
4Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Correspondence: jthorner@berkeley.edu

SUMMARY

We have come a long way in the 55 years since Edmond Fischer and the
late Edwin Krebs discovered that the activity of glycogen phosphorylase
is regulated by reversible protein phosphorylation. As the contents of
this book attest, many of the other fundamental molecular mechanisms
that operate in biological signaling have since been characterized and the
vast web of interconnected pathways that make up the cellular signaling
network has been mapped in considerable detail. Nonetheless, it is
important to consider how fast this field is still moving and the issues at
the current boundaries of our understanding. One must also appreciate
what experimental strategies have allowed us to attain our present level
of knowledge. Therefore, we summarize here some key issues (both
conceptual and methodological), raise unresolved questions, discuss
potential pitfalls, and highlight areas in which our understanding is still
 rudimentary. We hope these wide-ranging ruminations will be useful to
investigators who carry studies of signal transduction forward during the
rest of the 21st century.

Outline
1 Signaling in the atomic age
2 Seeing is believing
3 Signaling in the postgenomic era
4 Modularity in signaling
5 Posttranslational modifications and signaling
6 Integration of cell metabolism and signaling
7 Signal diversity
8 Prospectus
References

1 SIGNALING IN THE ATOMIC AGE

Our perceptions about signal transduction processes and the functions of the
molecules involved have blossomed as a consequence of technological
advances in genomics (Rogne and Taskén 2013), mass spectrometry
(Bensimon et al. 2012), structure determination (Chiu et al. 2006), and
computational power (Chakraborty and Das 2010). The increase in structural
biology, in particular, has been extremely important. It is often helpful,
especially for newcomers, to illustrate information flow, protein–protein
interactions, and other basic concepts by using schematic diagrams.
However, the acquisition of structural information about signaling molecules
at atomic resolution has given us unprecedented insights into the way
signaling proteins operate as nano-machines to control cellular processes.
This level of detail is vital to convey to the advanced student, the practitioner
at the bench, and those attempting to develop effective therapeutics. Starting
more than 20 years ago with acquisition of the first three-dimensional picture
of a protein kinase, cAMP-dependent protein kinase (also known as protein
kinase A, PKA) (Knighton et al. 1991) and elucidation of crystal structures of
the first noncatalytic folds found in many signaling proteins—SH2 domains
(which recognize phosphorylated tyrosine residues in various sequence
contexts) (Waksman et al. 1992), and SH3 domains (which recognize PxxP
motifs and variants thereof) (Musacchio et al. 1992)—the pace of advance
has been truly remarkable.
Structural information at atomic resolution has, for example, turned our
rather naïve initial views about how ligand-induced dimerization of receptor-
tyrosine kinases leads to activation into a highly nuanced view of the
articulation and dynamics of these molecules (Jura et al. 2011; Bessman and
Lemmon 2012; Endres et al. 2013). The same is true for the cytosolic protein-
tyrosine kinases (Bradshaw 2010; Kiu and Nicholson 2012). Similarly,
deconvolution of the structures of polytopic integral membrane proteins, in
particular G-protein-coupled receptors (GPCRs), which seemed an
insurmountable technological challenge less than 10 years ago, has yielded to
new methodology and produced a flood of high-resolution structures for
these molecules (Stenkamp et al. 2005; Granier and Kobilka 2012; Katritch et
al. 2013), and even a four-protein complex of one GPCR with its cognate
heterotrimeric G protein (Rasmussen et al. 2011). However, some molecules
critical for signaling remain recalcitrant to this approach. For example,
although structural information at atomic resolution has had an enormous
impact on our level of understanding of the function and regulation of certain
classes of ion channels (MacKinnon 2003), many that have critical roles in
vision, taste, mechanical sensation (hearing and touch), pain perception, and
other aspects of neurotransmission and neurosensation have not yet been
visualized in their native full-length form (Li et al. 2011; Lau et al. 2012;
Kumar and Mayer 2013). Nonetheless, arguably, the depth of our
understanding of detailed molecular mechanisms at the atomic level has come
farther and faster for proteins involved in signal transduction than for proteins
in many other areas of biology. That pace needs to be continued.
2 SEEING IS BELIEVING
Technological advances in imaging also deserve special mention because of
the critically important information that direct visualization provides about
the localization and movement of signaling molecules in individual cells. The
demonstration that a green fluorescent protein (GFP) from a jellyfish could
be fused to a protein of interest and the resulting chimera used to observe the
concentration and subcellular distribution of signaling molecules in live cells
in real time via fluorescence microscopy was a seminal breakthrough (Chalfie
et al. 1994). The discovery of GFP provoked a hunt for additional proteins
capable of generating a fluorophore internally, such as DsRed (Miyawaki
2002), or by coupling to endogenous chromophores such as biliverdin (Shu et
al. 2009; Piatkevich et al. 2013). Successful mutational strategies allowed
modification of the spectral properties of GFP itself (Zacharias and Tsien
2006; Tsien 2009) and its relatives (Shaner et al. 2008; Piatkevich and
Verkhusha 2010). Similarly, mutagenesis of small single-chain enzymes has
yielded variants that self-label covalently with a fluorophore substituent in
the course of one catalytic turnover when provided with an appropriate cell-
permeable, dye-derivatized substrate. This approach produced the so-called
SNAP (Corrêa et al. 2013), CLIP (Gautier et al. 2008), and Halo (Encell et al.
2012) tags. It is important to emphasize, however, that studies tracking
hybrid proteins must be confirmed by independent approaches (e.g., genetic
complementation) that show they retain biological function; sadly, such
constructs are often used and conclusions drawn without this necessary
validation.
There are additional tactics for bioorthogonal labeling of proteins (Debets
et al. 2013; Herner et al. 2013). For example, unnatural amino acid (UAA)
technology (Liu and Schultz 2010; Chin 2014) offers another route for
genetically encoding a protein with a site-specific fluorescent tag. By
extending the genetic code and making the necessary changes to the
translation machinery, one can incorporate at any specified position in a
protein a 21st amino acid (either a UAA with a fluorescent side chain [Kang
et al. 2013; Niu and Guo 2013] or a UAA whose side chain can be readily
coupled to a cell-permeable fluorophore [Borrmann et al. 2012], or even two
different UAAs at distinct sites in the same protein [Xiao et al. 2013a]).
Together, all of these methods provide a researcher with a broad palette for
fluorescent labeling, permitting simultaneous interrogation of the locations of
and/or conformational changes in multiple signaling proteins in the same cell.
Such labels have become even more useful because of concomitant
advances in fluorescence imaging that have taken us well beyond
conventional epifluorescence microscopy. These include dramatic
improvements to the quality of the microscopes themselves and, most
notably, the advent of various optical and computational methods to
determine the centroid of a point source of emitted fluorescence, such as
photoactivated localization microscopy (PALM), stochastic optical
reconstruction microscopy (STORM), and stimulated emission-depletion
microscopy (STED) (Sengupta et al. 2012; Coelho et al. 2013). These
techniques (collectively, referred to as super-resolution fluorescence
microscopy) achieve a spatial resolution better than the diffraction limit of
the illuminating light (∼200 nm), providing the capability to discriminate
between molecules separated by only ∼30 nm (Huang et al. 2009). Spinning-
disk confocal microscopy, deconvolution microscopy, and two-photon
microscopy allow image reconstruction in three dimensions, providing spatial
information. Depending on the nature of the molecule, the process, and the
cellular region being examined, techniques such as total internal reflection
fluorescence microscopy or selective plane illumination microscopy can be
used to follow the movement of single molecules by illuminating only a very
thin region of the cell.
Fluorescence speckle microscopy and photoactivation microscopy use
photoconvertible forms of fluorescent protein tags to permit monitoring of
the motion of individual molecules by limiting the number of detectable
molecules in the field of view. Fluorescence recovery after photobleaching
and fluorescence lifetime imaging, meanwhile, permit measurement of the
dynamics of the population of fluorescently labeled molecules in the cell.
Förster resonance energy transfer (FRET) between two different proteins
tagged with distinct fluorescent labels with appropriate spectral overlap can
be used to determine if they ever approach each other more closely than 10
nm (Schmid and Birbach 2007; Miyawaki 2011; Zhou et al. 2012). A related
method is bimolecular fluorescence complementation, in which the amino-
terminal half of a fluorescent protein is tethered to one protein, whereas the
carboxy-terminal half is tethered to another; if the two proteins interact, they
stabilize association of the two halves of the fluorescent reporter, allowing
acquisition of its fluorescent state (Kerppola 2009; Filonov and Verkhusha
2013). Collectively, application of such methods can provide crucial
information about the spatial and temporal behavior of molecules responsible
for signaling. Microfluidic devices in which individual cells are restrained
provide a convenient means to carefully manipulate their conditions and
monitor their resulting responses by such techniques (Cheong et al. 2009).
The ability to tag a protein that undergoes a marked conformational
change on ligand binding with a fluorescent protein that undergoes a
concomitant spectral change, or tag a protein at each end with two compatible
fluorophores that undergo a FRET change in response to ligand binding or a
posttranslational modification, has permitted construction of in vivo
biosensors that report responses in individual cells, such as an increase in
intracellular calcium ion concentration (Akerboom et al. 2012; Tong et al.
2013) or activation of a particular protein kinase (Kunkel et al. 2007; Harvey
et al. 2008; González-Vera 2012). Similarly, fusion of fluorescent labels to
ion channels (Guerrero and Isacoff 2001; Mutoh et al. 2011) and
photoinduced electron transfer to membrane-seeking chemical probes (Miller
et al. 2012) provide sensitive readouts for voltage changes in individual
neurons.
Conversely, fluorescently tagged ion channels whose functional
properties can be switched reversibly between an active and inactive state in
response to laser illumination allow nondestructive manipulation of cell
behavior by light of a particular wavelength (Gorostiza and Isacoff 2008;
Kramer et al. 2013). Using genetically encoded light-controlled proteins to
monitor and manipulate the behavior of live cells in real time has been
termed optogenetics (Lima and Miesenböck 2005; Miller 2006; Fenno et al.
2011). Other molecules that undergo dramatic conformational changes on
light absorption have also been exploited, such as flavin adenine
dinucleotide-binding light-, oxygen-, or voltage-sensing (LOV) domains
(Christie et al. 2012; Renicke et al. 2013). Likewise, following pioneering
work of Peter Quail (Leivar and Quail 2011) and Clark Lagarias (Rockwell et
al. 2006) on how plant and cyanobacterial phytochromes recruit protein
cofactors (PIFs) in a light-dependent manner to regulate transcription, the
light-gated dimerization of a protein fused to an approximately 900-residue
amino-terminal chromophore-containing fragment of a phytochrome and
another protein fused to an approximately 100-residue fragment of a PIF has
been exploited to control the initiation of signaling in animal cells (Levskaya
et al. 2009; Toettcher et al. 2013). Note, however, that the necessary segment
of the phytochrome can be difficult to work with because of its large size
(nearly 100,000 kDa) and the requirement to supply exogenously its cognate
linear tetrapyrrole chromophore (either phytochromobilin or
phycocyanobilin), which does not readily enter all cell types. These
approaches owe much to methods for small-molecule-driven protein–protein
association (Crabtree and Schreiber 1996; Farrar et al. 1996), for example,
those using the immunosuppressant (and mTORC1 inhibitor) rapamycin. In
the presence of rapamycin, a protein fused to the rapamycin-binding protein
FKBP12 will interact with a protein fused to the FKBP12–rapamycin-
complex-binding domain of mTOR, and both fusion proteins can also be
marked with fluorescent tags.
Advances in instrumentation have also made possible the imaging of
bioluminescent reporters, such as luciferase, in intact tissues and whole
animals (Sadikot and Blackwell 2008; Close et al. 2011). Microscopes
equipped with ultrasensitive charge-coupled device cameras optimized for the
near infrared range and upright parcentered and parfocal optical
configurations that provide a very long working distance and high numerical
aperture make the capture of this light possible. This form of optical imaging
is low cost and noninvasive and facilitates real-time in situ visualization of
signaling. For example, luciferase, either intact or split, when fused to a
protein(s) of interest can be used for analyzing where a protein is located or
in what cells a protein–protein interaction occurs, respectively (Stynen et al.
2012); and a luciferase gene placed downstream of another gene’s promoter
can serve as a transcriptional reporter to identify the cells that respond to a
signal (Ravnskjaer et al. 2013).
Improvements in imaging have not been limited to light microscopy.
Advances in electron microscopy (EM), including electron diffraction, cryo-
EM, electron tomography, and single-particle reconstruction, allow
visualization of the arrangement of the constituent polypeptides in large
multiprotein complexes (Baumeister and Steven 2000; Frank 2009; Lander et
al. 2012). Development of enzymic probes compatible with EM fixation and
sectioning methods, such as miniSOG (Shu et al. 2011) and APEX (Martell
et al. 2012), provide means to determine where a given signaling protein is
localized at the ultrastructural level. An additional advantage of miniSOG is
that this flavoprotein can also be visualized by light microscopy in living
cells, which can then be processed at any given time during a signaling
process for subsequent analysis at the EM level (Butko et al. 2012).
Our ability to obtain a snapshot of what is happening to individual
proteins in single cells in response to an external cue is not confined to
microscopy. Recently developed mass spectrometry methods allow protein
constituents in a single cell to be quantified, using heavy-atom-labeled
antibodies against those proteins and/or specific phospho-sites on those
proteins. This method has been dubbed “mass cytometry” because
fluorophore-tagged antibody reporters are replaced with isotopically tagged
ones, and depends on the use of an inductively coupled-plasma time-of-flight
mass spectrometer, which can resolve up to 100 different rare-earth-metal-
labeled antibody probes with single-cell sensitivity (Bandura et al. 2009;
Zivanovic et al. 2013). This technology has been used successfully to define
the state of 31 different proteins in particular cell types of the hematopoietic
lineage in bone marrow (Bendall et al. 2011), examine the effect of 27 small-
molecule protein kinase inhibitors on 14 different phosphorylation sites in
human peripheral blood mononuclear cells from eight donors (Bodenmiller et
al. 2012), and identify T cells specific for particular epitopes among 77
candidate rotaviral antigens (Newell et al. 2013). However, the sophisticated
instrumentation necessary is expensive and not widely available. Moreover,
the cells being analyzed are fixed, “stained” with the antibodies, and then
destroyed in the process. Thus, cells assigned to a given signaling state by
their mass cytometry signature cannot be recovered and followed
subsequently (unlike in conventional cytometry, in which the cells can
remain viable and be separated on the basis of their fluorescence signature by
a fluorescence-activated cell sorter).
Additional technological leaps make feasible observation of single
signaling molecules in action in vitro. Standard biochemical measurements
yield an ensemble average and, even for a purified preparation, it is
frequently difficult to discern what fraction of the molecules present are
properly folded and functional. In contrast, the use of optical traps
(“molecular tweezers”) allows measurement of the biophysical properties of
an individual protein in operation (Greenleaf et al. 2007; Moffitt et al. 2008).
Similarly, in silico molecular dynamic simulations based on crystal structure
coordinates and/or nuclear magnetic resonance constraints now allow us to
predict the accessible conformational states of a signaling protein (Baker
2006; Friedland and Kortemme 2010), or how it might dock with another
protein or small molecule (Kolb et al. 2009). This approach has been made
accessible by construction of computers with the necessary speed and
capacity (Dror et al. 2012) (and/or linking many computers together via the
Internet) (Beauchamp et al. 2012) and corresponding improvements in
algorithms for calculating the necessary energetics (Brooks et al. 2009; Lane
et al. 2013).

3 SIGNALING IN THE POSTGENOMIC ERA

Genetic methods have been just as important for the progress made in
deciphering signal transduction pathways. These biological regulatory
mechanisms have roots deep in evolutionary time. Thus, genetic studies in
single-celled eukaryotic microbes (e.g., budding [Thorner 2006] and fission
[Otsubo and Yamamato 2008] yeast) and experimentally tractable
invertebrates (e.g., fruit flies [Thompson 2010] and nematodes [Bargmann
and Kaplan 1998]), as well as in model vertebrates (including zebrafish
[Moro et al. 2013] and the mouse [Nardella et al. 2010; Guerra and Barbacid
2013]), have been invaluable. Genetic analysis of these organisms has
supplied critical information about gene products that represent, generate,
and/or propagate signals that control cell metabolism, growth, size, division,
differentiation, shape, and motility, and that, when defective, cause disease.
The development of instrumentation for facile DNA sequence determination
(Shendure et al. 2004; Ståhl and Lundeberg 2012) and effective algorithms
for comparative genomics (Venter et al. 2003; Dewey and Pachter 2006;
Jiang et al. 2009; Washietl et al. 2012) have permitted rapid decoding of
entire genomes. With this comprehensive information, the genetic findings
made in model organisms can illuminate related processes in all other living
things, including humans, because of the high degree of evolutionary
conservation in major signaling pathways. Moreover, interrogating the entire
biosphere will continue to be as important for understanding human biology
and disease as studies of human beings themselves. This viewpoint is readily
defensible especially when one considers that many clinically useful drugs
that modulate signaling are natural products. For example, rapamycin was
discovered only because someone chose to isolate a new aerobic Gram-
positive filamentous bacterium (Streptomyces hygroscopicus) from the soil of
Easter Island (Rapa Nui in Polynesian) (Vézina et al. 1975).
With the rise in genetics and genomics, sophisticated new tools for
genome engineering are allowing analysis of the function of genes in human
cells, including those involved in signaling, by many of the paradigmatic
methods initially developed for use in studies of model organisms. These
methods exploit nucleases and DNA- and RNA-binding proteins derived
from yet other microorganisms (Urnov et al. 2010; Carroll 2011; Gaj et al.
2013; Wei et al. 2013). At the same time, computational methods for
identifying conserved structural domains and other sequence motifs in
proteins have also advanced greatly (Doolittle 1995; Copley et al. 2002;
Galperin and Koonin 2012; Huang et al. 2013).

4 MODULARITY IN SIGNALING
One revelation derived from the explosion of sequence and structural
information is the extent to which signaling proteins appear to have arisen
during evolution by the shuffling and assembly of readily identifiable
modules. These stably folded domains, joined by flexible linkers, frequently
serve as recognition elements that mediate specific protein–protein
interactions that link them together in specific complexes. Understanding the
dynamics of the assembly of such complexes, how they direct signal
propagation, enhance signaling efficiency, and insulate pathways against
inadvertent stimulation continues to be an area of ongoing research.
Another important consequence of the modular nature of the proteins
involved in cellular regulation is that such an architecture allows the
constituent domains to evolve discrete and separable functions, which we are
just beginning to uncover and appreciate. For example, the p110β isoform of
the catalytic subunit of Class 1A phosphoinositide 3-kinase (PI3K) can
generate phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the plasma
membrane via its carboxy-terminal kinase domain in response to growth-
factor-initiated signaling by receptor-tyrosine kinases or GPCR activation
(Vadas et al. 2011; Dbouk et al. 2012; see also p. 87 [Hemmings and
Restuccia 2012]). However, on growth factor withdrawal, p110β dissociates
from receptors, interacts via its so-called helical domain with the small
GTPase Rab5 and helps stabilize the GTP bound state of Rab5 (Dou et al.
2013). This interaction stimulates autophagy because increasing the amount
of Rab5-GTP that decorates internal membranes results in recruitment of the
class III PI3K hVps34, which generates the phosphatidylinositol 3-phosphate
necessary for assembly of preautophagosomes (Parzych and Klionsky 2013).
The mitogen-activated protein kinase (MAPK) kinase MEK1 provides
another example. It was thought to be simply a dedicated component of the
Ras-Raf-MEK-ERK signaling cascade (English and Cobb 2002; Roskoski
2012; see also p. 81 [Morrison 2012]); however, we now know that once
feedback phosphorylated on its amino-terminal extension by the kinase ERK,
MEK1 forms a ternary complex with a multidomain adaptor protein called
MAGI-1, which is necessary for membrane recruitment of the PIP3-specific
phosphatase PTEN; MEK1 thereby promotes down-regulation of the PIP3-
dependent protein kinase Akt (Zmajkovicova et al. 2013). A related issue is
that inherently disordered regions in some proteins adopt alternative
structures when associated with different interaction partners, leading to
different outcomes. The p53 transcription factor provides a particularly
dramatic example of this (Dunker et al. 2008; Joerger and Fersht 2008;
Freed-Pastor and Prives 2012).
The phenomenon whereby a protein has multiple distinct functions has
been dubbed “moonlighting” (Jeffery 2009). The potential for this evolving is
greatest in multidomain proteins, but not restricted to them. For example, one
of the splice variants of the muscle form of the glycolytic enzyme pyruvate
kinase, PKM2 (Hitosugi et al. 2009), appears to have other roles. On the one
hand, PKM2, when proline-hydroxylated by prolyl hydroxylase 3, seems to
associate with the transcription factor HIF1α and act as a coactivator that
promotes expression of HIF1α-dependent genes (Luo et al. 2011). On the
other hand, once tyrosine phosphorylated in response to growth factors,
PKM2 may undergo a switch in both oligomerization state (from a tetramer
to a dimer) and catalytic function (from its glycolytic role to a protein
kinase), and affect transcription by phosphorylating both histones (e.g., T11
on histone H3, which promotes acetylation at K9, a modification that
stimulates transcription) (Yang et al. 2012) and transcription factors (e.g.,
Y705 in STAT3, which promotes its dimerization and transactivator
function) (Gao et al. 2012).
Such instances of moonlighting in signaling proteins may help explain
how the intricacies of human biology are achieved with only 21,000 or so
protein-coding genes (just four times as many as a yeast cell). Thus,
investigators need to be alert to the possibility that moonlighting could
contribute in unanticipated ways to the biological complexity observed in a
signaling process, above and beyond alternative pre-mRNA splicing,
differential protein processing, and other mechanisms for generating protein
diversity that we already understand.

5 POSTTRANSLATIONAL MODIFICATIONS AND

SIGNALING
For many of the stably folded domains that mediate specific protein–protein
interactions in signal transduction (see Ch. 2 [Lee and Yaffe 2014]), target
recognition depends on a posttranslational modification of an amino acid side
chain, from phosphorylation to methylation, acetylation, and ubiquitylation
(Pawson and Nash 2003; Bhattacharyya et al. 2006; Nash 2012; Sadowski
and Taylor 2013). Indeed, proteins can be covalently modified to form all
sorts of other adducts and more than 300 types are known (Krishna and Wold
1993; Sims and Reinberg 2008; Farley and Link 2009; Hart et al. 2011;
Scarpa et al. 2013). The protein–protein interactions dependent on such
modifications are generally dynamic because these groups are installed and
removed by enzymes whose own activity often depends on signaling—in
particular, protein phosphorylation (Hunter 2012; Jin and Pawson 2012)—
which makes the study of protein kinases and phosphoprotein phosphatases
central to our understanding of signal transduction (Cohen 2002; Fischer
2013). This area of research has driven development of new tools for globally
interrogating both the kinome (Knight et al. 2013) and the phosphoproteome
(Leitner et al. 2011), including position-oriented, combinatorial, synthetic
peptide libraries (Turk et al. 2006; Arsenault et al. 2011) and immobilized
whole proteome arrays (“protein chips”) (Ptacek and Snyder 2006) for
delineating kinase-substrate specificity, ever more specific small-molecule
kinase inhibitors (Cohen and Alessi 2013), genetic approaches to uniquely
sensitize a given kinase to inhibition (Elphick et al. 2007; Knight and Shokat
2007; Feldman and Shokat 2010; Kliegman et al. 2013), selective chemical
tags to covalently label particular kinases and phosphatases or their substrates
(Allen et al. 2007; Patricelli et al. 2007; Hertz et al. 2010; Sadowsky et al.
2011; Miller et al. 2013), yeast two-hybrid screens (Cook et al. 1996; Fukada
and Noda 2007; Sopko and Andrews 2008), and other strategies to trap
particular kinases and phosphatases in complexes with their targets
(Blanchetot et al. 2005; Boubekeur et al. 2011), as well as sophisticated mass
spectrometry instrumentation and corresponding methods for detecting and
cataloging phosphoproteins (Cohen and Knebel 2006; Chi et al. 2007;
Gevaert and Vandekerckhove 2009; Palumbo et al. 2011; Engholm-Keller
and Larsen 2013; Loroch et al. 2013; Roux and Thibault 2013).
In signaling that regulates cell growth and division (Ch. 6 [Rhind and
Russell 2012] and Ch. 5 [Duronio and Xiong 2013], respectively), the actions
of protein kinases are pivotal (Morgan 2007). The chemical constraints on
how these enzymes operate have underappreciated implications for pathway
logic and dynamics. Every catalytic turnover of a protein kinase requires that
the phosphoacceptor sequence and ADP dissociate from the jaws of the
active site to permit entry of a fresh molecule of ATP into the back of the
catalytic pocket for the next phosphotransfer event (Tarrant and Cole 2009;
Lassila et al. 2011). How then can the multifarious protein kinases present in
a cell at any given time avoid adventitious modification of inappropriate
targets? Likewise, how can a protein kinase phosphorylate a given substrate
processively at several sites, as is often the case?
Part of the answer may lie in condition-, developmental-stage-, and tissue-
specific expression of the genes encoding these protein kinases and their
corresponding substrates, which could ensure that they are available only in
the right cells under the right circumstances. RNA-seq experiments (for this
method, see McGettigan 2013) and studies using beads containing a mixture
of protein-kinase-binding inhibitors followed by identification of the bound
enzymes by mass spectrometry show, however, that at least certain
immortalized cell lines and some breast tumor tissue express the majority
(60%–75%) of the kinome repertoire (Duncan et al. 2012). But, kinase targets
may be more cell-type specific. For example, arachidonic acid, the precursor
for eisocanoids, is released from membrane phospholipids in tissues such as
spleen, gut, white fat, and macrophages by the action of the known kinase
target cytosolic phospholipase A2 (cPLA2). In humans, this enzyme exists as
six isoforms (cPLA2α, cPLA2β, cPLA2γ, cPLA2δ, cPLA2ε, and cPLA2ζ)
encoded by distinct genes. The most ubiquitously expressed and well-studied
isoform, cPLA2α, is phosphorylated at multiple sites by various different
protein kinases. Depending on the cell type and agonist examined,
phosphorylation seems to regulate both membrane binding and catalytic
activity of cPLA2α (Ghosh et al. 2006; Dennis et al. 2011). The biochemical
properties and tissue distributions of the other five isoforms suggest that
regulation of their phosphorylation is distinct from that of cPLA2α and may
also involve other mechanisms. Moreover, although cPLA2 is responsible for
arachidonate generation in many tissues, this fatty acid is supplied in brain,
liver, and lung via the action of a separate class of serine acylhydrolase,
MAGL, which further extends the regulatory possibilities (Savinainen et al.
2012; Mulvihill and Nomura 2013).
There are many other mechanisms that can dictate how a protein kinase
achieves its target specificity (see Ch. 2 [Lee and Yaffe 2014]). On the one
hand, various molecular matchmaker strategies (see below) have evolved to
ensure that a protein kinase encounters its proper substrates with high
probability and efficiency (Endicott et al. 2012). On the other hand, for
subsequent events to unfold, the rate of phosphorylation must exceed the rate
of dephosphorylation by ever-present and more promiscuous phosphoprotein
phosphatases (Cohen 1992). For example, because of just this sort of
antagonism, during the response to stimulation by epidermal growth factor
(EGF), the phosphorylated tyrosine sites on the cytosolic domain of the EGF
receptor turn over 100–1000 times before maximal receptor phosphorylation
is achieved (Kleiman et al. 2011). Thus, any elevation in phosphorylation
detected in response to a stimulus is not simply caused by modifications that
are installed and remain until the signal is terminated. Hence, the hydrolytic
activity of phosphatases ensures that inefficient or inadvertent
phosphorylation events have no physiological consequence.
Protein kinases that act on targets at a cellular membrane are either
integral membrane proteins (e.g., receptor tyrosine kinases) or possess
domains that permit association with receptors or receptor-associated proteins
(e.g., the JAK family of protein tyrosine kinases bound to the cytosolic
segments of cytokine receptors) and/or are posttranslationally modified with
substituents (e.g., N-myristoyl and S-palmitoyl groups) that strongly promote
partition into the membrane (e.g., the Src family of protein tyrosine kinases)
(Groves and Kuriyan 2010). In other instances, association of the kinase and
its substrate with a third partner (a scaffold, linker, or anchor protein) brings
about the necessary propinquity and, in addition, enhances reaction rate by
achieving a high local concentration of the reactants (Ferrell 2000; Kuriyan
and Eisenberg 2007). Thus, if biochemical analysis of the proteins associated
with a given protein kinase, or genetic analysis of a process, reveals another
gene in which mutation yields a phenotype similar to that resulting from loss
of the kinase, and the gene product in question has no obvious catalytic
function itself, then it may serve such a scaffolding role. Moreover, there is
emerging evidence that at least one mammalian protein involved in the innate
immune response to RNA virus infection serves as just such a signal-
activating platform when, in prion-like fashion, it is converted into an
amyloid-like fibril (Hou et al. 2011; Wu 2013).
In some cases, phosphorylation of a substrate by one protein kinase
converts a sequence motif into a high-affinity binding site for, and permits its
subsequent phosphorylation by, another protein kinase. The first of such two-
step modifications is termed “priming.” For example, the prior
phosphorylation of substrates by the cell cycle kinase cyclin-dependent
kinase (CDK) 1 can generate a phospho-epitope recognized by the Polo box
domains of the Polo family of protein kinases, which execute later cell cycle
events (Lowery et al. 2005; Strebhardt 2010). This feature can be exploited in
other ways. For example, a peptide library approach (Turk and Cantley 2003)
indicates GSK3β is a protein kinase that strongly prefers to phosphorylate a
primed substrate, one that possesses a serine or threonine residue in a
sequence with a phosphorylated serine residue at position +4. When S9 in
GSK3β is phosphorylated by Akt, however, this modification creates a
substrate-like sequence that moors its own amino terminus in its active site,
thereby blocking GSK3β action on other primed substrates (Frame and
Cohen 2001; Weston and Davis 2001). For both kinetic and thermodynamic
reasons, intramolecular interactions are favored over intermolecular
associations. Hence, investigators should always be aware that
phosphorylation sites present in a kinase may serve such roles.
In many cases, a protein kinase recognizes and interacts with its substrate
via at least one other association distinct and physically distant from the
active-site–phosphoacceptor-sequence interaction. Such secondary points of
enzyme-substrate binding are termed docking sites (Bardwell and Thorner
1996; Reményi et al. 2006; Goldsmith et al. 2007). The more docking
interactions between a kinase and its target, the higher the probability that the
substrate will not dissociate from the enzyme in the time it takes for the next
catalytic turnover event. Thus, multiple docking interactions ensure that the
enzyme will stay bound to the same substrate molecule (and phosphorylate
another phosphoacceptor site, if there is one) rather than jump to a new
substrate. Moreover, such docking interactions supply additional binding
energy that makes it possible for substrate phosphorylation to occur at
noncanonical (suboptimal, lower affinity) phosphorylation sites. Such
considerations help explain why an investigator may find, perhaps
perplexingly, nonconsensus sites in mass spectrometry data, even when they
are using a purified substrate and protein kinase.
A particularly illustrative example of the vital role of such docking
interactions in ensuring processive multisite phosphorylation that is now well
understood at the mechanistic level is CDK1-dependent phosphorylation of
the yeast CDK inhibitor Sic1 (Nash et al. 2001). In the case of Sic1, the
phosphomodifications are a prelude to its timely ubiquitylation by an SCF-
type ubiquitin ligase (Silverman et al. 2012) whose substrate recognition
subunit (Cdc4) interacts with phosphoepitopes (Tang et al. 2012). In Sic1,
there is a sequence motif (VLLPP) that binds with high affinity to a site in the
G1 cyclin (Cln2) in Cln2-CDK1-Cks1 complexes; similarly, there are four
interspersed RxL motifs that bind to a site in the S phase cyclin (Clb5) in
Clb5–CDK1-Cks1 complexes. The obligatory Cks1 subunit in both CDK
complexes contains a pocket that preferentially binds phospho-TP sites
(CDKs are generally highly selective for -SP- and -TP- sites); hence, once
phosphorylated at any such site, the phosphoepitope–Cks1 interaction
provides yet another docking interaction. After a sufficient amount of the
Cln2-bound CDK1-Cks1 complex builds up during G1 phase, it holds onto
Sic1 via two contacts (active-site–phosphoacceptor-site binding and VLLPP-
motif–Cln2-docking-pocket association); following the first phosphorylation,
there are now three such interactions possible (active-site–phosphoacceptor-
site binding, VLLPP-motif–Cln2-docking-pocket association, and
phosphoepitope–Cks1 interaction), which explains initiation and
establishment of processive phosphorylation (Kõivomägi et al. 2011).
Similarly, as the amount of Clb5-bound CDK1-Cks1 builds up in late-
G1/early-S phase, maintenance and completion of processive multisite
phosphorylation by the Clb5-CDK1-Cks1 enzyme is very efficient, given that
it has four times the probability of engaging an RxL motif than the Cln2-
CDK1-Cks1 complex does a single VLLPP motif (Venta et al. 2012).

6 INTEGRATION OF CELL METABOLISM AND

SIGNALING
Intermediates in metabolism may have been the first intracellular (and
intercellular) signaling molecules, acting as feedback or feed-forward
regulators (either allosteric effectors or covalent modifiers) of enzymes in
metabolic pathways and transcription factors that controlled expression of
those enzymes. Our focus on other levels of regulation has perhaps diverted
us from exploring these more ancestral control mechanisms because, in
metazoans, GPCR and protein kinase signaling in response to hormones and
growth factors override the selfish metabolic needs of any given cell, in favor
of the needs of the organism (see Ch. 14 [Hardie 2012]).
So, just as proteins can have moonlighting functions, we need to better
understand the manifold functions in signaling of what were formerly
considered merely metabolic intermediates. For example, until the
stimulatory function of histone lysine ε-N-acetylation in chromatin
remodeling and gene expression was uncovered (Racey and Byvoet 1971;
Turner 1991), acetyl-CoA was presumed to have only the less glamorous role
of conveying carbon from glycolytically generated pyruvate, or the
breakdown of fatty acids, to the tricarboxylic acid (TCA) cycle for energy
generation in the mitochondrion. We now know that acetyl-CoA has other
roles that impinge critically on the capacity of a cell to signal (Lin et al.
2013). For example, in neurons and some other cells, it is needed for
synthesis of the central neurotransmitter acetylcholine. Moreover, in all cells,
three molecules of acetyl-CoA can condense to form HMG-CoA for the
mevalonate pathway to make both isoprenoid compounds (including the
farnesyl and geranylgeranyl moieties attached to the carboxy-terminal CAAX
motifs in many small GTPases involved in signaling, such as H-Ras and K-
Ras) (Berndt et al. 2011; Resh 2012) and cholesterol (which is not only
important for the architecture and fluidity of the membrane in which
receptors reside, but is also attached to the carboxyl terminus of an important
developmental signaling protein, Hedgehog; Creanga et al. 2012; p. 107
[Ingham 2012]). Yet another role for acetyl-CoA that affects signaling has
recently turned up. The α-N-acetylation of Met1 in the Rub1/Nedd8-specific
E2 enzyme Ubc12 (although an irreversible modification, once installed)
increases the avidity of its binding to a hydrophobic pocket in its cognate E3
ligase Dcn1 and thereby stimulates Nedd8-ylation of the Cul1 scaffold
protein (Monda et al. 2013), which is necessary, in turn, for assembly and
activity of the SCF-type E3 ligases that control the ubiquitin-dependent
degradation of numerous signaling proteins (Deshaies et al. 2010). It is worth
noting that, simply through substrate availability (and dependent on the
relative Km values of the various ε-N-lysine acetyltransferases), the level of
acetyl-CoA will dictate the rate of acetylation of histones and other proteins
with concomitant consequences for gene expression and other cellular
functions (Starai et al. 2004; Londoño Gentile et al. 2013; Zhang et al. 2013).
Acetyl-CoA is just one prominent nexus linking cell metabolism and
signaling. The levels of other key metabolites have multiple effects that
influence both metabolism and cell signaling in ways that we are just
beginning to understand. For example, 2-oxoglutarate (2OG, also known as
α-ketoglutarate) is a central intermediate in the TCA cycle, but also needed
for amino acid interconversion and breakdown by transamination, which may
affect the activity level of a major amino acid sensor and growth regulator,
mTORC1 (Bar-Peled et al. 2012). In addition, 2OG is a critical cofactor for
the Jumonji class of histone methyl-N-lysine demethylases (Hou and Yu
2010), TET family 5-methylcytosine hydroxylases (Wu and Zhang 2011),
and EglN-type prolyl-4-hydroxylases (Freeman et al. 2003), all of which
likely affect the level of expression of genes encoding signaling proteins.
Moreover, neomorphic mutations in isocitrate dehydrogenase (both IDH1 and
IDH2) that prevent oxidative decarboxylation of isocitrate to 2OG, but
catalyze instead NADPH-dependent reduction of 2OG to (R)-2-
hydroxyglutarate (Dang et al. 2009), an antagonist of 2OG-dependent protein
and DNA demethylation, are strongly associated with certain cancers, clearly
implicating altered metabolism in tumorigenesis (Losman and Kaelin 2013).
Unsurprisingly, the major cellular methyl donor, S-adenosylmethionine
(AdoMet), is important not only for methylation of DNA bases and
epigenetic silencing (Guibert and Weber 2013), but also for protein
methylation that influences signaling. For example, AdoMet-dependent N-
methylation of an arginine residue in the inhibitory factor Smad6 dissociates
it from activated bone morphogenetic protein (BMP) receptors, alleviating
inhibition and enabling the type I subunit of the BMP receptor complex to
phosphorylate the transcription factors Smad1 and Smad5, which permits
their nuclear import and activates their gene regulatory function (Xu et al.
2013). Similarly, succinyl-CoA is another central intermediate in the TCA
cycle, and lysine succinylation and cysteine succination have both been
recently identified as posttranslational modifications of proteins that
influence the properties of the enzymes to which they are attached (Zhang et
al. 2011; Lin et al. 2012). An open-ended question for future study is how
many other cellular metabolites lie at the crossroads between central
metabolism and cell signaling or are involved in the fine-tuning of biological
processes that impinge on signaling.

7 SIGNAL DIVERSITY
During evolution, mechanisms have arisen that allow diverse cell types to
sense and respond to various stimuli. In vision, light photons are absorbed by
the rhodopsins (chromophore-containing GPCRs) and converted into
intracellular chemical and then electrochemical changes in the rod and cone
cells in the retinas of our eyes. Hearing depends on conversion of sound
waves into intracellular signals via the opening and closing of stretch-
activated ion channels that are mechanically coupled to ciliary bundles in
specialized hair cells in our ears. In a similar way, touch depends on
conversion of mechanical pressure or thermal differences into conformational
changes in ion channels that open to elevate the level of intracellular calcium
ions in specialized nerve fibers in our skin. Of course, other stimuli to which
our senses respond are chemical in nature. Taste depends on conversion of
the binding of various soluble compounds into intracellular changes in the
gustatory-receptor-containing cells in the papillae on our tongues. Smell
depends on conversion of the binding of various volatile compounds into
intracellular signals in the olfactory-receptor-containing cells on the roof of
our nasal cavity. Skin irritation caused by the common nettle in our gardens
has a similar source; its trichomes inject chemicals normally made in animal
cells, such as the neurotransmitter serotonin and the immune mediator
histamine, thus aggravating our nerves and provoking inflammation.
Indeed, the variety of chemical signals generated by cells and to which
they are able to respond is rather staggering. Many of the classical endocrine
and pituitary hormones were discovered and chemically identified in the
1800s and early 1900s. For example, Frederick Banting announced the
isolation of insulin in 1921 and Fred Sanger determined its structure in 1953
(Nobel Prizes being awarded for both accomplishments). However, new
types of hormones that impact cell and organismal physiology and
development are still being discovered at a surprising rate, which requires
that their cognate receptors and downstream mediators be identified. For
example, adipose-derived leptin and its receptor (Isse et al. 1995; Tartaglia et
al. 1995), neuropeptides orexin-A/hypocretin-1 and andorexin-B/hypocretin-
2 and their receptors (de Lecea et al. 1998; Sakurai et al. 1998), and stomach-
derived ghrelin and its receptor (Howard et al. 1996; Kojima et al. 1999),
which have such critical roles in controlling the interrelated processes of
energy metabolism and obesity, wakefulness and appetite, and hunger and
growth, were not characterized until the late 1990s. Indeed, there are still
many GPCRs and nuclear receptors that are “orphans,” in the sense that their
physiological ligands have not yet been determined (Civelli 2012; Pearen and
Muscat 2012).
The constellation of known signaling molecules continues to expand in
new and unanticipated directions. For example, it has recently been
appreciated that different bacterial species, some of which are intracellular
pathogens, synthesize unusual cyclic dinucleotides that control their own
transcription, including cyclic-di-AMP, cyclic-di-GMP, and the mixed cyclic-
GMP-AMP, in all cases linked 3′-5′ (Kalia et al. 2013). The presence of such
compounds in mammalian cells is sensed by their binding to an endoplasmic
reticulum (ER)-localized protein called STING (Woodward et al. 2010).
Once activated, STING stimulates a protein kinase (TANK-binding kinase 1),
which, in turn, phosphorylates and activates a transcription factor, IRF3, that
regulates interferon production (Chin et al. 2013). However, STING is
activated much more potently and elicits a more efficacious interferon
response when foreign DNA enters cells. The difference is due to the fact that
cytosolic DNA binds to the regulatory domain of a mammalian enzyme,
cGAMP synthase (cGAS), thereby stimulating production of cyclic-GMP-
AMP in which the linkage is cyclic-[G(2′-5′)pA(3′-5′)p] (Kranzusch et al.
2013; Shaw and Liu 2014). This endogenously generated signal potently
activates diverse hSTING variants, whereas not all of them respond well to
bacterial cyclic-[G(3′-5′)pA(3′-5′)p] (Diner et al. 2013). Thus, mammalian
cells have evolved a mechanism to generate a novel signal that permits the
innate immune system to distinguish infiltration by a naked DNA from entry
of a bacterial invader. Such interkingdom signaling, mediating interplay
between viruses, bacteria, and mammalian cells (Lim et al. 2009; Marks et al.
2013; Pluznick et al. 2013), is clearly prevalent, has important consequences
for human health, and warrants continued exploration (see also Ch. 20 [Alto
and Orth 2012]).
How a signal is deployed or displayed can also have information content
that needs to be considered. Extracellular signaling ligands not only are
released from cells via the classical secretory pathway in an autocrine,
juxtacrine, or endocrine manner, but can also pass “through” cells via the
process of paracytophagy and the generation of so-called argosomes (Greco
et al. 2001). Such mechanisms are also used for the entry and cell-to-cell
passage of many prokaryotic intracellular pathogens (Portnoy 2012). Some
mammalian cell types can even engulf an entire other cell by a
macroendocytic process dubbed entosis (Overholtzer et al. 2007; Florey and
Overholtzer 2012). We now appreciate that cells can also generate
“exosomes” (small ∼100-nm-diameter vesicles) as a means for quantal export
of ligands and other classes of informational molecules, including miRNAs,
because, once released into extracellular fluids, they can be taken up by other
cells (Bang and Thum 2012; Briscoe and Thérond 2013; Choi et al. 2013).
Cells know their place, at least in part, by making contacts with adjacent
cells and components of the extracellular matrix. However, many cells types
erect a single specialized projection, the primary cilium (Garcia-Gonzalo and
Reiter 2012; Nozawa et al. 2013), which constrains to this one location
certain classes of signaling receptors (such as the receptors for the Hedgehog
family of ligands) (Wong and Reiter 2008; Ch. 10 [Perrimon et al. 2012]),
presumably to confer the capacity to respond only to a highly polarized or
localized signal source. In contrast, other cells extend ultrafine processes and
specialized filopodial extensions (also referred to as cytonemes) that can
mediate cell-to-cell contacts and long-range transport of signal molecules
over a distance of many cell lengths (Roy et al. 2011; Sanders et al. 2013).
Similarly, juxtaposed cells in many epithelial layers are connected by gap
junctions that act as portholes through which certain intracellular signals,
such as an increase in cytosolic calcium ion concentration, can be spread
from cell to cell (Goodenough and Paul 2009). Clearly, we still have much to
learn about the interplay between signaling molecules and all levels of
cellular organization.

8 PROSPECTUS
For the foreseeable future, signal transduction research remains a field
confronted with a still vast frontier. As the contents of this book and issues
raised here make clear, unraveling signal transduction processes is a
multidisciplinary enterprise. Ultimately, understanding signaling will require
an appreciation for, understanding of, and the means to usefully grasp the
seamless interconnectedness among all the bits of information gleaned by
what were formerly considered disparate branches of the biological,
chemical, and physical sciences. Fortunately, as we highlight in this book,
there are recurrent themes, general mechanisms, common strategies, and
ubiquitous reactions in cell signaling that allow the complexity to be parsed
out productively. Indeed, we can already discern general design principles
that are allowing us to reengineer cellular responses to stimuli different from
those evolved in nature (Pryciak 2009; Burrill et al. 2011; Blount et al. 2012;
Lim et al. 2013).
Nonetheless, many unexpected discoveries that provide novel insights and
fresh paradigms will continue to be made, and these will open up new
avenues for understanding cell signaling. Some recent examples highlight
this point. Remarkably, proteins of the previously uncharacterized Fam20C
family turn out to be secreted atypical protein kinases that phosphorylate
casein and other extracellular substrates that have important physiological
roles in bone mineralization (Tagliabracci et al. 2013; Xiao et al. 2013b).
This discovery raises interesting questions about how these enzymes acquire
ATP in the lumen of the Golgi and whether such enzymes are made and have
important roles in the nervous system, in which ATP is stored in synaptic
vesicles (Zimmermann 2008) and released extracellularly to stimulate
purinergic receptors (Khakh and North 2012). Likewise, it has been
appreciated for decades that blood platelets also store ATP and other
compounds in storage vesicles that are released extracellularly when platelets
are activated at a site of injury (Da Prada et al. 1971; Higashii et al. 1985).
Does this released ATP also act, in part, through Fam20C-like extracellular
protein kinases? Another example is that very high serum levels of high-
molecular-weight hyaluronan dramatically protect the naked mole rat against
cancer (Tian et al. 2013). Hyaluronan is an extracellular proteoglycan and can
bind to various cell surface receptors. How does it suppress malignant growth
and/or enhance immune surveillance of precancerous tissue? And, do the
same mechanisms operate in humans?
It is also clear that we have just began to scratch the surface of how
microRNAs (Martinez and Gregory 2010; Mendell and Olson 2012) and
other RNAs encoded in our genomes (Hancks and Kazazian 2012; Batista
and Chang 2013) coordinately influence the levels of gene products involved
in signaling and are themselves controlled by signaling processes. Likewise,
an area of traditional biochemistry that clearly intersects with signaling in
many ways is the function of various classes of proteases; yet, we understand
the roles of many of these enzymes only very superficially and know even
less about their physiologically relevant enzyme–substrate relationships. For
example, in 2012, a new, circulating, exercise-induced regulatory hormone,
dubbed irisin, was described that converts white fat into more thermogenic
beige fat (Boström et al. 2012). However, irisin is identical to the ectodomain
of a small (212-residue) cell surface protein, fibronectin type III domain-
containing protein 5 (FNDC5), which is anchored in the plasma membrane
by a single carboxy-terminal hydrophobic transmembrane segment. In fact,
protease-mediated shedding of the extracellular domains of transmembrane
signaling proteins as separate entities with distinct functions is a common
phenomenon (Horiuchi 2013). Exercise stimulates FNDC5 expression in
skeletal muscle and the cleavage and release of its uniquely structured amino-
terminal fibronectin-III-like ectodomain as irisin (Erickson 2013;
Schumacher et al. 2013); however, the protease responsible for this shedding,
and whether it too is under any sort of regulation, is unknown.
Thus, if we take the long view, studies of signal transduction are still in
exponential phase with many important discoveries to come. Moreover, we
anticipate continued development of evermore sophisticated experimental
tools—from improvements in automated deep sequencing to characterize the
global transcriptome (Malone and Oliver 2011), to new mass spectrometry
instrumentation to catalog the cellular metabolome (Rubakhin et al. 2013), to
further refinement of mathematical, statistical, and computational theories
and methods to assist with display, interpretation, and modeling of the
complex networks of relationships involved in intra- and intercellular
signaling (Janes and Lauffenburger 2013; see also Ch. 4 [Azeloglu and
Iyengar 2014]). Continued advances of this sort will allow us to address
questions at an ever greater level of detail and resolution, providing answers
at the molecular level to long-standing mechanistic questions about the
myriad processes that comprise cell signaling.

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Cite this chapter as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a022913
Index

A
ABI1, 186
AC. See Adenylyl cyclase
ACC. See Acetyl-CoA carboxylase
ACE. See Angiotensin-converting enzyme
Acetylation, protein regulation mechanisms, 34–35
Acetylcholine receptor, nicotinic, 267
Acetyl-CoA, 170–171, 176–177, 393–394, 434–435
Acetyl-CoA carboxylase (ACC), 287
ACL. See ATP-citrate lyase
Acrosome reaction, 336
ActA, 399
ACTH. See Adrenocorticotrophic hormone
Actin
cell migration and polymerization, 184–186
pathogen modifiers
elongation factors, 400
nucleation factors, 399–400
Activators of G-protein-mediated signaling (AGSs), 14
ADAM1b, 336
ADAM2, 336
ADAM3, 336
ADAM10, 111
ADAM17 (TACE), 111
Adenylyl cyclase (AC), 99–101, 335
Adiponectin, energy homeostasis role, 281
Adipose tissue triglyceride lipase (ATGL), 289
Adrenocorticotrophic hormone (ACTH), energy homeostasis role, 278
AF2, 130–131
AGS proteins, 14
AGSs. See Activators of G-protein-mediated signaling
AKAPs. See A-kinase anchoring proteins
A-kinase anchoring proteins (AKAPs), 43, 53–54, 101, 254, 335
Akt
cancer signaling, 408–416, 418–421
glucose metabolism signaling
G-protein-coupled receptor signaling, 13
lymphocyte signaling, 321–323
lymphocyte signaling, 322
mTORC1 target, 175
phosphoinositide, 3-kinase pathway overview, 87–89
PI3K/Akt, 168, 170–171
PIP3 signaling, 57–59
subcellular localization, 42
ALDH1A1, 335
α-Catenin, 20, 136
AMBRA1, 378
Amino acids, metabolism signaling cascades, 173, 175
2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptor
(AMPAR), learning and memory role, 249–251, 253, 256
AMPAR. See, 2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic
acid receptor
AMPK
energy homeostasis role, 278–279, 283–284, 287, 289
mTORC1 regulation, 176
Amyloid-β, 295
Anaphase. See Mitosis
Angiogenesis, cancer, 420
Angiomotin, 135
Angiotensin receptor, 273
Angiotensin-converting enzyme (ACE), 273
AP1, 318
APAF1, 368, 376–377
APC, 411
Apoptosis
cancer, 412
caspases
activation, function, and regulation, 367–368, 376
caspase-1/-5/-11 activation in inflammasome pathway, 374
caspase-2 activation in PIDDosome pathway, 374–375
caspase-8 activation in death receptor pathway, 371, 373–374
caspase-9 activation in mitochondrial pathway, 368–371
kinases, 375
overview, 366–367
TNFR1 induction, 303
Approximated, 134
Arp2/3, 186, 255, 305, 399–400
Arpp19, 155
ASC, 300, 374
ASK1, 351
ATF1, 101
ATF2, 349
ATF4, 349
ATF6, unfolded protein response, 347–349
ATG proteins. See Autophagy
ATM, 417
DNA damage checkpoint, 158–159
recruitment, 35
ATP-citrate lyase (ACL), 414
ATR, DNA damage checkpoint, 158–159
Aurora B, 41
Autophagy
 cell death role, 379
signaling overview, 366, 377–379

B
Bacteria. See Infection
Bad, 372, 378
BAFF, 319–320, 322
Bak, 369
Bax, 369
B-cell receptor (BCR)
adaptor molecules, 316
ITAM, 315–316
signaling
calcium, 316–317
diacylglycerol, 316–318
ERK1/2, 319
inhibitory signals, 323
nuclear factor of activated T cells, 317–318
overview, 125–127, 317
protein kinase C, 318–319
Ras, 319
structure and function, 314–315
Bcl2, 356, 367, 369–371, 378–379, 412
BCL6, 334
Bcl10, 307, 319
BCR. See B-cell receptor
BCR-ABL, 407
Beclin, 1, 378–379
BEN. See Biased excitable network
β-Arrestin, G-protein-coupled receptor complex, 14, 107
β-Catenin, 20, 104, 417
β-TrCP, 370
Biased excitable network (BEN), 193
Bid, 370, 372
BIK, 372
Bim, 356, 370, 372, 412
BK channel, 62
BLNK, 316
Bmf, 356, 373
BMP. See Bone morphogenetic protein
Bnip3, 373, 378
BOC, 107
Bone morphogenetic protein (BMP)
BMPRII, 114
embryonic patterning, 223, 225
signaling overview, 113–114
BRCA, 418
Brinker, 226

C
Cadherins, signaling, 20
Calcineurin, learning and memory role, 253–254
Calcium
binding motifs, 61
buffering, 61–62
channels and regulation of levels, 61
history of study, 95–96
lymphocyte signaling, 316–317
signaling overview, 59–61, 95–97
smooth muscle sensitization, 273
spatiotemporal organization of signaling, 62
termination of signal, 62
Calcium/calmodulin-dependent protein kinase II (CaMKII), learning and
memory role, 251–253, 258
Calcium-induced calcium release (CICR), 62, 269
Calcium release-activated channel (CRAC), 305, 316–317
Calmodulin (CaM), calcium signaling, 97
Calsequestrin (CSQ), 268–269, 271
CaM. See Calmodulin
CAMKII, caspase activation role, 375
CaMKII. See Calcium/calmodulin-dependent protein kinase II
cAMP. See Cyclic AMP
CAMs. See Cell adhesion molecules
Cancer
cell polarity signaling, 210
dysregulation
angiogenesis, 420
apoptosis, 412
cell fate and differentiation, 417
cell migration, 415–417
cell polarity, 415–417
cell proliferation, 409–412
extracellular matrix, 418
genomic instability, 417–418
inflammation, 420–421
metabolism, 412–415
microenvironment, 418
fibroblasts, 421
gene mutations
overview, 407
signaling pathways, 407–408, 419
inflammation, 307
progression, 406–407
prospects for study, 421–422
CAR. See Constitutive active/androstane receptor
Carbohydrate. See Glucose, metabolism signaling
Carbon monoxide (CO), signal transduction, 25
Carboxy-terminal Src kinase (CSK), 315
Cardiac muscle. See Muscle contraction
Carma1, 319
Carnitine:palmitoyl transferase (CPT), 284
Casein kinase II (CK2), 370
Caspases. See Apoptosis; Necrosis
Catecholaminergic polymorphic ventricular tachycardia (CPVT), 271
CATSPER, 336
Caveolae, signaling, 43
Cbl, 8
Cdc2, 146, 153
Cdc4, 145
Cdc13, 146
Cdc20, 148, 160–161
Cdc25, 155, 157–159, 330
Cdc34, 146
Cdc42, 82, 354, 368, 98
cell migration role, 186, 188
cell polarity role, 200–201, 207
CDKs. See Cyclin-dependent kinases
CDO, 107
Cdr2, 157
C/EBPα, 131
C/EBPβ, 297
CED3, 376
CED4, 376
CED9, 376
Cell adhesion molecules (CAMs). See also specific molecules
cadherin-dependent adhesions, 20
signaling, 19–21
Cell cycle
cancer, 409–412
cyclin-dependent kinase inhibitors
overview, 143–144
transcriptional regulation by inhibitors, 144–145
G1 regulation
cyclin D, 141–142
 cyclin E, 142–143
cyclin-dependent kinases, 141, 143
retinoblastoma protein, 140–141
ubiquitinylation, 145–147, 147–148
G2/M transition. See Mitosis
meiosis. See Meiosis
overview, 140–141
Cell fate. See Cancer; Embryonic patterning, Drosophila
Cell migration
actin polymerization, 184–186
adhesions, 186–188
cancer, 415–417
chemotaxis signaling
adaptation and excitation–global inhibition models, 193
excitability of networks, 192–193
myosin contraction, 186
overview, 184
polarization, 188
prospects for study, 193–195
signaling
focal adhesion kinase, 188–189
genetic analysis, 191–192
paxillin, 188–189
phosphoinositide, 3-kinase, 189–191
Rho GTPases, 188
Cell polarity. See also Par proteins
cancer, 415–417
cell migration, 188
machinery
intercellular junctions, 203
Par proteins, 202–203
symmetry breaking and positive-feedback loops, 200–202
Par protein localization
active exclusion, 205–207
 membrane phospholipid attachment, 204
membrane protein anchoring, 204–205
messenger RNA localization, 205
oligomerization, 204
signaling
cancer, 210
Hippo pathway, 209–210
overview, 200
Par3–Par6–protein kinase C signaling, 207–209
Wnt signaling cross talk, 209
Ceramide
hydrolysis, 59
signaling overview, 56
stress response, 59
cGMP. See Cyclic GMP
CHBP, 400
Chemotaxis. See Cell migration
Chk1, 158–159, 417
Chk2, 158–159, 417
Cholecystokinin, receptor, 273
Cholera toxin, 390
Cholesterol
Hedgehog coupling, 107
liver metabolism, 287–289
CHOP, 350, 352
CICR. See Calcium-induced calcium release
Cif, 400
CIN85, 316
CK1, 317
CKII. See Casein kinase II
CKS1, 147
Cks1, 434
Clb5, 146, 434
Cln2, 434
CO. See Carbon monoxide
Colony-stimulating factor, 1 (CSF1), 420
Complement, C5A receptor in innate immunity, 304–305
Computational models, signaling networks
dynamical models, 71–72
graph theory for signaling network models, 69, 72–74
network models, 70–71
Constitutive active/androstane receptor (CAR), 23–24
Cortisol, energy homeostasis role, 278–279
Cos, 107
COX2, 297
CPI-17, 273
CPT. See Carnitine:palmitoyl transferase
CPVT. See Catecholaminergic polymorphic ventricular tachycardia
CRAC. See Calcium release-activated channel
Crb2, 159
CRE. See Cyclic AMP response element
CREB, 274, 297, 334
CRIB domain, 398
Crk, 8
CRL4, 148
Crumbs, 135, 204, 416
CSF. See Cytostatic factor
CSF1. See Colony-stimulating factor, 1
CSK. See Carboxy-terminal Src kinase
CSL complex, 110–111
CSQ. See Calsequestrin
CTLA4, 319, 323
Ctp1, 158
Cyclic AMP (cAMP)
G-protein-coupled receptor signaling, 13, 53
muscle relaxation, 265
phosphodiesterases, 55
protein kinase A as target, 53–54
 signaling overview, 99–101
targets, 55
Cyclic AMP response element (CRE), 287
Cyclic GMP (cGMP)
muscle relaxation, 265
phosphodiesterases, 55
protein kinase G as target, 55
targets, 55
Cyclic GMP-dependent protein kinase (PKG), 55
Cyclin B, mitosis entry role, 154–156
Cyclin D
G1 regulation, 141–142
ubiquitinylation, 145–147
Cyclin-dependent kinases (CDKs)
activating kinase, 154
CDK1
activation in mitosis entry, 152–157
caspase activation, 375–377
oocyte maturation role, 331
G1 entry regulation, 141
inhibitors
overview, 143–144
transcriptional regulation
INK4, 144–145
p21, 144
posttranscriptional regulation, 143
Cyclin E, G1 regulation, 142–143
Cyclophilin D (CypD), 380
Cyclosporin A, 318
CYLD, 380
CypD. See Cyclophilin D
Cytochrome c, 376
Cytokine receptor family, overview, 4, 6
Cytokinesis, mitosis coordination, 161–162
Cytostatic factor (CSF), 328

D
Dachsous, 134
DAI, 380
Dcn1, 434
DCP1, 376
Death receptors. See specific receptors
Deltex, 110
Development. See Embryonic patterning, Drosophila
Diabetes type, 2, overnutrition, 289–290
Diacylglycerol (DAG)
lymphocyte signaling, 316–318
muscle calcium sensitization, 273
protein kinase C as target, 55, 57
signaling overview, 57
DIAP1, 376
DISC, 374
Discs large, 135
DKK, 104
Dlg, 204
DLG1, 57
DNA damage checkpoint, 157–159
DNA-PK, DNA damage checkpoint, 158
DNA replication checkpoint, 159
DNMT1, 407
DOCK180, 19
Dock2, 305
Dorsal, 217
Double-strand break (DSB), 157–158
Double-stranded RNA-dependent kinase (PKR), 349, 351
DrrA, 398
DSB. See Double-strand break
DUSP, 355

E
E2F, 141, 143
E4orf6, 401
E-cadherin, 20
Ect1, 207
EGF. See Epidermal growth factor
eIF2, 349
Electron microscopy, signaling studies, 430
Elk1, 40
Embryonic patterning, Drosophila
induction of cell fate, 216
signaling
bone morphogenetic protein, 223, 225
epidermal growth factor, 222–224, 229
fibroblast growth factor, 222–223, 229
Hedgehog, 222, 225
integration of pathways, 227–230
linear signaling, 226
long-range ligand distribution, 225–226
negative-feedback switches, 226
Notch, 220–222
overview, 216–218
threshold generation, 226–227
Wnt, 225
transcriptional cascade interactions with signaling, 217, 219
Emi2, 331, 338
EMT. See Epithelial-to-mesenchymal transition
Endoplasmic reticulum. See Unfolded protein response
Endoplasmic reticulum stress element (ERSE), 347
Endosomal sorting complex required for transport (ESCRT), 9, 111
Energy homeostasis. See also Fatty acid metabolism; Glucose, metabolism
 signaling
AMPK role, 278–279, 283–284, 287, 289
brown adipose tissue fatty acid oxidation, 289
diabetes type, 2 and insulin resistance, 289–290
hormonal control
adipose tissue, 281
adrenal gland, 278
hypothalamic-pituitary axis, 278
pancreas, 280
thyroid gland, 279–280
liver
carbohydrate metabolism acute regulation, 284–286
gluconeogenesis long-term regulation, 286–287
lipid metabolism, 287–289
muscle
exercise adaptation, 284
fatty acid oxidation, 284
glucose uptake and glycogen synthesis, 282–284
glycogen breakdown, 281–282
prospects for study, 290–291
ENTPD5, 168
EphB6, 8
Epidermal growth factor (EGF)
embryonic patterning, 222–224, 229
receptor
cancer, 9
dimerization, 7
phosphorylation, 433
Epithelial-to-mesenchymal transition (EMT), cancer, 416–417
EPSP. See Excitatory postsynaptic potential
ERK. See Extracellular signal-regulated kinase
ERO1, 349
ERR. See Estrogen-related receptor
ERSE. See Endoplasmic reticulum stress element
ESCRT. See Endosomal sorting complex required for transport
E-selectin, innate immunity role, 303–304
EspG, 398
Estrogen-related receptor (ERR), 21, 23
Ets, 334
Excitatory postsynaptic potential (EPSP), 248
ExoS, 396
Extracellular signal-regulated kinase (ERK)
cancer signaling, 408–410, 412, 415–416, 418, 421
development role, 223, 226–227, 230
learning and memory role, 258
lymphocyte signaling, 319
lymphocyte signaling by ERK1/2, 319
overview, 81–82
stress signaling
activation cascade, 354–355
inactivation, 355
overview, 353–354
physiological roles
cell death, 356
inflammation, 356–357
metabolism, 357
scaffold protein function, 355–356
substrates, 41

F
FADD, 16, 300, 302–303, 373–374
FAK. See Focal adhesion kinase
Far1, 146
Farnesoid X receptor (FXR), 23
Fas, 15
Fat, 134
Fatty acid metabolism
 brown fat oxidation, 289
muscle oxidation, 284
FBW7, 146
Fbxo proteins, 146
Fc receptors
FcεR1 and immunoglobulin E binding, 316
innate immunity
FcεRI, 305–307
FcγR, 307
Fertilization. See Reproduction
FGF. See Fibroblast growth factor
Fibroblast growth factor (FGF)
cancer dysregulation, 417
embryonic patterning, 222–223, 229
receptor
dimerization, 7
stabilization, 40
FIDOP domain, 397
FIP200, 379
FK506, 318
FKBP12, 430
FLIP, 303, 373–375, 380, 412
Fluorescence resonance energy transfer (FRET), signaling studies, 429
FNDC5, 437
Focal adhesion kinase (FAK), 18, 20, 188–189, 418
Follicle-stimulating hormone (FSH), 334–335
Formyl peptide receptor (FPR), innate immunity, 304–305
4E-BP1, 44
Fos, 41, 274
Foxo
cancer signaling, 414–415
FOXO1, 87
FOXO3A, 371
lymphocyte signaling, 322
FPR. See Formyl peptide receptor
FRET. See Fluorescence resonance energy transfer
Frizzled, 10, 103, 209
FSH. See Follicle-stimulating hormone
FUNDC1, 379
Fus3, 146
FXR. See Farnesoid X receptor
Fyb, 392
Fyn, 20

G
G protein
GTPase cycle, 37
heterotrimeric G proteins, 38–39
receptor specificity, 11–12
small G proteins, 37–38
G-protein-coupled receptors (GPCRs)
activation, 11
classes and structure, 10–11
G-protein specificity, 11–12
guanine nucleotide exchange factors, 37, 39
innate immunity, 304–305
kinase networks, 13–14
ligand-induced conformational change, 12–13
lipid messengers, 57
overview, 10
pathogen signaling corruption in host
bacteria protein mimics
G proteins, 398
GTPase-activating proteins, 396
guanine-nucleotide exchange factors, 395–395
G-protein modifiers, 397–398
overview, 395
 Yersinia, 396–397
protein–protein interactions, 14
second messengers, 13
sensory receptors. See Sensory receptors
signal integration, 15
signal termination, 14–15
G1. See Cell cycle
G2/M transition. See Mitosis
GADD34, 350
GADD45, 355
GATA4, 269
Gcn5, 176, 284
GDNF. See Glial-derived neurotrophic factor
Genomic instability, cancer, 417–418
Germ-cell nuclear receptor, 24
Germinal vesicle breakdown (GVBD), 330
GFP. See Green fluorescent protein
GFRA1, 334
GH. See Growth hormone
Gli1, 107–108
Gli2, 107
Gli3, 107
Glial-derived neurotrophic factor (GDNF), 8, 333–334
Glucocorticoid response element (GRE), 286
Glucose
liver
carbohydrate metabolism acute regulation, 284–286
gluconeogenesis long-term regulation, 286–287
metabolism signaling
cancer, 412–415
endoplasmic reticulum signals, 351–353
hypoxia-inducible factor, 1, 171–173
PI3K/Akt, 168, 170–171
pyruvate kinase metabolic switch, 173–174
 muscle uptake and glycogen synthesis, 282–284
transporters, 168, 282, 284
Glycogen, muscle
breakdown, 281–282
glucose uptake and synthesis, 282–284
Glycosylation, protein regulation mechanisms, 36–37
GPCRs. See G-protein-coupled receptors
GPR3, 329
Graph theory, signaling network models, 69, 72–74
Grb2, 18, 320
GRE. See Glucocorticoid response element
Green fluorescent protein (GFP), signaling studies, 428
Growth hormone (GH), receptor dimerization, 7
GRP78, 347, 351
GRP94, 347
GSK3, 104, 145–146, 284, 317, 370, 411, 433
Guanylyl cyclase, 24–25
Gustation. See Sensory receptors
GVBD. See Germinal vesicle breakdown

H
H2AX, 158–159
HAT. See Histone acetyltransferase
HCN. See Hyperpolarization-activated cyclic nucleotide-gated channel
HDAC. See Histone deacetylase
Hearing. See Sensory receptors
Heart failure. See Muscle contraction
Hedgehog (Hh)
embryonic patterning, 222–223, 225
signaling overview, 107–108
Heme oxygenase (HO), 25
Hemese, embryonic patterning, 219–220
HES1, 227
Hexokinase, 168
Hh. See Hedgehog
HIF1. See Hypoxia-inducible factor, 1
Hippo
cell polarity signaling, 209–210
signaling overview, 131–136
Histone acetyltransferase (HAT), 35
Histone deacetylase (HDAC), 35, 130, 268
HMG-CoA reductase, 287–288
HMGB1, 294
HNF4, 131
Hormone-sensitive lipase (HSL), 289
HRK, 373
HSL. See Hormone-sensitive lipase
HuR, 356
2-Hydroxyglutarate aciduria, 176–177
Hyperpolarization-activated cyclic nucleotide-gated channel (HCN), 55
Hypoxia-inducible factor, 1 (HIF1)
glucose metabolism signaling, 171–171
oxygen signaling, 24

I
ICAM. See Intercellular cell adhesion molecule
ICP0, 400–401
IDH. See Isocitrate dehydrogenase
IKK, 45, 123, 297–299, 303, 307, 351, 393
induced genes, 298
Toll-like receptor activation, 295–297
IL-2 receptor, 320–321
IL-3
metabolism regulation, 176
receptor, 176
IL-4 receptor, 321
IL-7 receptor, 322
IL-12 receptor, 320
ILK. See Integrin-linked kinase
Immunoreceptor tyrosine activation motif (ITAM), 303, 315–317
Immunoreceptor tyrosine inhibition motif (ITIM), 323
Infection
actin modifiers
elongation factors, 400
nucleation factors, 399–400
bacteria virulence factors, 390–392
G-protein-coupled receptor signaling corruption
overview, 395
bacteria protein mimics
G proteins, 398
GTPase-activating proteins, 396
guanine-nucleotide exchange factors, 395–395
G-protein modifiers, 397–398
Yersinia, 396–397
lipid signaling highjacking, 398–399
mitogen-activated protein kinase signaling corruption
anthrax, 393
overview, 392
Shigella, 394
Yersinia, 393–394
oncoproteins of viruses, 390
ubiquitylation disruption, 400–401
Inflammation
cancer, 307, 420–421
caspase-1/-5/-11 activation in inflammasome pathway of apoptosis, 374
mitogen-activated protein kinase stress signaling, 356–357
unfolded protein response, 350–351
Information flow, signaling networks
computational models. See Computational models, signaling networks
contextual nature of information, 75
 emergent properties of networks
bistability, 74
oscillations, 75
redundancy, 75
ultrasensitivity, 74
graph theory models, 69, 72–74
networks of pathways, 66, 68–69
noise filtering, 76
overview, 66–67
versatility of responses, 75–76
INK4, transcriptional regulation, 144–145
Innate immunity. See also specific receptors
E-selectin, 303–304
Fc receptors
FcεRI, 305–307
FcγR, 307
G-protein-coupled receptors, 304–305
inflammation and cancer, 307
integrin receptors, 303–304
Nod-like receptors, 300
pattern recognition receptor ligands, 294–295
RIG-I-like receptors, 298–300
TNFR1 signaling
cell death induction, 303
MAPK, 301–303
nuclear factor-κB, 301–303
Toll-like receptor
interferon induction, 297–298
ligands, 295
TAK1 and IKK activation, 295–296
TLR4 signaling, 296
TRIF in signaling, 298
Inositol hexaphosphate (IP6), 57
Inositol tetraphosphate (IP4), 57
Inositol-requiring enzyme, 1 (IRE1), unfolded protein response, 347–348,
350
Inositol trisphosphate (IP3)
G-protein-coupled receptor signaling, 13, 57, 96
receptors, 60, 273, 337
Insulin
energy homeostasis role, 280
resistance and overnutrition, 289–290
response unit, 287
Insulin receptor substrate (IRS), 87
Integrin receptors
innate immunity, 303–304
ligands, 18
mechanosensing signaling, 17–19
Integrin-linked kinase (ILK), 18–19, 189
Intercellular cell adhesion molecule (ICAM), 17, 303
IP3. See Inositol trisphosphate
IP4. See Inositol tetraphosphate
IP6. See Inositol hexaphosphate
IpgD, 398–399
IRAK1, 123, 295, 298
IRE1. See Inositol-requiring enzyme, 1
IRF1, 298
IRF7, 297–298, 401
IRS. See Insulin receptor substrate
IRS1, 351
Ishihara test, 243
Isocitrate dehydrogenase (IDH), 176, 178–179, 407, 415, 436
ITAM. See Immunoreceptor tyrosine activation motif
ITIM. See Immunoreceptor tyrosine inhibition motif
IZUMO, 336

J
JAK/STAT signaling
cytokine signaling in lymphocytes, 320–321
overview, 117–119
JAM. See Junctional adhesion molecule
Janus kinase. See JAK/STAT signaling
JIP1, 356
JIP2, 356
JIP3, 356
JIP4, 356
JNK. See Jun N-terminal kinase
Jun N-terminal kinase (JNK)
overview, 82–83
stress signaling
activation cascade, 354–355
inactivation, 355
overview, 353–354
physiological roles
cell death, 356
inflammation, 356–357
metabolism, 357
scaffold protein function, 355–356
Junctional adhesion molecule (JAM), 204–205

K
KIP1, 147
Kit, 9, 334
KitL. See Stem cell factor
KSR1, 356
Ku70, 158
Ku80, 158

L
Lactate dehydrogenase (LDH), 414
LAMP1, 378
LAMP2, 378
LAT, 126, 316
LATS kinases, 162
LC3, 379
Lck, 315
LDH. See Lactate dehydrogenase
Learning and memory
GTPases in synaptic plasticity, 257–259
Hebbian behavior of synapses, 248–249
postsynaptic density scaffold proteins, 254–256
prospects for study, 259
spine synapse signaling in brain
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid
receptor, 249–251, 253, 256
calcium-regulated signaling in postsynaptic density
calcium/calmodulin-dependent protein kinase II, 251–253, 258
calcineurin, 253–254
N-methyl-D-aspartate receptor, 249–251
overview, 249–251
LEF/TCF, 114
LEGI model, 193
Leptin, 281
Lethal factor, anthrax, 393
Leukotrienes, signaling overview, 59
Leydig cell, 332
Lgl, 204–205, 210
LGP2, 298–299
LH. See Luteinizing hormone
LIM kinase, 186
Linear ubiquitin chain assembly complex (LUBAC), 295, 303
Lipid raft, signaling, 43
Lipids. See Fatty acid metabolism; specific lipids
Liver X receptor (LXR), 23, 130–131
LKB1, 415
LMP1, 407
Long-term depression (LTD), 249, 251–253
Long-term potentiation (LTP), 249
Lowfat, 134
LRP4, 8
LRP6, 228
LTD. See Long-term depression
LTP. See Long-term potentiation
LUBAC. See Linear ubiquitin chain assembly complex
Luteinizing hormone (LH), 334
LXR. See Liver X receptor

M
MAD, 134, 227
Magnesium
signaling overview, 59–60, 62–63
transport, 63
MAGUKS, 254
MALT1, 307, 319, 420
MAM. See Mitochondria-associated endoplasmic reticulum membrane
MAML, 111
Mammalian target of rapamycin (mTOR)
complexes. See mTORC1; mTORC2
G-protein-coupled receptor signaling, 13
history of study, 92–93
MAPK. See Mitogen-activated protein kinase
Maturation promoting factor (MPF), 328, 330
MCC. See Mitotic checkpoint complex
Mcl1, 356, 369–370, 379, 412
Mdc1, 158
mDia1, 186, 305
mDia2, 186
MDM2, 43–44, 411
Mechanosensation. See Sensory receptors
MEF2, 219, 268
Meiosis
oocyte
meiosis I, 328–331
meiosis II arrest, 331–332
spermatocyte meiosis and release, 335
Memory. See Learning and memory
MEN. See Mitotic exit networks
Messenger RNA (mRNA), stability, 44
Met1, 434
Methylation, protein regulation mechanisms, 35–36
MGA5, 298–300
MHCK. See Myosin heavy-chain kinase
MicroRNA, signaling, 44
Microtubule-organizing center (MTOC), 188, 318
Migration. See Cell migration
Mitochondria-associated endoplasmic reticulum membrane (MAM), 346–347
Mitochondrial outer membrane permeabilization (MOMP), 368–371, 376
Mitogen-activated protein kinase (MAPK). See also specific kinases
activation, 35
cascade, 81
classification, 81–83
computational models, 72–73
cyclin D transcription control, 141
development role, 217
embryonic patterning, 217
G-protein-coupled receptor signaling, 13
interacting kinases, 410–411
learning and memory role, 258
motifs, 40
pathogen signaling corruption in host
 anthrax, 393
overview, 392
Shigella, 394
Yersinia, 393–394
scaffolds, 43, 85
stress signaling
activation cascade, 354–355
inactivation, 355
overview, 353–354
physiological roles
cell death, 356
inflammation, 356–357
metabolism, 357
scaffold protein function, 355–356
subcellular localization, 41
synaptic plasticity role, 258
TNFR1 signaling, 301–303
unfolded protein response, 346
Mitosis
anaphase entry, 160
CDK1 activation, 152–157, 160
cytokinesis coordination, 161–162
DNA damage checkpoint, 157–159
DNA replication checkpoint, 159
G2/M transition regulation, 157
network dissection, 160
spindle assembly checkpoint, 160–161
table of proteins in control, 154
transitions, 152–153
Mitotic checkpoint complex (MCC), 161
Mitotic exit network (MEN), 161–162
MLCK. See Myosin light-chain kinase
MLCP. See Myosin light-chain phosphatase
MLKL, 303, 380
MMSET, 36
Modularity, signaling studies, 431–432
MOMP. See Mitochondrial outer membrane permeabilization
Mos, 330–331
M-phase promoting factor (MPF), 153
MPF. See Maturation promoting factor; M-phase promoting factor
Mrc1, 150
MRN complex, 158
mRNA. See Messenger RNA
MscC, 238
MscL, 238–239, 245
MscS, 238–239
MSK1, 303, 394
MSK2, 394
MTOC. See Microtubule-organizing center
mTOR. See Mammalian target of rapamycin
mTORC1, 88, 174–175
activation and regulation, 88, 92–93, 176
metabolic signaling, 178
translational control, 44
unfolded protein response studies, 350
mTORC2, 92–93, 175
Muscle contraction
cardiac muscle
contraction, 269
heart failure, 271
hypertrophy
exercise-induced, 269–270
pathophysiological, 270–271
energy homeostasis
exercise adaptation, 284
fatty acid oxidation, 284
glucose uptake and glycogen synthesis, 282–284
glycogen breakdown, 281–282
 signaling overview, 264–266
skeletal muscle
contraction, 266–268
fiber types and exercise response, 268–269
malignant hyperthermia, 269
smooth muscle
calcium sensitization, 273
contraction, 272–273
types, 271
vascular disease, 273–274
Myc, 41, 173, 408, 411, 415, 418
MyD88, 123, 295–298, 300
Myopic, 134
Myosin
cell migration and contraction, 186
myosin II, 186
Myosin heavy-chain kinase (MHCK), 191
Myosin light-chain kinase (MLCK), 186, 265, 268, 273, 304
Myosin light-chain phosphatase (MLCP), 265, 273
MYPT1, 273
Myristoylation, membrane proteins, 42
Myt1, 330

N
NBR1, 379
Nbs1, 158
NCAM. See Neural cell adhesion molecule
Nck, 8, 189
NCS1, 271
Necrosis
caspase control, 380
excitotoxicity, 381
Nox1 induction, 380–381
 overview, 366
types, 379–380
Nedd4, 110, 113
Nedd8, 45
Nek2, 41
NEMO, 45, 123
Nerve growth factor (NGF), receptor dimerization, 7
Neural cell adhesion molecule (NCAM), signaling, 20–21
Neuromuscular junction (NMJ), 267
NF-κB. See Nuclear factor-κB
NF1, 411
NF2, 134
NFAT. See Nuclear factor of activated T cells
NGF. See Nerve growth factor
Nitric oxide (NO)
muscle relaxation, 265
signal transduction, 24–25, 37
Nitrosylation, protein regulation mechanisms, 37
NIX, 373, 379
NLR. See Nod-like receptor
NLRC4, 300, 303
NLRP1, 300, 303
NLRP3, 300, 303, 351
NMDAR. See N-Methyl-D-aspartate receptor
N-Methyl-D-aspartate receptor (NMDAR), learning and memory role, 249–
251
NMJ. See Neuromuscular junction
NO. See Nitric oxide
Nod-like receptor (NLR), signaling, 300
NOD1, 300
NOD2, 300
Noise filtering, signaling networks, 76
Notch
development role, 220–221
 embryonic patterning, 220–222
intracellular domain, 111–112, 220
proteolysis and activation, 9–10
signaling overview, 109–111
Nox1, necrosis induction, 380–381
Noxa, 372
NPAS2, 25
NR4A receptors, 24
NR5A receptors, 24
NRF1, 284
NRF2, 284
NSK1, 303
Nuclear factor of activated T cells (NFAT), 127, 268, 274, 307, 317–318, 416
Nuclear factor-κB (NF-κB)
induced genes, 298
lymphocyte signaling, 127
TLR signaling, 297
TNFR1 signaling, 301–303
Nuclear receptors. See also specific receptors
activation, 21
classification, 21–22
orphan receptors, 24
overview, 21, 129–132
promoter binding
heterodimers, 22
homodimers, 22
monomers, 22–23
structure, 21–22, 130
types, 23–24, 130
Numb, cell polarity role, 207–207

O
Oct4, 173
Odd paired, embryonic patterning, 219
Olfaction. See Sensory receptors
Omi, 369
Oocyte. See also Reproduction
activation on fertilization, 336–338
maturation
meiosis I, 328–331
meiosis II arrest, 331–332
overview, 328
OSM, 356
OspF, 394
Oxidative stress, unfolded protein response, 349
Oxygen, signal transduction, 24

P
p16, 144–145, 411
p18, 144–145
p21, 144, 143–144, 146–148, 411, 417
p27, 144, 411
p38 stress-activated protein kinase (SAPK)
cell cycle checkpoint, 159
overview, 84–85
stress signaling
activation cascade, 354–355
inactivation, 355
overview, 353–354
physiological roles
cell death, 356
inflammation, 356–357
metabolism, 357
scaffold protein function, 355–356
p53, 43–44, 417, 420
p57, 144, 147
PAK, 113, 189, 258, 398
PAK2, 368
PAK3, 193
Palmitoyl acyltransferase (PAT), 42
Palmitoylation, membrane proteins, 42–43
Pals1, 204
PAR1. See Protease-activated receptor, 1
Par proteins
cell polarity role, 202–203
localization
active exclusion, 205–207
membrane phospholipid attachment, 204
membrane protein anchoring, 204–205
messenger RNA localization, 205
oligomerization, 204
Par1, 205
Par2, 202
Par3, 202–203, 205–208
Par3–Par6–protein kinase C signaling, 207–209
Par5, 205
Par6, 202–208
Parvin, 189
PAT. See Palmitoyl acyltransferase
Patched, 107–108
Pathogens. See Infection
Patj, 204, 210
PAX2, 229
Paxillin, cell migration role, 188–189
PCNA, 411
PD-1, 319
PDEs. See Phosphodiesterases
PDGF. See Platelet-derived growth factor
PDK. See Pyruvate dehydrogenase kinase
PEA15, 355
Peli1, 298
PEPCK. See Phosphoenolpyruvate carboxykinase
PERK, 347–348, 350, 352
Perlipin1, 289
Permeability transition pore (PTP), 63
Peroxisome proliferator-activated receptor (PPAR), 23, 132
PFK. See Phosphofructokinase
PGC1α, 268, 270, 274, 284
PHAPI, 371
PHD. See Prolyl hydroxylase
Phosphatidylinositol bisphosphate (PIP2)
bacteria hydrolysis, 398–399
signaling overview, 55–57, 63
Phosphatidylinositol trisphosphate (PIP3)
Akt signaling, 57–59, 87
lymphocyte signaling, 322–323
Phosphodiesterases (PDEs), 99
oocyte PDE3, 329
overview, 55
Phosphoenolpyruvate carboxykinase (PEPCK), 286–287
Phosphofructokinase (PFK), 168, 285–286
Phosphoinositide, 3-kinase (PI3K)
activation, 52, 58, 87
Akt pathway overview, 87–89
cancer signaling, 408–416, 418–421
cell migration role, 189–191
G-protein-coupled receptor signaling, 13–14
glucose metabolism signaling, 168, 170–171
lymphocyte signaling, 126–127
lymphocyte signaling, 321–323
mTORC1 target, 175
recruitment, 42
subcellular localization, 42
Phospholamban, 269
Phospholipase A2 (PLA2), isoforms, 432–433
Phospholipase C (PLC)
activation, 52
FcR signaling, 307
feedback control, 9
fertilization role, 337–338
LAT recruitment, 316
lymphocyte signaling, 126
messenger generation, 56–57
muscle calcium sensitization, 273
Phosphorylation, protein regulation mechanisms, 33–34
PI3K. See Phosphoinositide, 3-kinase
PIDD, 374–375
PIF, 429
Pins, cell polarity role, 205–206
PIP2. See Phosphatidylinositol bisphosphate
PIP3. See Phosphatidylinositol trisphosphate
PIXα, 305
PKA. See Protein kinase A
PKBR1, 191–192
PKC. See Protein kinase C
PKD. See Protein kinase D
PKG. See Cyclic GMP-dependent protein kinase
PKG. See Protein kinase G
PKI. See Protein kinase inhibitor
PKR. See Double-stranded RNA-dependent kinase
PLA2. See Phospholipase A2
Platelet-derived growth factor (PDGF)
chemokine activity, 193
receptor, 9
PLC. See Phospholipase C
Plk1. See Polo-like kinase, 1
Plx1, 338
Plzf, 334
PML-RAR fusion, 417
Polarity. See Cell polarity
Polo-like kinase, 1 (Plk1), 40–41, 338
Pom1, 157
Pop1, 146
Postsynaptic density (PSD)
calcium-regulated signaling
calcineurin, 253–254
calcium/calmodulin-dependent protein kinase II, 251–253, 258
scaffold proteins, 254–256
PP1. See Protein phosphatase, 1
PP2A. See Protein phosphatase, 2A
PPAR. See Peroxisome proliferator-activated receptor
PRAS40, 88
Pregnane X receptor (PXR), 23–24
Prex1, 305
Prolyl hydroxylase (PHD), 171–173
Prostaglandins, signaling overview, 59
Protease-activated receptor, 1 (PAR1), 10
Protein kinase A (PKA)
cyclic AMP target, 53–54, 101
isozymes, 57
myristoylation, 43
regulation, 101
substrates, 101
Protein kinase B. See Akt
Protein kinase C (PKC)
atypical PKC, 135, 188, 202–203, 205–208
diacylglycerol target, 55, 57
feedback control, 9
lipid messengers, 57
lymphocyte signaling, 318–319
muscle calcium sensitization, 273
Par protein localization, 205–207
 polarity signaling, 207–209
receptor feedback, 9
Protein kinase D (PKD), 318
Protein kinase G (PKG), cyclic GMP target, 55
Protein kinase inhibitor (PKI), 101
Protein levels, equation, 45
Protein phosphatase, 1 (PP1)
learning and memory role, 254
PKA as substrate, 101
Protein phosphatase, 2A (PP2A)
CDK1 as substrate, 155
learning and memory role, 254
oocyte maturation role, 331
PKA as substrate, 101
PSD. See Postsynaptic density
PSD93, 254
PSD95, 254–256
P-selectin, 303
PSGL1, 303
PTB domain, protein–protein interactions, 39–40
PTEN, 59, 89, 176, 189–191, 407–408, 418
PTP. See Permeability transition pore
Puma, 372
PXR. See Pregnane X receptor
Pyk2, 18
Pyrin domain, 368
Pyruvate dehydrogenase kinase (PDK), 284, 414
Pyruvate kinase, 173–174, 415, 431

Q
Q30, 400

R
RA. See Retinoic acid
Rab, 104, 387, 431, 82, 141, 188–189, 396–398
cell migration role, 188–189
G-protein-coupled receptor signaling, 13
Rac1, 354, 368
RACK1, 350
Rad17, 159
Raf1, synaptic plasticity role, 258
Rag, 175
RAIDD, 374–375
RAMPs. See Receptor activity-modifying proteins
Rap
learning and memory role, 259
synaptic plasticity role, 258–259
Rap1, 318
RAR. See Retinoic acid receptor
Ras
cancer signaling, 408–410, 412, 415–416, 418, 421
innate immunity, 305
learning and memory role, 259
lymphocyte signaling, 319
prenylation, 42–43
synaptic plasticity role, 257–259
RasC, 192
Rb. See Retinoblastoma protein
Receptor activity-modifying proteins (RAMPs), 14
Receptor tyrosine kinases (RTKs)
cell adhesion molecule interactions, 21
coreceptors, 8
dimerization, 5–7
downstream signaling, 8
endocytosis, 9
feedback and amplification, 9
mutations and disease, 9
 overview, 4–5
proteolysis, 10
Regulators of G-protein-coupled receptor signaling (RGS), 15
Replication protein A (RPA), 158
Reproduction. See also Meiosis; Oocyte; Sperm
fertilization
acrosome reaction, 336
gamete fusion and egg activation, 336–338
prospects for study, 339
zygote formation, 338
Ret, 333–334
Retinoblastoma protein (Rb)
cancer, 408
cell cycle control, 140–141
Retinoic acid (RA), sperm maturation role, 335
Retinoic acid receptor (RAR), 22, 317
Retinoid X receptor (RXR), 21–22, 130
REV-ERB, 24–25
RGS. See Regulators of G-protein-coupled receptor signaling
Rheb, 175, 322
RHIM, 298, 303, 380
Rho, G-protein-coupled receptor signaling, 13
Rho1, 207
RhoA, 396
cell migration role, 188
cytoskeleton regulation, 188
Par6 regulation, 208
Rhodopsin, 239–240
RhoG, 305
RIG-I-like receptor (RLR), signaling, 298–300
RIP1, 298, 300, 303, 355, 373–374, 380
RIP2, 300
RIP3, 303, 366, 373, 379–380
RIPK, 380
RLR. See RIG-I-like receptor
ROCK, 186
Ror, 106
RPA. See Replication protein A
RSK, 330–331, 411
Rsr1, 200
RTKs. See Receptor tyrosine kinases
Rub1, 434
Rum1, 147
RXR. See Retinoid X receptor
Ryanodine receptor (RyR), 264–265, 267–269, 271, 274
Ryk, 8, 105
RyR. See Ryanodine receptor

S
S6 kinase, 88
S144, 205
SAP102, 254
SAP97, 254
SAPK. See p38 stress-activated protein kinase
SARA. See Smad anchor for receptor activation
SCF. See Stem cell factor
Scribble, 135, 204, 210, 416
SDF1, 37
Second messengers. See also specific molecules
cyclic nucleotides, 53–55
ions, 59–83
lipids, 55–59
overview, 52–53
Secretion systems, bacteria, 391–39
Sensory receptors
evolution, 242–244
G-protein-coupled receptors, 234–235, 238–239, 241
 olfaction, 242
photoreceptors, 239, 241, 243
prospects for study, 244–245
receptor activation, 238–240
signaling overview, 234–238
thermosensation, 242
Septation initiation network (SIN), 161–162
SERCA, 62, 268–270, 274
Serine/threonine kinase receptors
activation, 7
downstream signaling, 8
mutations and disease, 9
overview, 4, 6
Serpent, embryonic patterning, 219
Sevenless, 230
SF. See Sperm cytosolic factor
SH2 domain
motifs, 40–41
protein–protein interactions, 39–40
SH3 domain
motifs, 41
protein–protein interactions, 39–40
SHANK, 254–256
Shc, 8
SHIP, 323, 338
Ship1, 190
SHP1, 323
Sic1, 146, 434
SIK1, 287
Sildenafil, 55
SIN. See Septation initiation network
SIRT1, 284, 287, 375
Skeletal muscle. See Muscle contraction
Ski, 113
SKP2, 146–147
SLP76, 128, 316
SMAC. See Supramolecular activation cluster
Smac, 369
Smad, transforming growth factor-β signaling, 113–114
Smad anchor for receptor activation (SARA), 113
Smo, 107
Smooth muscle. See Muscle contraction
SNAREs, 62, 336
SNARK/NUAK2, 283
SNoN, 113
SOCE. See Store-operated calcium entry
Sodium/potassium ATPase, 59
SopE, 395–396
Sperm cytosolic factor (SF), 338
Sperm. See also Reproduction
capacitation and calcium channels, 335–336
maturation
overview, 332–334
stem cell proliferation and maintenance, 334–335
spermatocyte meiosis and release, 335
Sphingomyelin, signaling, 56
Sphingosine, hydrolysis, 59
Spindle assembly checkpoint, 160–161
SPIRE, 399
SptP, 396
Src, 19, 189, 315, 411
SREBP. See Sterol response element-binding protein
SRF, 274
STATs. See JAK/STAT signaling
Stem cell factor (SCF), 145, 147, 333
Sterol response element-binding protein (SREBP), 288, 414
STIM1, 307, 316
STIM2, 316
STING, 436
Store-operated calcium entry (SOCE), 337
Stress-activated protein kinase. See p38 stress-activated protein kinase
SuFu, 107
SUMO, 45, 131
Supramolecular activation cluster (SMAC), 318
Swe1, 157
Syk, 4, 315–316
SynGAP, 258

T
TAB2, 355
TAB3, 355
TACE. See ADAM17
TAK1, 45, 295–297, 300, 303, 355
Talin, 18, 187
TAO1, 135
Target of rapamycin. See Mammalian target of rapamycin
Taste. See Sensory receptors
TAZ, 115, 230
TBC1D1, 283
TBC1D4, 282
TBK1, 123, 298, 300
T cell
classification, 314
costimulatory molecules, 319–320
cytokine signaling, 320–321
PI3K/Akt signaling, 321–323
receptor. See T-cell receptor
T-cell receptor (TCR)
adaptor molecules, 316
ITAM, 315–316
signaling
 calcium, 316–317
diacylglycerol, 316–318
ERK1/2, 319
inhibitory signals, 323
nuclear factor of activated T cells, 317–318
overview, 125–127, 317
protein kinase C, 318–319
Ras, 319
structure and function, 314–315
TCF, 227, 319
TCF/LEF, 103–104
TCR. See T-cell receptor
Tel1, 158
Tem1, 162
TET1, 407
TGF-β. See Transforming growth factor-β
Thrombospondin (Tsp1), 420
Thyroid hormone
energy homeostasis role, 279–280
receptor, 22
TIGAR, 415
TIMP3, 420
Tinman, 219
Tip60, 35
TLR. See Toll-like receptor
TNFRs. See Tumor necrosis factor receptors
Toll-like receptor (TLR)
interferon induction, 297–298
ligands, 295
signaling overview, 121–123
TAK1 and IKK activation, 295–296
TLR4 signaling, 296
TRIF in signaling, 298
TopBP1, 159
TorC2, 191–192
Torso, development role, 217
Tp12, 297
TRADD, 16, 298, 300, 302, 371, 373–374
TRAF, 15
TRAF2, 59, 350–351, 355, 374
TRAF3, 123, 297–298
TRAF6, 45, 114, 295, 297, 307
TRAIL, 373, 412
Transcriptional regulation, protein levels, 43–44
Transforming growth factor-β (TGF-β)
receptor types, 8
signaling overview, 114–115
Translational regulation, protein levels, 44
TRB3, 350
TRIF, Toll-like receptor signaling, 298
Troponin C, 62
TRPA1, 242
TRPC6, 272
TRPM6, 63
TRPM7, 63
TRPM8, 239, 242
TRPV1, sensory role, 237, 239, 242, 244
TSC1, 93, 350
TSC2, 88, 93, 350, 411, 413, 416
Tsp1. See Thrombospondin
TTP, 356
Tumor necrosis factor receptors (TNFRs)
activation, 15
caspase-8 activation in death receptor pathway, 371, 373–374
ligand diversity, 15
pathology, 17
signaling, 15–17
structure, 15–16
 TNFR1 signaling
cell death induction, 303
MAPK, 301–303
nuclear factor-κB, 301–303
TXNIP, 351

U
UbcH proteins, 295, 301
Ubiquitylation
G1 regulation
CIP/KIP degradation, 147–148
cyclin degradation, 145–147
pathogen disruption, 400–401
protein degradation, 44–47
UCP. See Uncoupling protein
ULK1, 378–379
Uncoupling protein (UCP), 289
Unfolded protein response (UPR)
canonical signaling, 346–349
noncanonical aspects, 349
physiological roles
cell survival and death responses, 350
inflammation, 350–351
metabolic responses, 351–353
overview, 349–350
UPR. See Unfolded protein response

V
VAMP8, 378
Vascular cell adhesion molecule (VCAM), 17, 303
Vascular endothelial growth factor (VEGF)
cancer angiogenesis, 420
 hypoxia signaling, 24
VCAM. See Vascular cell adhesion molecule
VEGF. See Vascular endothelial growth factor
VHL, 171–172, 420
Vinculin, 18
VirG, 399
Virus. See Infection
Vision. See Sensory receptors
VOCC. See Voltage-operated calcium channel
Voltage-operated calcium channel (VOCC), 59, 62, 337
VopA/P, 394
VopS, 397
VPA0450, 399
Vps15, 378
Vps34, 378

W
WASP, 398
WAVE, 186, 40
Wee1, 155, 157, 329–330
WH2 domain, 399–400
WIP1, 355
Wnt
canonical signaling, 104
cell polarity signaling cross talk, 209
embryonic patterning, 225
noncanonical signaling, 104–105
signaling overview, 103–105
WTS, 134

X
XBP1, 347, 349–351, 353
XIAP, 369, 375, 412

Y
YAP, 115, 230
YK1. See Yorkie
YopE, 396
YopH, 392
YopJ, 393–394
YopT, 396
Yorkie (YKI), 134, 143, 298, 230
YpkA, 396

Z
Zap70, 4
ZO1, 20
ZO2, 20