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mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6

Nature Cell Biology volume 15, pages 406–416 (2013)Cite this article

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Abstract

Autophagy is important in the basal or stress-induced clearance of bulk cytosol, damaged organelles, pathogens and selected proteins by specific vesicles, the autophagosomes. Following mTOR (mammalian target of rapamycin) inhibition, autophagosome formation is primed by the ULK1 and the beclin-1–Vps34–AMBRA1 complexes, which are linked together by a scaffold platform, the exocyst. Although several regulative steps have been described along this pathway, few targets of mTOR are known, and the cross-talk between ULK1 and beclin 1 complexes is still not fully understood. We show that under non-autophagic conditions, mTOR inhibits AMBRA1 by phosphorylation, whereas on autophagy induction, AMBRA1 is dephosphorylated. In this condition, AMBRA1, interacting with the E3-ligase TRAF6, supports ULK1 ubiquitylation by LYS-63-linked chains, and its subsequent stabilization, self-association and function. As ULK1 has been shown to activate AMBRA1 by phosphorylation, the proposed pathway may act as a positive regulation loop, which may be targeted in human disorders linked to impaired autophagy.

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Figure 1: AMBRA1 interacts with the kinase ULK1 in mammalian cells.
Figure 2: AMBRA1 is important for ULK1 stability and kinase activity.
Figure 3: AMBRA1 is also important for ULK1 Lys-63-linked ubiquitylation.
Figure 4: AMBRA1 regulates ULK1 ubiquitylation by forming a complex with the TRAF6 ubiquitin ligase.
Figure 5: AMBRA1–TRAF6 interaction is necessary for ULK1 Lys-63-linked ubiquitylation.
Figure 6: ULK1 Lys-63-linked ubiquitylation is necessary for ULK1 self-association.
Figure 7: mTOR phosphorylates AMBRA1 at Ser 52, inhibiting its role in ULK1 modification.
Figure 8: Proposed model of AMBRA1 regulation in ULK1 ubiquitylation.

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Acknowledgements

We thank the Animal Facility (STA) of the University of Rome ‘Tor Vergata’ for the mouse work, M. Acuña Villa and M. W. Bennett for editorial and secretarial work, and G. Basile and M. Corrado for research assistance. We are indebted to S. A. Tooze (Cancer Research Institute London, UK), D. H. Kim (University of Minnesota Cancer Center, USA), and D. M. Sabatini and R. Zoncu (Whitehead Institute for Biomedical Research, USA) for kindly providing us with ULK1 and ATG13 constructs and HEK293 cells stably expressing RAPTOR–FLAG, respectively. This work was supported in part by grants from the Telethon Foundation (GGP10225), AIRC (IG2010 and IG2012 to FC and MP), FISM (2009), the Italian Ministry of University and Research (PRIN 2009 and FIRB Accordi di Programma 2011) and the Italian Ministry of Health (Ricerca Finalizzata and Ricerca Corrente to F.C., M.P. and G.M.F.).

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Authors and Affiliations

  1. Dulbecco Telethon Institute at the Department of Biology, University of Rome ‘Tor Vergata’, 00133 Rome, Italy

    Francesca Nazio, Flavie Strappazzon, Valentina Cianfanelli, Matteo Bordi & Francesco Cecconi

  2. Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, 00143 Rome, Italy

    Francesca Nazio, Flavie Strappazzon, Pamela Bielli, Valentina Cianfanelli, Matteo Bordi & Francesco Cecconi

  3. National Institute for Infectious Diseases IRCCS ‘L. Spallanzani’, 00149 Rome, Italy

    Manuela Antonioli, Mauro Piacentini & Gian Maria Fimia

  4. Freiburg Institute for Advanced Studies (FRIAS), School of Life Sciences-LifeNet, University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany

    Christine Gretzmeier & Joern Dengjel

  5. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany

    Christine Gretzmeier & Joern Dengjel

  6. Department of Biology, University of Rome ‘Tor Vergata’, 00133 Rome, Italy

    Mauro Piacentini

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Contributions

F.N. performed most experiments with crucial help from F.S. (immunofluorescence and confocal analyses), M.A. (mutagenesis and cloning), V.C. (immunoprecipitation analysis), M.B. (real-time PCR) and P.B. (kinase assay in vitro). C.G. and J.D. performed the mass spectrometry analysis; G.M.F. provided critical reagents. F.N. and F.C. wrote the manuscript with the help and suggestions of G.M.F. and M.P.; F.C. conceived and designed the research. All authors discussed the results and commented on the manuscript.

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Correspondence to Francesco Cecconi.

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Nazio, F., Strappazzon, F., Antonioli, M. et al. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat Cell Biol 15, 406–416 (2013). https://doi.org/10.1038/ncb2708

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