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Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins

Nature Immunology volume 5, pages 255–265 (2004)Cite this article

Abstract

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca2+-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-θ and PLC-γ1. T cells from Itch- and Cbl-b–deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function–associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

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Figure 1: Decreased PLC-γ1 levels correlate with T cell anergy.
Figure 2: Decreased PLC-γ1 and impaired Ca2+ mobilization correlate with T cell anergy.
Figure 3: E3 ubiquitin ligases of the HECT type induce ubiquitination and degradation of PLC-γ1.
Figure 4: Upregulation of E3 ligases in T cells subjected to sustained Ca2+ signaling.
Figure 5: Ionomycin-anergized T cells show decreased stability of the immunological synapse.
Figure 6: Cblb- and Itch-deficient T cells are resistant to anergy induction.

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References

  1. Hunter, T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225–236 (1995).

    Article  CAS  Google Scholar 

  2. Sun, H., Charles, C.H., Lau, L.F. & Tonks, N.K. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75, 487–493 (1993).

    Article  CAS  Google Scholar 

  3. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  4. Sorkin, A. & Von Zastrow, M. Signal transduction and endocytosis: close encounters of many kinds. Nat. Rev. Mol. Cell. Biol. 3, 600–614 (2002).

    Article  CAS  Google Scholar 

  5. Valitutti, S., Muller, S., Salio, M. & Lanzavecchia, A. Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation. J. Exp. Med. 185, 1859–1864 (1997).

    Article  CAS  Google Scholar 

  6. Schwartz, R.H. T cell anergy. Annu. Rev. Immunol. 21, 305–334 (2003).

    Article  CAS  Google Scholar 

  7. Macian, F., Lopez-Rodriguez, C. & Rao, A. Partners in transcription: NFAT and AP-1. Oncogene 20, 2476–2489 (2001).

    Article  CAS  Google Scholar 

  8. Macian, F. et al. Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109, 719–731 (2002).

    Article  CAS  Google Scholar 

  9. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

    Article  CAS  Google Scholar 

  10. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

    Article  CAS  Google Scholar 

  11. Huppa, J.B., Gleimer, M., Sumen, C. & Davis, M.M. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nat. Immunol. 4, 749–755 (2003).

    Article  CAS  Google Scholar 

  12. Lee, K.H. et al. T cell receptor signaling precedes immunological synapse formation. Science 295, 1539–1542 (2002).

    Article  CAS  Google Scholar 

  13. Murphy, L.O., Smith, S., Chen, R.H., Fingar, D.C. & Blenis, J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat. Cell. Biol. 4, 556–564 (2002).

    Article  CAS  Google Scholar 

  14. Lee, K.H. et al. The immunological synapse balances T cell receptor signaling and degradation. Science 302, 1218–1222 (2003).

    Article  CAS  Google Scholar 

  15. Fang, D. et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat. Immunol. 3, 281–287 (2002).

    Article  CAS  Google Scholar 

  16. Naramura, M. et al. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand- induced TCR down-modulation. Nat. Immunol. 3, 1192–1199 (2002).

    Article  CAS  Google Scholar 

  17. Anandasabapathy, N. et al. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535–547 (2003).

    Article  CAS  Google Scholar 

  18. Katzmann, D.J., Odorizzi, G. & Emr, S.D. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell. Biol. 3, 893–905 (2002).

    Article  CAS  Google Scholar 

  19. Perry, W.L. et al. The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a18H mice. Nat. Genet. 18, 143–146 (1998).

    Article  CAS  Google Scholar 

  20. Chiang, Y.J. et al. Cbl-b regulates the CD28 dependence of T-cell activation. Nature 403, 216–220 (2000).

    Article  CAS  Google Scholar 

  21. Bachmaier, K. et al. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 403, 211–216 (2000).

    Article  CAS  Google Scholar 

  22. Healy, J.I. et al. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6, 419–428 (1997).

    Article  CAS  Google Scholar 

  23. Glynne, R. et al. How self-tolerance and the immunosuppressive drug FK506 prevent B-cell mitogenesis. Nature 403, 672–676 (2000).

    Article  CAS  Google Scholar 

  24. Irvin, B.J., Williams, B.L., Nilson, A.E., Maynor, H.O. & Abraham, R.T. Pleiotropic contributions of phospholipase C-γ1 (PLC-γ1) to T- cell antigen receptor-mediated signaling: reconstitution studies of a PLC-γ1-deficient Jurkat T-cell line. Mol. Cell. Biol. 20, 9149–9161. (2000).

    Article  CAS  Google Scholar 

  25. Wells, A.D. et al. Regulation of T cell activation and tolerance by phospholipase Cγ-1-dependent integrin avidity modulation. J. Immunol. 170, 4127–4133 (2003).

    Article  CAS  Google Scholar 

  26. Kaji, T., Hachimura, S., Ise, W. & Kaminogawa, S. Proteome analysis reveals caspase activation in hyporesponsive CD4 T lymphocytes induced in vivo by the oral administration of antigen. J. Biol. Chem. 278, 27836–27843 (2003).

    Article  CAS  Google Scholar 

  27. Rizo, J. & Sudhof, T.C. C2-domains, structure and function of a universal Ca2+-binding domain. J. Biol. Chem. 273, 15879–15882 (1998).

    Article  CAS  Google Scholar 

  28. Rotin, D., Staub, O. & Haguenauer-Tsapis, R. Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J. Membr. Biol. 176, 1–17 (2000).

    Article  CAS  Google Scholar 

  29. Plant, P.J. et al. Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J. Cell. Biol. 149, 1473–1484 (2000).

    Article  CAS  Google Scholar 

  30. Seto, E.S., Bellen, H.J. & Lloyd, T.E. When cell biology meets development: endocytic regulation of signaling pathways. Genes Dev. 16, 1314–1336 (2002).

    Article  CAS  Google Scholar 

  31. Katzmann, D.J., Babst, M. & Emr, S.D. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145–155 (2001).

    Article  CAS  Google Scholar 

  32. Rao, N., Dodge, I. & Band, H. The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system. J. Leukoc. Biol. 71, 753–763 (2002).

    CAS  PubMed  Google Scholar 

  33. Dustin, M.L., Bromley, S.K., Kan, Z., Peterson, D.A. & Unanue, E.R. Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc. Natl. Acad. Sci. USA 94, 3909–3913 (1997).

    Article  CAS  Google Scholar 

  34. Negulescu, P.A., Krasieva, T.B., Khan, A., Kerschbaum, H.H. & Cahalan, M.D. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4, 421–430 (1996).

    Article  CAS  Google Scholar 

  35. Dustin, M.L. & Springer, T.A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619–624 (1989).

    Article  CAS  Google Scholar 

  36. Sun, Z. et al. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature 404, 402–407 (2000).

    Article  CAS  Google Scholar 

  37. Yokoi, N. et al. Cblb is a major susceptibility gene for rat type 1 diabetes mellitus. Nat. Genet. 31, 391–394 (2002).

    Article  CAS  Google Scholar 

  38. Courbard, J.R. et al. Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH. J. Biol. Chem. 277, 45267–45275 (2002).

    Article  CAS  Google Scholar 

  39. Kowanetz, K. et al. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and downregulation of EGF receptors. J. Biol. Chem. 278, 39735–39746 (2003).

    Article  CAS  Google Scholar 

  40. Lechner, O. et al. Fingerprints of anergic T cells. Curr. Biol. 11, 587–595 (2001).

    Article  CAS  Google Scholar 

  41. Korthauer, U. et al. Anergic T lymphocytes selectively express an integrin regulatory protein of the cytohesin family. J. Immunol. 164, 308–318 (2000).

    Article  CAS  Google Scholar 

  42. Kolanus, W. et al. αLβ2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86, 233–242 (1996).

    Article  CAS  Google Scholar 

  43. Boussiotis, V.A., Freeman, G.J., Berezovskaya, A., Barber, D.L. & Nadler, L.M. Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science 278, 124–128 (1997).

    Article  CAS  Google Scholar 

  44. Reedquist, K.A. et al. The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J. Cell. Biol. 148, 1151–1158 (2000).

    Article  CAS  Google Scholar 

  45. Sebzda, E., Bracke, M., Tugal, T., Hogg, N. & Cantrell, D.A. Rap1A positively regulates T cells via integrin activation rather than inhibiting lymphocyte signaling. Nat. Immunol. 3, 251–258 (2002).

    Article  CAS  Google Scholar 

  46. Katagiri, K. et al. Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase. Mol. Cell. Biol. 20, 1956–1969 (2000).

    Article  CAS  Google Scholar 

  47. Katagiri, K., Hattori, M., Minato, N. & Kinashi, T. Rap1 functions as a key regulator of T-cell and antigen-presenting cell interactions and modulates T-cell responses. Mol. Cell. Biol. 22, 1001–1015 (2002).

    Article  CAS  Google Scholar 

  48. Suga, K. et al. CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1. FEBS Lett. 489, 249–253 (2001).

    Article  CAS  Google Scholar 

  49. Ishida, D. et al. Antigen-driven T cell anergy and defective memory T cell response via deregulated Rap1 activation in SPA-1-deficient mice. Proc. Natl. Acad. Sci. USA 100, 10919–10924 (2003).

    Article  CAS  Google Scholar 

  50. Fang, D. et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat. Immunol. 3, 281–287 (2002).

    Article  CAS  Google Scholar 

  51. Gajewski, T.F., Qian, D., Fields, P. & Fitch, F.W. Anergic T-lymphocyte clones have altered inositol phosphate, calcium, and tyrosine kinase signaling pathways. Proc. Natl. Acad. Sci. USA 91, 38–42 (1994).

    Article  CAS  Google Scholar 

  52. Khoshnan, A., Bae, D., Tindell, C.A. & Nel, A.E. The physical association of protein kinase C theta with a lipid raft-associated inhibitor of κB factor kinase (IKK) complex plays a role in the activation of the NF-κB cascade by TCR and CD28. J. Immunol. 165, 6933–6940 (2000).

    Article  CAS  Google Scholar 

  53. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  Google Scholar 

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Acknowledgements

We thank members of the Rao and Dustin laboratories for discussions, and T. Starr for the preparation of ICAM-1 and I-Ek for planar bilayer experiments. We also thank A. Altman, H. Band, J. Brugge, C. Joazeiro, M. Katan for advice and reagents. Supported by National Institutes of Health grants RO1-AI48213, RO1-AI40127 and RO3-HD39685 (to A.R.), RO1-AI50280 and R21-AI48542 (to Y.-C.L.) and AI-43542; an Irene Diamond Foundation grant (to M.L.D.); EMBO (V.H.); and the Cancer Research Institute (S.-H.I. and S.F.).

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Author notes

  1. Fernando Macián

    Present address: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA

  2. Sin-Hyeog Im

    Present address: Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-ku, Kwangju, 500-712, Korea

Authors and Affiliations

  1. Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Vigo Heissmeyer, Fernando Macián, Sin-Hyeog Im, Stefan Feske & Anjana Rao

  2. Program in Molecular Pathogenesis and Department of Pathology, Skirball Institute of Molecular Medicine, New York University School of Medicine, New York, 10016, New York, USA

    Rajat Varma & Michael L Dustin

  3. Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, 92121, California, USA

    K Venuprasad & Yun-Cai Liu

  4. Department of Microbiology, Columbia University, College of Physicians and Surgeons, 701 West 168th Street, HHSC, New York, 10032, New York, USA

    Hua Gu

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Heissmeyer, V., Macián, F., Im, SH. et al. Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat Immunol 5, 255–265 (2004). https://doi.org/10.1038/ni1047

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