Article | Published:

K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8+ T cell activation

Nature Immunology volume 16, pages 12531262 (2015) | Download Citation

Abstract

The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger–type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase–like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8+ T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8+ T cells but not that of naive CD4+ T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    & T cell receptor signalling networks: branched, diversified and bounded. Nat. Rev. Immunol. 13, 257–269 (2013).

  2. 2.

    , & Tailoring T-cell receptor signals by proximal negative feedback mechanisms. Nat. Rev. Immunol. 8, 699–712 (2008).

  3. 3.

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

  4. 4.

    et al. K33-linked polyubiquitination of T cell receptor-ζ regulates proteolysis-independent T cell signaling. Immunity 33, 60–70 (2010).

  5. 5.

    et al. The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells. Nat. Immunol. 15, 657–666 (2014).

  6. 6.

    , & E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance. Proc. Natl. Acad. Sci. USA 106, 16770–16775 (2009).

  7. 7.

    et al. The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation. Immunity 32, 670–680 (2010).

  8. 8.

    , , & Altered thymic positive selection and intracellular signals in Cbl-deficient mice. Proc. Natl. Acad. Sci. USA 95, 15547–15552 (1998).

  9. 9.

    , & Perturbed regulation of ZAP-70 and sustained tyrosine phosphorylation of LAT and SLP-76 in c-Cbl-deficient thymocytes. J. Immunol. 162, 7133–7139 (1999).

  10. 10.

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

  11. 11.

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

  12. 12.

    & Proteolysis-independent regulation of PI3K by Cbl-b-mediated ubiquitination in T cells. Nat. Immunol. 2, 870–875 (2001).

  13. 13.

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

  14. 14.

    et al. Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex. Nat. Immunol. 7, 57–66 (2006).

  15. 15.

    et al. The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity. Nat. Immunol. 12, 1002–1009 (2011).

  16. 16.

    et al. Peli1 promotes microglia-mediated CNS inflammation by regulating Traf3 degradation. Nat. Med. 19, 595–602 (2013).

  17. 17.

    et al. Roquin paralogs 1 and 2 redundantly repress the Icos and Ox40 costimulator mRNAs and control follicular helper T cell differentiation. Immunity 38, 655–668 (2013).

  18. 18.

    , & Immunity by ubiquitylation: a reversible process of modification. Nat. Rev. Immunol. 5, 941–952 (2005).

  19. 19.

    & The role of E3 ligases in autoimmunity and the regulation of autoreactive T cells. Curr. Opin. Immunol. 19, 665–673 (2007).

  20. 20.

    et al. Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 306, 271–275 (2004).

  21. 21.

    et al. A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3ζ-chain degradation. Immunity 29, 33–43 (2008).

  22. 22.

    et al. Regulation of ZAP-70 activation and TCR signaling by two related proteins, Sts-1 and Sts-2. Immunity 20, 37–46 (2004).

  23. 23.

    & Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3. Proc. Natl. Acad. Sci. USA 99, 14843–14848 (2002).

  24. 24.

    , , , & RNF41 (Nrdp1) controls type 1 cytokine receptor degradation and ectodomain shedding. J. Cell Sci. 124, 921–932 (2011).

  25. 25.

    et al. The E3 ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp1) promotes M2 macrophage polarization by ubiquitinating and activating transcription factor CCAAT/enhancer-binding Protein β (C/EBPβ). J. Biol. Chem. 287, 26740–26748 (2012).

  26. 26.

    et al. Loss of Nrdp1 enhances ErbB2/ErbB3-dependent breast tumor cell growth. Cancer Res. 66, 11279–11286 (2006).

  27. 27.

    et al. The diabetes susceptibility gene Clec16a regulates mitophagy. Cell 157, 1577–1590 (2014).

  28. 28.

    et al. The E3 ubiquitin ligase Nrdp1 'preferentially' promotes TLR-mediated production of type I interferon. Nat. Immunol. 10, 744–752 (2009).

  29. 29.

    , & CD8+ T cells in multiple sclerosis. Expert Opin. Ther. Targets 17, 1053–1066 (2013).

  30. 30.

    et al. A phosphatase activity of Sts-1 contributes to the suppression of TCR signaling. Mol. Cell 27, 486–497 (2007).

  31. 31.

    , , & Sts-2 is a phosphatase that negatively regulates ζ-associated protein (ZAP)-70 and T cell receptor signaling pathways. J. Biol. Chem. 286, 15943–15954 (2011).

  32. 32.

    , , , & The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70. J. Biol. Chem. 272, 33140–33144 (1997).

  33. 33.

    et al. Cbl promotes ubiquitination of the T cell receptor ζ through an adaptor function of Zap-70. J. Biol. Chem. 276, 26004–26011 (2001).

  34. 34.

    et al. The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. J. Immunol. 164, 4616–4626 (2000).

  35. 35.

    , , , & Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement. Proc. Natl. Acad. Sci. USA 98, 9611–9616 (2001).

  36. 36.

    , & Role of CD8+ T cells in murine experimental allergic encephalomyelitis. Science 256, 1213–1215 (1992).

  37. 37.

    et al. Less mortality but more relapses in experimental allergic encephalomyelitis in CD8−/− mice. Science 256, 1210–1213 (1992).

  38. 38.

    et al. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat. Immunol. 5, 516–523 (2004).

  39. 39.

    et al. Mice with a disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156, 5–7 (1996).

  40. 40.

    & Interferon-γ confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26, 1641–1646 (1996).

  41. 41.

    , , , & IFN-γ plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157, 3223–3227 (1996).

  42. 42.

    , , , & Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192, 117–122 (2000).

  43. 43.

    , & Failure to suppress the expansion of the activated CD4 T cell population in interferon γ-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192, 123–128 (2000).

  44. 44.

    et al. E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKCζ. J. Exp. Med. 208, 2099–2112 (2011).

  45. 45.

    et al. Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO. Nat. Immunol. 15, 612–622 (2014).

  46. 46.

    et al. Constitutive MHC class I molecules negatively regulate TLR-triggered inflammatory responses via the Fps-SHP-2 pathway. Nat. Immunol. 13, 551–559 (2012).

  47. 47.

    , , , & Heat shock protein 70, released from heat-stressed tumor cells, initiates antitumor immunity by inducing tumor cell chemokine production and activating dendritic cells via TLR4 pathway. J. Immunol. 182, 1449–1459 (2009).

  48. 48.

    & Active induction of experimental allergic encephalomyelitis. Nat. Protoc. 1, 1810–1819 (2006).

  49. 49.

    & Passive induction of experimental allergic encephalomyelitis. Nat. Protoc. 1, 1952–1960 (2006).

Download references

Acknowledgements

We thank H. Shen (University of Pennsylvania School of Medicine) for virulent recombinant L. monocytogenes (LM-OVA) and attenuated recombinant L. monocytogenesactA-LM-OVA); H. Shen, X. Zhu and Z. Li for technical assistance; L. Lu for discussions; and Q. Guo for assistance with confocal microscopy. Supported by the National Key Basic Research Program of China (2010CB911903 and 2013CB530500), the National Natural Science Foundation of China (81222039, 81172851, 81471566, 31170863, 81123006 and 31390431), the National Excellent Doctoral Dissertation of China (200775) and the Shanghai Committee of Science and Technology (11QH1402900).

Author information

Author notes

    • Mingjin Yang
    •  & Taoyong Chen

    These authors contributed equally to this work.

Affiliations

  1. National Key Laboratory of Medical Molecular Biology & Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.

    • Mingjin Yang
    •  & Xuetao Cao
  2. National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China.

    • Mingjin Yang
    • , Taoyong Chen
    • , Xuelian Li
    • , Chen Wang
    • , Yan Gu
    • , Yanfang Liu
    • , Sheng Xu
    •  & Xuetao Cao
  3. Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China.

    • Zhou Yu
    • , Songqing Tang
    •  & Jianli Wang
  4. Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.

    • Weihua Li
    •  & Xuemin Zhang

Authors

  1. Search for Mingjin Yang in:

  2. Search for Taoyong Chen in:

  3. Search for Xuelian Li in:

  4. Search for Zhou Yu in:

  5. Search for Songqing Tang in:

  6. Search for Chen Wang in:

  7. Search for Yan Gu in:

  8. Search for Yanfang Liu in:

  9. Search for Sheng Xu in:

  10. Search for Weihua Li in:

  11. Search for Xuemin Zhang in:

  12. Search for Jianli Wang in:

  13. Search for Xuetao Cao in:

Contributions

M.Y., T.C., X.L., Z.Y., S.T. and C.W. did the main experiments and analyzed data; M.Y., Y.G., Y.L. and S.X. performed flow cytometry assays; W.L. and X.Z. assisted with the liquid chromatography–mass spectrometry; J.W. contributed materials; and X.C. and T.C. designed and supervised the experiments and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Taoyong Chen or Xuetao Cao.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–3

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ni.3258

Further reading