Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28

Nature Immunology volume 12, pages 1105–1112 (2011)Cite this article

Subjects

Abstract

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (TH2 cells) and interleukin 17–producing helper T cells (TH17 cells) but not of T helper type 1 cells (TH1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell–mediated inflammatory diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Requirement of PKC-θ V3 for localization to the immunological synapse, cSMAC and signaling.
Figure 2: The PKC-θ V3 domain is required and sufficient for CD28 interaction.
Figure 3: A proline-rich motif in the V3 domain of PKC-θ determines its localization to the immunological synapse and interacts with CD28.
Figure 4: Importance of the proline-rich motif in the V3 domain of PKC-θ for localization to the immunological synapse and CD28 interaction.
Figure 5: The interaction between CD28 and the V3 domain of PKC-θ is Lck dependent.
Figure 6: The V3 domain interferes with PKC-θ-mediated signaling and T cell differentiation.
Figure 7: V3 inhibits TH2- but not TH1-mediated lung inflammation.

Similar content being viewed by others

References

  1. Dustin, M.L. The cellular context of T cell signaling. Immunity 30, 482–492 (2009).

    Article  CAS  Google Scholar 

  2. Dustin, M.L., Chakraborty, A.K. & Shaw, A.S. Understanding the structure and function of the immunological synapse. Cold Spring Harb. Perspect. Biol. 2, a002311 (2010).

    Article  CAS  Google Scholar 

  3. Monks, C.R., Kupfer, H., Tamir, I., Barlow, A. & Kupfer, A. Selective modulation of protein kinase C-θ during T-cell activation. Nature 385, 83–86 (1997).

    Article  CAS  Google Scholar 

  4. 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 

  5. Pfeifhofer, C. et al. Protein kinase C θ affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197, 1525–1535 (2003).

    Article  CAS  Google Scholar 

  6. 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 

  7. Hayashi, K. & Altman, A. Protein kinase C θ (PKCθ): a key player in T cell life and death. Pharmacol. Res. 55, 537–544 (2007).

    CAS  Google Scholar 

  8. Coudronniere, N., Villalba, M., Englund, N. & Altman, A. NF-κB activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-θ. Proc. Natl. Acad. Sci. USA 97, 3394–3399 (2000).

    CAS  PubMed  Google Scholar 

  9. Baier-Bitterlich, G. et al. Protein kinase C-θ isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16, 1842–1850 (1996).

    Article  CAS  Google Scholar 

  10. Altman, A. et al. Positive feedback regulation of PLCγ1/Ca2+ signaling by PKCθ in restimulated T cells via a Tec kinase-dependent pathway. Eur. J. Immunol. 34, 2001–2011 (2004).

    Article  CAS  Google Scholar 

  11. Lin, X., O'Mahony, A., Geleziunas, R. & Greene, W.C. Protein kinase C θ participates in NF-κB/Rel activation induced by CD3/CD28 costimulation through selective activation of IκB β (IKKβ). Mol. Cell. Biol. 20, 2933–2940 (2000).

    Article  CAS  Google Scholar 

  12. Manicassamy, S., Sadim, M., Ye, R.D. & Sun, Z. Differential roles of PKC-θ in the regulation of intracellular calcium concentration in primary T cells. J. Mol. Biol. 355, 347–359 (2006).

    Article  CAS  Google Scholar 

  13. Marsland, B.J., Soos, T.J., Spath, G., Littman, D.R. & Kopf, M. Protein kinase C θ is critical for the development of in vivo T helper Th2 cell but not Th1 cell responses. J. Exp. Med. 200, 181–189 (2004).

    Article  CAS  Google Scholar 

  14. Salek-Ardakani, S., So, T., Halteman, B.S., Altman, A. & Croft, M. Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase C θ. J. Immunol. 173, 6440–6447 (2004).

    Article  CAS  Google Scholar 

  15. Salek-Ardakani, S., So, T., Halteman, B.S., Altman, A. & Croft, M. Protein kinase Cθ controls Th1 cells in experimental autoimmune encephalomyelitis. J. Immunol. 175, 7635–7641 (2005).

    Article  CAS  Google Scholar 

  16. Anderson, K. et al. Mice deficient in PKC θ demonstrate impaired in vivo T cell activation and protection from T cell-mediated inflammatory diseases. Autoimmunity 39, 469–478 (2006).

    Article  CAS  Google Scholar 

  17. Tan, S.L. et al. Resistance to experimental autoimmune encephalomyelitis and impaired IL-17 production in protein kinase C θ-deficient mice. J. Immunol. 176, 2872–2879 (2006).

    Article  CAS  Google Scholar 

  18. Berg-Brown, N.N. et al. PKCθ signals activation versus tolerance in vivo. J. Exp. Med. 199, 743–752 (2004).

    Article  CAS  Google Scholar 

  19. Giannoni, F., Lyon, A.B., Wareing, M.D., Dias, P.B. & Sarawar, S.R. Protein kinase C θ is not essential for T-cell-mediated clearance of murine gammaherpesvirus 68. J. Virol. 79, 6808–6813 (2005).

    Article  CAS  Google Scholar 

  20. Marsland, B.J. et al. Innate signals compensate for the absence of PKCθ during in vivo CD8+ T cell effector and memory responses. Proc. Natl. Acad. Sci. USA 102, 14374–14379 (2005).

    Article  CAS  Google Scholar 

  21. Marsland, B.J. et al. TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-θ signaling and promote autoimmune myocarditis. J. Immunol. 178, 3466–3473 (2007).

    Article  CAS  Google Scholar 

  22. Valenzuela, J.O. et al. PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice. J. Clin. Invest. 119, 3774–3786 (2009).

    Article  CAS  Google Scholar 

  23. Keranen, L.M. & Newton, A.C. Ca2+ differentially regulates conventional protein kinase Cs′ membrane interaction and activation. J. Biol. Chem. 272, 25959–25967 (1997).

    Article  CAS  Google Scholar 

  24. Huang, J., Sugie, K., La Face, D.M., Altman, A. & Grey, H.M. TCR antagonist peptides induce formation of APC-T cell conjugates and activate a Rac signaling pathway. Eur. J. Immunol. 30, 50–58 (2000).

    Article  CAS  Google Scholar 

  25. Tseng, S.Y., Waite, J.C., Liu, M., Vardhana, S. & Dustin, M.L. T cell-dendritic cell immunological synapses contain TCR-dependent CD28–CD80 clusters that recruit protein kinase C θ. J. Immunol. 181, 4852–4863 (2008).

    Article  CAS  Google Scholar 

  26. Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C θ translocation. Immunity 29, 589–601 (2008).

    Article  CAS  Google Scholar 

  27. Melowic, H.R. et al. Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cθ. J. Biol. Chem. 282, 21467–21476 (2007).

    Article  CAS  Google Scholar 

  28. Kay, B.K., Williamson, M.P. & Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 14, 231–241 (2000).

    Article  CAS  Google Scholar 

  29. Dodson, L.F. et al. Targeted knock-in mice expressing mutations of CD28 reveal an essential pathway for costimulation. Mol. Cell. Biol. 29, 3710–3721 (2009).

    Article  CAS  Google Scholar 

  30. Miller, J. et al. Two pathways of costimulation through CD28. Immunol. Res. 45, 159–172 (2009).

    Article  CAS  Google Scholar 

  31. Sanchez-Lockhart, M. et al. Cutting edge: CD28-mediated transcriptional and posttranscriptional regulation of IL-2 expression are controlled through different signaling pathways. J. Immunol. 173, 7120–7124 (2004).

    Article  CAS  Google Scholar 

  32. Sadra, A. et al. Identification of tyrosine phosphorylation sites in the CD28 cytoplasmic domain and their role in the costimulation of Jurkat T cells. J. Immunol. 162, 1966–1973 (1999).

    CAS  PubMed  Google Scholar 

  33. Hofinger, E. & Sticht, H. Multiple modes of interaction between Lck and CD28. J. Immunol. 174, 3839–3840 (2005).

    Article  CAS  Google Scholar 

  34. Liu, Y. et al. Regulation of protein kinase Cθ function during T cell activation by Lck-mediated tyrosine phosphorylation. J. Biol. Chem. 275, 3603–3609 (2000).

    Article  CAS  Google Scholar 

  35. Bi, K. et al. Antigen-induced translocation of PKC-θ to membrane rafts is required for T cell activation. Nat. Immunol. 2, 556–563 (2001).

    Article  CAS  Google Scholar 

  36. Huang, J. et al. CD28 plays a critical role in the segregation of PKC θ within the immunologic synapse. Proc. Natl. Acad. Sci. USA 99, 9369–9373 (2002).

    Article  CAS  Google Scholar 

  37. Acuto, O. & Michel, F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 3, 939–951 (2003).

    Article  CAS  Google Scholar 

  38. Rudd, C.E., Taylor, A. & Schneider, H. CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol. Rev. 229, 12–26 (2009).

    Article  CAS  Google Scholar 

  39. Sanchez-Lockhart, M., Graf, B. & Miller, J. Signals and sequences that control CD28 localization to the central region of the immunological synapse. J. Immunol. 181, 7639–7648 (2008).

    Article  CAS  Google Scholar 

  40. Villalba, M. et al. A novel functional interaction between Vav and PKCθ is required for TCR-induced T cell activation. Immunity 12, 151–160 (2000).

    Article  CAS  Google Scholar 

  41. D'Ambrosio, D., Cantrell, D.A., Frati, L., Santoni, A. & Testi, R. Involvement of p21ras in T cell CD69 expression. Eur. J. Immunol. 24, 616–620 (1994).

    Article  CAS  Google Scholar 

  42. Spitaler, M., Emslie, E., Wood, C.D. & Cantrell, D. Diacylglycerol and protein kinase D localization during T lymphocyte activation. Immunity 24, 535–546 (2006).

    Article  CAS  Google Scholar 

  43. Stahelin, R.V. et al. Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cδ. J. Biol. Chem. 279, 29501–29512 (2004).

    Article  CAS  Google Scholar 

  44. Carrasco, S. & Merida, I. Diacylglycerol-dependent binding recruits PKCθ and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes. Mol. Biol. Cell 15, 2932–2942 (2004).

    Article  CAS  Google Scholar 

  45. Thuille, N. et al. Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCθ in T lymphocytes. EMBO J. 24, 3869–3880 (2005).

    Article  CAS  Google Scholar 

  46. Colon-Gonzalez, F. & Kazanietz, M.G. C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim. Biophys. Acta 1761, 827–837 (2006).

    Article  CAS  Google Scholar 

  47. Boschelli, D.H. Small molecule inhibitors of PKCθ as potential antiinflammatory therapeutics. Curr. Top. Med. Chem. 9, 640–654 (2009).

    Article  CAS  Google Scholar 

  48. Holst, J. et al. Generation of T-cell receptor retrogenic mice. Nat. Protoc. 1, 406–417 (2006).

    Article  CAS  Google Scholar 

  49. Becart, S. et al. Tyrosine-phosphorylation-dependent translocation of the SLAT protein to the immunological synapse is required for NFAT transcription factor activation. Immunity 29, 704–719 (2008).

    Article  CAS  Google Scholar 

  50. Canonigo-Balancio, A.J., Fos, C., Prod'homme, T., Becart, S. & Altman, A. SLAT/Def6 plays a critical role in the development of Th17 cell-mediated experimental autoimmune encephalomyelitis. J. Immunol. 183, 7259–7267 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Littman (New York University) for Prkcq−/− mice; T. So, W. Duan and M. Croft for the MCC-specific T hybridoma DCEK fibroblast system and advice on the allergic airway inflammation model; S. Becart, C. Fos and Y. Harada for discussions and suggestions; and K. Hayashi for some of the PKC-θ constructs. Supported by the US National Institutes of Health (CA35299), by The Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for Scientific Research on Innovative Areas 'Fluorescence Live imaging' 23113521) and the United States-Israel Binational Science Foundation. This is manuscript number 1392 from the La Jolla Institute for Allergy and Immunology.

Author information

Authors and Affiliations

  1. Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California

    Kok-Fai Kong, Ann J Canonigo-Balancio & Amnon Altman

  2. USA,

    Kok-Fai Kong, Ann J Canonigo-Balancio & Amnon Altman

  3. Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan

    Tadashi Yokosuka & Takashi Saito

  4. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Tadashi Yokosuka

  5. The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Be'er Sheva, Israel

    Noah Isakov

  6. Laboratory of Cell Signaling, World Premiere International Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan

    Takashi Saito

Authors
  1. Kok-Fai Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tadashi Yokosuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ann J Canonigo-Balancio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Noah Isakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takashi Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Amnon Altman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.-F.K. and A.A. designed the experiments and wrote the manuscript; K.-F.K. generated and analyzed data; T.S. and T.Y. provided expertise in cell imaging; T.S. participated in discussion of the data; T.Y. generated the PKC-GFP and CD28–cyan fluorescent protein fusion vectors; N.I. participated actively in discussions leading to this work and in experimental design; and A.J.C.-B. did various experiments and animal work.

Corresponding author

Correspondence to Amnon Altman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9. (PDF 3339 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kong, KF., Yokosuka, T., Canonigo-Balancio, A. et al. A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28. Nat Immunol 12, 1105–1112 (2011). https://doi.org/10.1038/ni.2120

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2120

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing