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.

  • Resource
  • Published:

Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes

Nature Immunology volume 12, pages 352–361 (2011)Cite this article

Subjects

Abstract

Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

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: Analysis of the basal and TCR-regulated phosphoproteome in CTLs.
Figure 2: Identification of consistent phosphorylations in CTLs by Ingenuity Pathway Analysis software.
Figure 3: Phosphorylated chromatin regulators in CTLs.
Figure 4: Subcellular distribution of HDAC7 in CTLs.
Figure 5: Exclusion of HDAC7 from the nucleus is required for normal CTL function.
Figure 6: Exclusion of HDAC7 from the nucleus is required for expression of the high-affinity IL-2 receptor.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Matthews, S.A. & Cantrell, D.A. New insights into the regulation and function of serine/threonine kinases in T lymphocytes. Immunol. Rev. 228, 241–252 (2009).

    Article  CAS  Google Scholar 

  2. Lin, J.X., Spolski, R. & Leonard, W.J. Critical role for Rsk2 in T-lymphocyte activation. Blood 111, 525–533 (2008).

    Article  CAS  Google Scholar 

  3. Salmond, R.J., Emery, J., Okkenhaug, K. & Zamoyska, R. MAPK, phosphatidylinositol 3-kinase, and mammalian target of rapamycin pathways converge at the level of ribosomal protein S6 phosphorylation to control metabolic signaling in CD8 T cells. J. Immunol. 183, 7388–7397 (2009).

    Article  CAS  Google Scholar 

  4. Sinclair, L.V. et al. Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking. Nat. Immunol. 9, 513–521 (2008).

    Article  CAS  Google Scholar 

  5. Waugh, C., Sinclair, L., Finlay, D., Bayascas, J.R. & Cantrell, D. Phosphoinositide (3,4,5)-triphosphate binding to phosphoinositide-dependent kinase 1 regulates a protein kinase B/Akt signaling threshold that dictates T-cell migration, not proliferation. Mol. Cell. Biol. 29, 5952–5962 (2009).

    Article  CAS  Google Scholar 

  6. Rao, R.R., Li, Q., Odunsi, K. & Shrikant, P.A. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin. Immunity 32, 67–78 (2010).

    Article  Google Scholar 

  7. Araki, K. et al. mTOR regulates memory CD8 T-cell differentiation. Nature 460, 108–112 (2009).

    Article  CAS  Google Scholar 

  8. Pearce, E.L. et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature 460, 103–107 (2009).

    Article  CAS  Google Scholar 

  9. Finlay, D.K. et al. Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes. J. Exp. Med. 206, 2441–2454 (2009).

    Article  CAS  Google Scholar 

  10. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  11. Brockmeyer, C. et al. T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies themis as a new TCR signalosome component. J. Biol. Chem. published online, doi:10.1074//jbc.M110.201236 (28 December 2010).

  12. Mayya, V. et al. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2, ra46 (2009).

    Article  Google Scholar 

  13. Nguyen, V. et al. A new approach for quantitative phosphoproteomic dissection of signaling pathways applied to T cell receptor activation. Mol. Cell. Proteomics 8, 2418–2431 (2009).

    Article  CAS  Google Scholar 

  14. Cornish, G.H., Sinclair, L.V. & Cantrell, D.A. Differential regulation of T-cell growth by IL-2 and IL-15. Blood 108, 600–608 (2006).

    Article  CAS  Google Scholar 

  15. Weninger, W., Crowley, M.A., Manjunath, N. & von Andrian, U.H. Migratory properties of naive, effector, and memory CD8+ T cells. J. Exp. Med. 194, 953–966 (2001).

    Article  CAS  Google Scholar 

  16. Kalia, V. et al. Prolonged interleukin-2Rα expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity 32, 91–103 (2010).

    Article  CAS  Google Scholar 

  17. Pipkin, M.E. et al. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity 32, 79–90 (2010).

    Article  CAS  Google Scholar 

  18. Malek, T.R. & Castro, I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 33, 153–165 (2010).

    Article  CAS  Google Scholar 

  19. Muller, M.R. & Rao, A. NFAT, immunity and cancer: a transcription factor comes of age. Nat. Rev. Immunol. 10, 645–656 (2010).

    Article  Google Scholar 

  20. Wabnitz, G.H., Nebl, G., Klemke, M., Schroder, A.J. & Samstag, Y. Phosphatidylinositol 3-kinase functions as a Ras effector in the signaling cascade that regulates dephosphorylation of the actin-remodeling protein cofilin after costimulation of untransformed human T lymphocytes. J. Immunol. 176, 1668–1674 (2006).

    Article  CAS  Google Scholar 

  21. Marklund, U. et al. Serine 16 of oncoprotein 18 is a major cytosolic target for the Ca2+/calmodulin-dependent kinase-Gr. Eur. J. Biochem. 225, 53–60 (1994).

    Article  CAS  Google Scholar 

  22. Wang, Z. et al. Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes. Cell 138, 1019–1031 (2009).

    Article  CAS  Google Scholar 

  23. Pflum, M.K., Tong, J.K., Lane, W.S. & Schreiber, S.L. Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation. J. Biol. Chem. 276, 47733–47741 (2001).

    Article  CAS  Google Scholar 

  24. Martin, M., Kettmann, R. & Dequiedt, F. Class IIa histone deacetylases: regulating the regulators. Oncogene 26, 5450–5467 (2007).

    Article  CAS  Google Scholar 

  25. Dequiedt, F. et al. HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis. Immunity 18, 687–698 (2003).

    Article  CAS  Google Scholar 

  26. Dequiedt, F. et al. Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor-induced Nur77 expression and apoptosis. J. Exp. Med. 201, 793–804 (2005).

    Article  CAS  Google Scholar 

  27. Parra, M., Kasler, H., McKinsey, T.A., Olson, E.N. & Verdin, E. Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation. J. Biol. Chem. 280, 13762–13770 (2005).

    Article  CAS  Google Scholar 

  28. Kao, H.Y. et al. Mechanism for nucleocytoplasmic shuttling of histone deacetylase 7. J. Biol. Chem. 276, 47496–47507 (2001).

    Article  CAS  Google Scholar 

  29. Zhang, C.L. et al. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell 110, 479–488 (2002).

    Article  CAS  Google Scholar 

  30. McKinsey, T.A., Zhang, C.L., Lu, J. & Olson, E.N. Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106–111 (2000).

    Article  CAS  Google Scholar 

  31. McKinsey, T.A., Zhang, C.L. & Olson, E.N. Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14–3-3 to histone deacetylase 5. Proc. Natl. Acad. Sci. USA 97, 14400–14405 (2000).

    Article  CAS  Google Scholar 

  32. Matthews, S.A. et al. Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell. Biol. 26, 1569–1577 (2006).

    Article  CAS  Google Scholar 

  33. Martin, M., Kettmann, R. & Dequiedt, F. Class IIa histone deacetylases: conducting development and differentiation. Int. J. Dev. Biol. 53, 291–301 (2009).

    Article  CAS  Google Scholar 

  34. Fischle, W. et al. Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR. Mol. Cell 9, 45–57 (2002).

    Article  CAS  Google Scholar 

  35. Li, X., Song, S., Liu, Y., Ko, S.H. & Kao, H.Y. Phosphorylation of the histone deacetylase 7 modulates its stability and association with 14–3-3 proteins. J. Biol. Chem. 279, 34201–34208 (2004).

    Article  CAS  Google Scholar 

  36. Kasler, H.G. & Verdin, E. Histone deacetylase 7 functions as a key regulator of genes involved in both positive and negative selection of thymocytes. Mol. Cell. Biol. 27, 5184–5200 (2007).

    Article  CAS  Google Scholar 

  37. Dubois, F. et al. Differential 14–3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling. Mol. Cell. Proteomics 8, 2487–2499 (2009).

    Article  CAS  Google Scholar 

  38. Gao, J., Opiteck, G.J., Friedrichs, M.S., Dongre, A.R. & Hefta, S.A. Changes in the protein expression of yeast as a function of carbon source. J. Proteome Res. 2, 643–649 (2003).

    Article  CAS  Google Scholar 

  39. Gao, C. et al. CRM1 mediates nuclear export of HDAC7 independently of HDAC7 phosphorylation and association with 14–3-3s. FEBS Lett. 580, 5096–5104 (2006).

    Article  CAS  Google Scholar 

  40. Yang, X.J. & Seto, E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol. 9, 206–218 (2008).

    Article  CAS  Google Scholar 

  41. Parra, M., Mahmoudi, T. & Verdin, E. Myosin phosphatase dephosphorylates HDAC7, controls its nucleocytoplasmic shuttling, and inhibits apoptosis in thymocytes. Genes Dev. 21, 638–643 (2007).

    Article  CAS  Google Scholar 

  42. Martin, M. et al. Protein phosphatase 2A controls the activity of histone deacetylase 7 during T cell apoptosis and angiogenesis. Proc. Natl. Acad. Sci. USA 105, 4727–4732 (2008).

    Article  CAS  Google Scholar 

  43. Tamas, P. et al. LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells. Eur. J. Immunol. 40, 242–253 (2010).

    Article  CAS  Google Scholar 

  44. Fox, C.J., Hammerman, P.S. & Thompson, C.B. The Pim kinases control rapamycin-resistant T cell survival and activation. J. Exp. Med. 201, 259–266 (2005).

    Article  CAS  Google Scholar 

  45. Chang, S., Bezprozvannaya, S., Li, S. & Olson, E.N. An expression screen reveals modulators of class II histone deacetylase phosphorylation. Proc. Natl. Acad. Sci. USA 102, 8120–8125 (2005).

    Article  CAS  Google Scholar 

  46. Seki, Y. et al. TIF1β regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner. Proc. Natl. Acad. Sci. USA 107, 10926–10931 (2010).

    Article  CAS  Google Scholar 

  47. Chang, C.W. et al. Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1β/KAP1. BMC Mol. Biol. 9, 61 (2008).

    Article  Google Scholar 

  48. McNulty, D.E. & Annan, R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol. Cell. Proteomics 7, 971–980 (2008).

    Article  CAS  Google Scholar 

  49. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  50. Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121 (2001).

    Article  CAS  Google Scholar 

  51. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  Google Scholar 

Download references

Acknowledgements

We thank members of the Biological Services Unit, R. Clarke of the Flow Cytometry Facility and members of the Cantrell laboratory for critical reading of the manuscript; and the Finnish DNA Microarray Centre at the Centre for Biotechnology (Turku, Finland) and N. Schurch for microarray analysis. Supported by the Wellcome Trust (065975/Z/01/A) and the Medical Research Council (J.G.).

Author information

Authors and Affiliations

  1. Division of Immunology and Cell Biology, The College of Life Sciences, The University of Dundee, Dundee, Scotland, UK

    Maria N Navarro, Jurgen Goebel, Carmen Feijoo-Carnero & Doreen A Cantrell

  2. The Beatson Institute for Cancer Research, Glasgow, Scotland, UK

    Nick Morrice

Authors
  1. Maria N Navarro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jurgen Goebel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carmen Feijoo-Carnero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nick Morrice
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Doreen A Cantrell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.N.N., J.G. and C.F.-C. did the experiments and analyzed the results; N.M. supervised SILAC methodology and bioinformatic analysis; and M.N.N. and D.A.C. designed the experiments, analyzed the results and wrote the paper.

Corresponding author

Correspondence to Doreen A Cantrell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Methods (PDF 10880 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Navarro, M., Goebel, J., Feijoo-Carnero, C. et al. Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes. Nat Immunol 12, 352–361 (2011). https://doi.org/10.1038/ni.2008

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2008

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