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Protein microarrays for multiplex analysis of signal transduction pathways

Abstract

We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4+CD25+ regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.

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Figure 1: RPP microarray performance characteristics and validation.
Figure 2: Application of RPP microarrays in the study of signaling kinetics and pathway delineation.
Figure 3: Protein microarray screen with a panel of 62 phospho-specific antibodies.
Figure 4: CD3 crosslinking induces Raf-1 Ser259 dephosphorylation in both Jurkat and primary human T cells.
Figure 5: Differential STAT protein phosphorylation in IL-2-stimulated CD4+CD25+ TR cells.

References

  1. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    Article  CAS  Google Scholar 

  2. Robinson, W.H. et al. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat. Med. 8, 295–301 (2002).

    Article  CAS  Google Scholar 

  3. Robinson, W.H. et al. Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis. Nat. Biotechnol. 21, 1033–1039 (2003).

    Article  CAS  Google Scholar 

  4. Nielsen, U.B., Cardone, M.H., Sinskey, A.J., MacBeath, G. & Sorger, P.K. Profiling receptor tyrosine kinase activation by using Ab microarrays. Proc. Natl. Acad. Sci. USA 100, 9330–9335 (2003).

    Article  Google Scholar 

  5. Haab, B.B., Dunham, M.J. & Brown, P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2, RESEARCH0004 (2001).

  6. MacBeath, G. Protein microarrays and proteomics. Nat. Genet. 32 Suppl., 526–532 (2002).

    Article  CAS  Google Scholar 

  7. Paweletz, C.P. et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20, 1981–1989 (2001).

    Article  CAS  Google Scholar 

  8. Espina, V. et al. Protein microarrays: molecular profiling technologies for clinical specimens. Proteomics 3, 2091–2100 (2003).

    Article  CAS  Google Scholar 

  9. Liotta, L.A. et al. Protein microarrays: meeting analytical challenges for clinical applications. Cancer Cell 3, 317–325 (2003).

    Article  CAS  Google Scholar 

  10. Irish, J.M. et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228 (2004).

    Article  CAS  Google Scholar 

  11. Bobrow, M.N., Litt, G.J., Shaughnessy, K.J., Mayer, P.C. & Conlon, J. The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J. Immunol. Methods 150, 145–149 (1992).

    Article  CAS  Google Scholar 

  12. Wang, Z., Gluck, S., Zhang, L. & Moran, M.F. Requirement for phospholipase C-gamma1 enzymatic activity in growth factor-induced mitogenesis. Mol. Cell. Biol. 18, 590–597 (1998).

    Article  CAS  Google Scholar 

  13. Kane, L.P., Andres, P.G., Howland, K.C., Abbas, A.K. & Weiss, A. Akt provides the CD28 costimulatory signal for up-regulation of IL-2 and IFN-gamma but not TH2 cytokines. Nat. Immunol. 2, 37–44 (2001).

    Article  CAS  Google Scholar 

  14. Zhang, J. et al. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J. Immunol. 162, 3819–3829 (1999).

    CAS  PubMed  Google Scholar 

  15. Cantrell, D. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14, 259–274 (1996).

    Article  CAS  Google Scholar 

  16. Matthews, S.A., Rozengurt, E. & Cantrell, D. Protein kinase D. A selective target for antigen receptors and a downstream target for protein kinase C in lymphocytes. J. Exp. Med. 191, 2075–2082 (2000).

    Article  CAS  Google Scholar 

  17. Yuan, J., Bae, D., Cantrell, D., Nel, A.E. & Rozengurt, E. Protein kinase D is a downstream target of protein kinase C theta. Biochem. Biophys. Res. Commun. 291, 444–452 (2002).

    Article  CAS  Google Scholar 

  18. Kleijn, M. & Proud, C.G. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes. BMC Biochem. 3, 11 (2002).

    Article  Google Scholar 

  19. Michaud, N.R., Fabian, J.R., Mathes, K.D. & Morrison, D.K. 14–3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14–3-3- and Ras-independent manner. Mol. Cell. Biol. 15, 3390–3397 (1995).

    Article  CAS  Google Scholar 

  20. Zimmermann, S. & Moelling, K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741–1744 (1999).

    Article  CAS  Google Scholar 

  21. Jaumot, M. & Hancock, J.F. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14–3-3 interactions. Oncogene 20, 3949–3958 (2001).

    Article  CAS  Google Scholar 

  22. Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22, 531–562 (2004).

    Article  CAS  Google Scholar 

  23. Almeida, A.R., Legrand, N., Papiernik, M. & Freitas, A.A. Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J. Immunol. 169, 4850–4860 (2002).

    Article  Google Scholar 

  24. Furtado, G.C., Curotto de Lafaille, M.A., Kutchukhidze, N. & Lafaille, J.J. Interleukin 2 signaling is required for CD4+ regulatory T cell function. J. Exp. Med. 196, 851–857 (2002).

    Article  CAS  Google Scholar 

  25. Antov, A., Yang, L., Vig, M., Baltimore, D. & Van Parijs, L. Essential role for STAT5 signaling in CD25+CD4+ regulatory T cell homeostasis and the maintenance of self-tolerance. J. Immunol. 171, 3435–3441 (2003).

    Article  CAS  Google Scholar 

  26. Snow, J.W. et al. Loss of tolerance and autoimmunity affecting multiple organs in STAT5A/5B-deficient mice. J. Immunol. 171, 5042–5050 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank W.H. Robinson and M. Kattah for insightful discussions; and A. Zhang for technical assistance. S.M.C. is supported by the Stanford Medical Scientist Training Program. P.J.U. is supported by grants from the Dana Foundation, Northern California Chapter of the Arthritis Foundation, the Stanford Program in Molecular and Genetic Medicine (PMGM), NIH Grants DK61934, AI50854, AI50865, and AR49328, and NHLBI Proteomics Contract N01-HV-28183.

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Correspondence to Paul J Utz.

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Supplementary information

Supplementary Fig. 1

Representative image of a RPP microarray in an 8-pad subarray format. (PDF 132 kb)

Supplementary Fig. 2

Kinetics of Akt (Ser473), p38 MAPK and SAPK/JNK phosphorylation. (PDF 153 kb)

Supplementary Fig. 3

Differential STAT protein phosphorylation in T regulatory cells in response to IL-2. (PDF 66 kb)

Supplementary Fig. 4

Proof-of-concept experiment demonstrating the use of RPP microarrays for screening compounds with kinase inhibition activity. (PDF 54 kb)

Supplementary Table 1

List of 62 phospho-specific antibodies used in Figure 4 (PDF 8 kb)

Supplementary Note (PDF 6 kb)

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Chan, S., Ermann, J., Su, L. et al. Protein microarrays for multiplex analysis of signal transduction pathways. Nat Med 10, 1390–1396 (2004). https://doi.org/10.1038/nm1139

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