Phosphatase Modulators pp 241-270

Part of the Methods in Molecular Biology book series (MIMB, volume 1053)

Evaluating Effects of Tyrosine Phosphatase Inhibitors on T Cell Receptor Signaling

  • Souad Rahmouni
  • Laurence Delacroix
  • Wallace H. Liu
  • Lutz Tautz
Protocol

Abstract

The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) function. Contrary to earlier assumptions, it is now clear that both PTKs and PTPs are highly specific, non-redundant, and tightly regulated enzymes. Hematopoietic cells express particularly high numbers of PTKs and PTPs, and aberrant function of these proteins have been linked to many hematopoietic disorders. While PTK inhibitors are among FDA approved drugs for the treatment of leukemia and other cancers, efforts to develop therapeutics that target specific PTPs are still in its infancy. Here, we describe methods on how to evaluate effects of PTP inhibitors on T cell receptor signaling. Moreover, we provide a comprehensive strategy for compound prioritization, applicable to any drug discovery project involving T cells. We present a testing funnel that starts with relatively high-throughput luciferase reporter assays, followed by immunoblot, calcium flux, flow cytometry, and proliferation assays, continues with cytokine bead arrays, and finishes with specificity assays that involve RNA interference. We provide protocols for experiments in the Jurkat T cell line, but more importantly give detailed instructions, paired with numerous tips, on how to prepare and work with primary human T cells.

Key words

T cells PBMC NFAT/AP-1 assay Phosphotyrosine blot Calcium flux Cytokine bead array (CBA) 3H-thymidine incorporation siRNA CD25 CD69 TCR PTP Small molecule inhibitors 

References

  1. 1.
    Mustelin T, Vang T, Bottini N (2005) Protein tyrosine phosphatases and the immune response. Nat Rev Immunol 5:43–57PubMedCrossRefGoogle Scholar
  2. 2.
    Alonso A, Sasin J, Bottini N et al (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699–711PubMedCrossRefGoogle Scholar
  3. 3.
    Mustelin T, Alonso A, Bottini N et al (2004) Protein tyrosine phosphatases in T cell physiology. Mol Immunol 41:687–700PubMedCrossRefGoogle Scholar
  4. 4.
    Mustelin T, Rahmouni S, Bottini N et al (2003) Role of protein tyrosine phosphatases in T cell activation. Immunol Rev 191:139–147PubMedCrossRefGoogle Scholar
  5. 5.
    Goebel-Goody SM, Baum M, Paspalas CD et al (2012) Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in neuropsychiatric disorders. Pharmacol Rev 64:65–87PubMedCrossRefGoogle Scholar
  6. 6.
    Julien SG, Dube N, Hardy S et al (2011) Inside the human cancer tyrosine phosphatome. Nat Rev Cancer 11:35–49PubMedCrossRefGoogle Scholar
  7. 7.
    Pulido R, Hooft van Huijsduijnen R (2008) Protein tyrosine phosphatases: dual-specificity phosphatases in health and disease. FEBS J 275:848–866PubMedCrossRefGoogle Scholar
  8. 8.
    Rhee I, Veillette A (2012) Protein tyrosine phosphatases in lymphocyte activation and autoimmunity. Nat Immunol 13:439–447PubMedCrossRefGoogle Scholar
  9. 9.
    Tautz L, Pellecchia M, Mustelin T (2006) Targeting the PTPome in human disease. Expert Opin Ther Targets 10:157–177PubMedCrossRefGoogle Scholar
  10. 10.
    Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7:833–846PubMedCrossRefGoogle Scholar
  11. 11.
    Vang T, Miletic AV, Arimura Y et al (2008) Protein tyrosine phosphatases in autoimmunity. Annu Rev Immunol 26:29–55PubMedCrossRefGoogle Scholar
  12. 12.
    Barr AJ (2010) Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development. Future Med Chem 2:1563–1576PubMedCrossRefGoogle Scholar
  13. 13.
    Bialy L, Waldmann H (2005) Inhibitors of protein tyrosine phosphatases: next-generation drugs? Angew Chem Int Ed Engl 44:3814–3839PubMedCrossRefGoogle Scholar
  14. 14.
    Vintonyak VV, Antonchick AP, Rauh D et al (2009) The therapeutic potential of phosphatase inhibitors. Curr Opin Chem Biol 13:272–283PubMedCrossRefGoogle Scholar
  15. 15.
    Vang T, Liu WH, Delacroix L et al (2012) LYP inhibits T-cell activation when dissociated from CSK. Nat Chem Biol 8:437–446PubMedCrossRefGoogle Scholar
  16. 16.
    Negro R, Gobessi S, Longo PG et al (2012) Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT. Blood 119:6278–6287PubMedCrossRefGoogle Scholar
  17. 17.
    Tautz L, Mustelin T (2007) Strategies for developing protein tyrosine phosphatase inhibitors. Methods 42:250–260PubMedCrossRefGoogle Scholar
  18. 18.
    Abraham RT, Weiss A (2004) Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol 4:301–308PubMedCrossRefGoogle Scholar
  19. 19.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  20. 20.
    Sherf BA, Navarro SL, Hannah RR (1996) Dual-luciferase™ reporter assay: an advanced co-reporter technology integrating firefly and renilla luciferase assays. Promega Notes 57:02Google Scholar
  21. 21.
    Sergienko E, Bobkova E, Vasile S et al (2010) Selective HePTP inhibitors: probe 1. Probe Reports from the Molecular Libraries Program, Bethesda (MD): National Center for Biotechnology Information (US)Google Scholar
  22. 22.
    Sergienko E, Xu J, Liu WH et al (2012) Inhibition of hematopoietic protein tyrosine phosphatase augments and prolongs ERK1/2 and p38 activation. ACS Chem Biol 7:367–377PubMedCrossRefGoogle Scholar
  23. 23.
    Hogan PG, Lewis RS, Rao A (2010) Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu Rev Immunol 28:491–533PubMedCrossRefGoogle Scholar
  24. 24.
    Cebrian M, Yague E, Rincon M et al (1988) Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes. J Exp Med 168:1621–1637PubMedCrossRefGoogle Scholar
  25. 25.
    Cosulich ME, Rubartelli A, Risso A et al (1987) Functional characterization of an antigen involved in an early step of T-cell activation. Proc Natl Acad Sci USA 84:4205–4209PubMedCrossRefGoogle Scholar
  26. 26.
    Hara T, Jung LK, Bjorndahl JM et al (1986) Human T cell activation. III. Rapid induction of a phosphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA 1) by 12-o-tetradecanoyl phorbol-13-acetate, mitogens, and antigens. J Exp Med 164:1988–2005PubMedCrossRefGoogle Scholar
  27. 27.
    Reddy M, Eirikis E, Davis C et al (2004) Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function. J Immunol Methods 293:127–142PubMedCrossRefGoogle Scholar
  28. 28.
    Porebski G, Gschwend-Zawodniak A, Pichler WJ (2011) In vitro diagnosis of T cell-mediated drug allergy. Clin Exp Allergy 41:461–470PubMedCrossRefGoogle Scholar
  29. 29.
    Liao W, Lin JX, Leonard WJ (2011) IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol 23:598–604PubMedCrossRefGoogle Scholar
  30. 30.
    Morgan E, Varro R, Sepulveda H et al (2004) Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 110:252–266PubMedCrossRefGoogle Scholar
  31. 31.
    Weiss WA, Taylor SS, Shokat KM (2007) Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat Chem Biol 3:739–744PubMedCrossRefGoogle Scholar
  32. 32.
    Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA interference. Nat Rev Genet 8:173–184PubMedCrossRefGoogle Scholar
  33. 33.
    Walker JM (1984) Gradient SDS polyacrylamide gel electrophoresis. Methods Mol Biol 1:57–61PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Souad Rahmouni
    • 1
  • Laurence Delacroix
    • 1
  • Wallace H. Liu
    • 2
  • Lutz Tautz
    • 2
  1. 1.Immunology and Infectious Diseases, GIGA-Signal TransductionUniversity of LiègeLiègeBelgium
  2. 2.Infectious and Inflammatory Disease CenterSanford-Burnham Medical Research InstituteLa JollaUSA

Personalised recommendations