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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Notes
- 1.
The amount of antibody will depend on the abundance of protein in the sample, and the affinity of the protein to the antibody. Determine the optimal amount of primary antibody by running test IPs with several different concentrations of antibody. Therefore, separate each IP, together with the flow-through, by SDS-PAGE and reveal the levels of the protein by western blot analysis. An optimal antibody concentration will lead to a complete depletion of the protein in the flow-through.
References
Mustelin T, Vang T, Bottini N (2005) Protein tyrosine phosphatases and the immune response. Nat Rev Immunol 5:43–57
Alonso A, Sasin J, Bottini N et al (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699–711
Mustelin T, Alonso A, Bottini N et al (2004) Protein tyrosine phosphatases in T cell physiology. Mol Immunol 41:687–700
Mustelin T, Rahmouni S, Bottini N et al (2003) Role of protein tyrosine phosphatases in T cell activation. Immunol Rev 191:139–147
Goebel-Goody SM, Baum M, Paspalas CD et al (2012) Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in neuropsychiatric disorders. Pharmacol Rev 64:65–87
Julien SG, Dube N, Hardy S et al (2011) Inside the human cancer tyrosine phosphatome. Nat Rev Cancer 11:35–49
Pulido R, Hooft van Huijsduijnen R (2008) Protein tyrosine phosphatases: dual-specificity phosphatases in health and disease. FEBS J 275:848–866
Rhee I, Veillette A (2012) Protein tyrosine phosphatases in lymphocyte activation and autoimmunity. Nat Immunol 13:439–447
Tautz L, Pellecchia M, Mustelin T (2006) Targeting the PTPome in human disease. Expert Opin Ther Targets 10:157–177
Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7:833–846
Vang T, Miletic AV, Arimura Y et al (2008) Protein tyrosine phosphatases in autoimmunity. Annu Rev Immunol 26:29–55
Barr AJ (2010) Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development. Future Med Chem 2:1563–1576
Bialy L, Waldmann H (2005) Inhibitors of protein tyrosine phosphatases: next-generation drugs? Angew Chem Int Ed Engl 44:3814–3839
Vintonyak VV, Antonchick AP, Rauh D et al (2009) The therapeutic potential of phosphatase inhibitors. Curr Opin Chem Biol 13:272–283
Vang T, Liu WH, Delacroix L et al (2012) LYP inhibits T-cell activation when dissociated from CSK. Nat Chem Biol 8:437–446
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 119:6278–6287
Tautz L, Mustelin T (2007) Strategies for developing protein tyrosine phosphatase inhibitors. Methods 42:250–260
Abraham RT, Weiss A (2004) Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol 4:301–308
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Sherf BA, Navarro SL, Hannah RR (1996) Dual-luciferase™ reporter assay: an advanced co-reporter technology integrating firefly and renilla luciferase assays. Promega Notes 57:02
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)
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 7:367–377
Hogan PG, Lewis RS, Rao A (2010) Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu Rev Immunol 28:491–533
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 168:1621–1637
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 84:4205–4209
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 164:1988–2005
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 293:127–142
Porebski G, Gschwend-Zawodniak A, Pichler WJ (2011) In vitro diagnosis of T cell-mediated drug allergy. Clin Exp Allergy 41:461–470
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 23:598–604
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 110:252–266
Weiss WA, Taylor SS, Shokat KM (2007) Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat Chem Biol 3:739–744
Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA interference. Nat Rev Genet 8:173–184
Walker JM (1984) Gradient SDS polyacrylamide gel electrophoresis. Methods Mol Biol 1:57–61
Acknowledgment
This work was supported by NIH grants R03MH095532, R03MH084230, and R21CA132121 (to L.T.), by the Belgian National Funds for Scientific Research (FRS-FNRS), and by a grant from the University of Liège (Belgium) (to S.R.).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Rahmouni, S., Delacroix, L., Liu, W.H., Tautz, L. (2013). Evaluating Effects of Tyrosine Phosphatase Inhibitors on T Cell Receptor Signaling. In: Millán, J. (eds) Phosphatase Modulators. Methods in Molecular Biology, vol 1053. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-562-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-562-0_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-561-3
Online ISBN: 978-1-62703-562-0
eBook Packages: Springer Protocols