Protocol

Phosphatase Modulators

Volume 1053 of the series Methods in Molecular Biology pp 241-270

Date:

Evaluating Effects of Tyrosine Phosphatase Inhibitors on T Cell Receptor Signaling

  • Souad RahmouniAffiliated withImmunology and Infectious Diseases, GIGA-Signal Transduction, University of Liège
  • , Laurence DelacroixAffiliated withImmunology and Infectious Diseases, GIGA-Signal Transduction, University of Liège
  • , Wallace H. LiuAffiliated withInfectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute
  • , Lutz TautzAffiliated withInfectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute

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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