Measuring Protein Tyrosine Phosphatase Activity Dependent on SH2 Domain-Mediated Regulation.

Src-homology-2 (SH2) domains bind selectively to phosphotyrosine (pTyr) residues located in target binding proteins; therefore, they are key elements in pTyr-mediated signaling pathways. The binding of an SH2 domain to a pTyr acts as a docking mechanism that attracts proteins into signaling hubs, and in some cases, it can also regulate the catalytic activity of signaling enzymes such as protein kinases or protein phosphatases. Therefore, compounds that selectively bind SH2 domains can be potentially used to modulate the activity of such SH2 domain-containing enzymes. This chapter describes how to measure the regulation of protein tyrosine phosphatase activity through allosteric binding of peptides to SH2 domains, and uses human recombinant protein tyrosine phosphatase SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase 2) purified from bacteria as a case example. The phosphatase activity against the artificial substrate DiFMUP (6, 8-Difluoro-4-Methylumbelliferyl Phosphate) is measured over time in the presence of a peptide that selectively binds and activates SHP2 at different concentrations to determine the half maximal effective concentration (EC).