Identification of tyrosine phosphatases that dephosphorylate the insulin receptor - A brute force approach based on "substrate-trapping" mutants

Many pharmacologically important receptors, including all cytokine receptor s, signal via tyrosine (auto) phosphorylation, followed by resetting to the ir original state through the action of protein tyrosine phosphatases (PTPs ). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics, We have set up a sy stematic approach for finding PTPs that are specific for a receptor and hav e validated this approach with the insulin receptor kinase, We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substr ate-trapping" PTP mutants. These results, combined with secondary dephospho rylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstra te that this approach can rapidly reduce the number of PTPs that have a par ticular receptor or other phosphoprotein as their substrate.