THIOPHOSPHORYLATED SUBSTRATE-ANALOGS ARE POTENT ACTIVE-SITE-DIRECTED INHIBITORS OF PROTEIN-TYROSINE PHOSPHATASES

Thiophosphotyrosyl protein and peptide substrate analogs were found to be potent and specific protein-tyrosine phosphatase inhibitors with IC(50)s in the range of 0.2-30 mu M. The analogs were based on highly reactive substrates and included thiophosphotyrosyl forms of reduced carboxamidomethylated and maleylated lysozyme and peptides based on tyrosine phosphorylation sites of lysozyme, alpha(s2)-casein, and platelet-derived growth factor receptor. These analogs inhibited protein-tyrosine phosphatases from both the intracellular and transmembrane classes and from a variety of species ranging from a prokaryote (Yersinia enterolitica) to man. The extent of inhibition of phosphatase activity by a given analog varied with the phosphatase species. In contrast, protein kinases and protein-serine/threonine phosphatases were not significantly affected by these analogs. The mechanism of inhibition was investigated using rat brain protein-tyrosine phosphatase-1 as a prototype. These studies indicated that the inhibition was rapid and reversible and was competitive in nature. The K-i for inhibition by various thiophosphotyrosyl analogs was generally proportional to the apparent K-m for the corresponding phosphorylated substrates. Unphosphorylated substrate molecules were generally much weaker inhibitors than the corresponding thiophosphotyrosyl substrate analogs. Taken together these results point to an active site-directed mechanism for inhibition. These specific inhibitory probes could be used to study substrate binding mechanisms as well as physiological roles of various protein-tyrosine phosphatases. (C) 1994 Academic Press, Inc.