SEQUENCE SPECIFICITY IN RECOGNITION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR BY PROTEIN-TYROSINE PHOSPHATASE-1B

Protein tyrosine phosphatases all contain a conserved cysteine that fo rms an intermediate thiophosphate ester bond during tyrosine phosphate hydrolysis. A bacterial glutathione S-transferase fusion protein cont aining rat brain phosphatase PTP1b was constructed in which this conse rved cysteine was mutated to serine. The resulting catalytically inact ive enzyme was labeled in vivo to high specific activity with S-35, an d the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealed a nonlinear Scatch ard plot, with a K(d) for high affinity binding of approximately 100 n M. A number of glutathione S-transferase fusion proteins containing sr c homology 2 (SH2) domains attenuated phosphatase binding in a concent ration-dependent manner. Phospholipase C (PLC)gamma and the GTPase-act ivating protein of ras were the most potent inhibitors. Tyrosine-phosp horylated EGF receptor peptide fragments were evaluated for specific i nhibition of PTP1b and PLCgamma SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine 992, blocked the b inding of both fusion proteins. Another phosphopeptide, modeled on tyr osine 1148, inhibited the binding of PTP1b but not the PLCgamma fusion protein. This site specificity was confirmed by analysis of equilibri um binding of the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence speci ficity in the binding of proteins involved in the regulation of intrac ellular signaling by receptor tyrosine kinases.