CHARACTERIZATION OF PROTEIN-TYROSINE-PHOSPHATASE SH-PTP2 - STUDY OF PHOSPHOPEPTIDE SUBSTRATES AND POSSIBLE REGULATORY ROLE OF SH2 DOMAINS

The src homology 2 (SH2) domain containing protein-tyrosine-phosphatas e SH-PTP2, was over-expressed in Escherichia coil for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates. The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(Delta SH2)), exhibited Mic haelis-Menten kinetics, and demonstrated striking substrate preference s on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation. For example, while a K-M of 59 mu M and k(cat)/K-M of 1.1 x 10(5) were obtained using SH-PTP2(Delta SH2) a nd PDGFR(Y1021), a phosphorylation site within the platelet-derived gr owth factor receptor, other peptides revealed no detectable phosphate release. PDGFR(Y1009), modeled after a sequence identified as an in vi vo binding site for SH-PTP2, was also a good substrate for this enzyme . The truncated form, lacking the SH2 domains demonstrated higher cata lytic efficiency than the full-length enzyme. Interestingly, soluble S H2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner. There was also evidence of a non-pho sphotyrosine-mediated association between the two domains, These obser vations suggested that the SH2 domains have a direct role in regulatin g the catalytic activity of SH(-)PTP2.