IDENTIFICATION OF A PUTATIVE SYP SUBSTRATE, THE PDGF-BETA RECEPTOR

Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate, We tested this hypothesis by determining whether recombinant Syp (rSyp) and a contro l PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the be ta PDGFR, The beta PDGFR was phosphorylated at multiple tyrosine resid ues in an in vitro kinase assay and then incubated with increasing con centrations of rSyp or rPTP1B, While the receptor was nearly completel y dephosphorylated by high concentrations of rPTP1B, receptor dephosph orylation by rSyp plateaued at approximately 50%. Two-dimensional phos phopeptide maps of the beta PDGFR demonstrated that rSyp displayed a c lear preference for certain receptor phosphorylation sites; the most e fficiently dephosphorylated sites were phosphotyrosines (Tyr(P))-771 a nd -751, followed by Tyr(P)-740, while Tyr(P)-1021 and Tyr(P)-1009 wer e very poor substrates, In contrast, rPTP1B displayed no selectivity f or the various beta PDGFR tyrosine phosphorylation sites and dephospho rylated all of them with comparable efficiency, A Syp construct that l acked the SH2 domains was still able to discriminate between the vario us receptor phosphorylation sites, although less effectively than full -length Syp. These in vitro studies predicted that Syp can dephosphory late the receptor in vivo, Indeed, we found that a beta PDGFR mutant ( F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor, In additio n, the GTPase-activating protein of Pas (GAP) and phosphatidylinositol 3-kinase were less stably associated with the mild type beta PDGFR th an with the F1009 receptor, These findings are consistent with the in vitro experiments showing that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol S -kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studie s reveal that Syp is a substrate-selective PTP and that both the catal ytic domain and the SH2 domains contribute to Syp's ability to choose substrates, Furthermore, it appears that Syp plays a role in PDGF-depe ndent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of r eceptor-associated signal relay enzymes.