Identification of a Putative Syp Substrate, the PDGFβ Receptor

Abstract

Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor ␤ receptor (␤PDGFR), the ␤PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the ␤PDGFR. The ␤PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the ␤PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P))-771 and -751, followed by Tyr(P)-740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various ␤PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp.These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a ␤PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type ␤PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showing that Syp prefers to dephosphorylate sites on the ␤PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the ␤PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.Tyrosine phosphorylation modulates the activity of many enzymes involved in regulation of a wide variety of cellular events and is a reversible process governed by the opposing effects of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs) 1 (1-3). In addition to a conserved catalytic domain, most PTPs also contain highly variable noncatalytic sequences, which can regulate the activity and/or subcellular location of the PTP (1, 3). The initially purified PTPs failed to show a high degree of substrate specificity (4), and this led to the hypothesis that subcellular targeting is the primary determinant in selection of a substrate. More recent evidence indicates that an i...

SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF β-receptor

“…Since a mutant PDGF receptor lacking the ability to associate with and activate SHP-2 showed a considerably higher phosphorylation of Tyr771 than wild-type receptor expressing cells, SHP-2 is likely to be important for the dephosphorylation of Y771. The ability of SHP-2 to dephosphorylate Tyr771 in the PDGF b-receptor has previously been demonstrated by Klingho er and Kazlauskas (1995). The possibility that a di erence in substrate speci®city between the a-and b-receptor contributes to the di erence in phosphorylation of Tyr771 in homo-and heterodimeric complexes, is unlikely since no di erence in phosphorylation of Tyr771 was observed in chimeric constructs of a-and b-receptors (data not shown).…”

“…The SH2 domain containing PTPase SHP-2 has previously been suggested to be able to dephosphorylate Tyr771 (Klingho er and Kazlauskas, 1995). We and others (Kazlauskas et al, 1993;RoÈ nnstrand et al, 1999) have demonstrated that association of SHP-2 with the PDGF b-receptor is dependent on phosphorylation of Tyr763 and Tyr1009 in the PDGF b-receptor.…”

SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis

“…It has been shown that SHP-2 can dephosphorylate the autophosphorylated PDGF b-receptor, and possibly also its substrates, and thereby act in a negative manner in signaling (Klingho er and Kazlauskas, 1995). Saxton et al (1997) found that murine ®broblasts carrying a mutated version of SHP-2, lacking the aminoterminal SH2 domain and with a non-functional carboxyterminal SH2 domain, responded to PDGF-BB with increased MAP kinase activation compared to wild-type cells, consistent with a negative control function of SHP-2 in PDGFmediated activation of MAP kinase.…”

Tyrosine phosphatase SHP-2 dephosphorylates the platelet-derived growth factor receptor but enhances its downstream signalling

“…The intensive phosphorylation of the receptor itself and Myr-SHP-2(C-S) observed in this study is indicative of the activation of the kinase, which presumably leads to the phosphorylation of most, if not all, of the sites. SHP-2, in contrast, might attack only certain sites as indicated by the incomplete dephosphorylation of PDGF-R. Studies in itro do indeed suggest that SHP-2 has a clear preference for certain receptor phosphorylation sites [29]. The most efficiently dephosphorylated sites are Tyr-771 (a GTPase-activating protein (GAP)-binding site), Tyr-751 (a Nck-and phosphoinositide 3-kinase-binding site), followed by Tyr-740 (a phosphoinositide 3-kinase-binding site).…”