Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

Abstract

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the platelet-derived growth factor (jPDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the OLPDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-y. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that oaPDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.

Phosphorylation of Tyrosine 720 in the Platelet-Derived Growth Factor α Receptor Is Required for Binding of Grb2 and SHP-2 but Not for Activation of Ras or Cell Proliferation

“…While it is problematic to compare the signaling properties of receptors expressed in different cell lines, studies to date have shown that individually removing PI3K, SHP-2, and PLC␥ does not diminish the ability of the ␣PDGFR to initiate DNA synthesis (15,(68)(69)(70). This sharply contrasts with the observation that removing the binding sites for PI3K, SHP-2, or PLC␥ at least partially affect the ␤PDGFR's ability to transduce a signal to initiate DNA synthesis in most of the cell types tested (26).…”

“…While PI3K, PLC␥, and Src have been shown to be required for initiation of DNA synthesis by the ␤PDGFR, the ␣PDGFR does not seem to require either PI3K or PLC␥ for mitogenic signaling (15,17,30,59,60,68,69). The involvement of Src in ␣PDGFR signal relay has not yet been investigated.…”

“…As in the case of the ␤PDGFR, ligand binding induces dimerization of the ␣PDGFR subunits and results in phosphorylation of the receptor at multiple tyrosine residues. As a result, the ␣PDGFR associates with numerous SH2 domain-containing proteins, including phospholipase C␥-1 (PLC␥-1), the regulatory subunit (p85) of the phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2 (previously called SH-PTP2, PTP-1D, and Syp), Grb2, Src, and an as-yet-unidentified 120-kDa protein (3,69). Unlike the ␤PDGFR, the ␣PDGFR does not associate with or trigger tyrosine phosphorylation of the GTPase-activating protein of Ras RasGAP) (3,20).…”

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…Autophosphorylation of the PDGF b-receptor leads to direct binding of phosphatidylinositol 3'-kinase (PI3K), phospholipase C-g (PLC-g), Src family tyrosine kinases, GTPase-activating protein for Ras (RasGAP), the protein tyrosine phosphatase SHP-2, and the adaptor molecules Grb2 and Shc, and the respective binding sites have been identi®ed (Claesson-Welsh, 1994). Among these signal transduction molecules, at least PI3K, PLC-g and SHP-2 also bind to the areceptor (Baznet et al, 1995;Eriksson et al, 1992;Yu et al, 1991). During an analysis of autophosphorylation sites in the a-receptor involved in stimulatory and inhibitory signals for chemotaxis (Yokote et al, 1966), we noticed that Tyr-762 in the receptor is an autophosphorylation site.…”

The oncogenic FIP1L1-PDGFRαfusion protein displays skewed signaling properties compared to its wild-type PDGFRαcounterpart

“…Y 731 and Y 742 have been identified as recruitment site for PI3-kinase. 12 Y 1018 and Y 988 have been shown to mediate association with PLC-g1 5 and cCbl. 13 Finally, Y 849 an autophosphorylation site in the activation loop, is required for kinase activity.…”