Tyrosines 1021 and 1009 Are Phosphorylation Sites in the Carboxy Terminus of the Platelet-Derived Growth Factor Receptor β Subunit and are Required for Binding of Phospholipase Cγ and a 64-Kilodalton Protein, Respectively

Abstract

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) β subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase Cγ (PLCγ), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLCγ and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLCγ and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLCγ, since a phosphorylated carboxy-terminal fusion protein selectively bound PLCγ. To determine the biological consequences of failure to associate with PLCγ, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLCγ were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLCγ enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLCγ were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLCγ tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLCγ tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLCγ and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLCγ to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.