Gap suppresses pdgf-dependent activation of pi3 kinase by inhibiting ras
Diagnostic Techniques and Procedures
Upon binding of platelet derived growth factor (PDGF), the PDGF β receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and, as a result, binds several SH2 domain-containing signal relay enzymes including PI3 kinase (PI3K), phospholipase Cγ (PLCγ), the GTPase activating protein of ras(GAP), and the tyrosine phosphatase Syp. Previous studies have defined PI3K and PLCγ as positive mediators of PDGFR signaling but defined roles for GAP and Syp have remained more elusive. In this study we have investigated the hypothesis that Syp and/or GAP exert(s) an effect on PDGFR signaling by modulating the activity of PI3K. To do so we constructed a series of PDGFR mutants which contain the binding sites for PI3K as well as either GAP, Syp, or PLCγ. Utilizing this series of receptors, stably expressed in HepG2 cells, we found that the ability of GAP to associate with the PDGFR, but not Syp, correlates with a dramatic inhibition of the production of phosphatidylinositol-3,4,5-trisphosphate, the major biological product of PI3K. Biochemical characterization of these receptors revealÂed that kinase activity and PI3K association are all normal, however, PDGF-dependent Ras activation in the cells expressing the PI3K/GAP double add-back receptor was completely inhibited. These results indicate that activation Âof PI3K in a PDGF-stimulated cell requires more than just binding to the activated PDGFR, it appears that accumulation of active Ras is also a requirement. In addition, GAP contributes to PDGFR signal relay by preventing the accumulation of active Ras, which correlates tightly with failure to activate PI3K.