Platelet-derived growth factor-induced H2O2 production requires the activation of phosphatidylinositol 3-kinase

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor t riggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol S-kina se (PI3K), the GTPase-activating protein of Pas (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-yl (PLC-gamma 1), to specific phosp hotyrosine residues. The roles of these various effecters in PDGF-induced g eneration of H2O2 have now been investigated in HepG2 cells expressing vari ous PDGF receptor mutants. These mutants included a kinase-deficient recept or and receptors in which various combinations of the tyrosine residues req uired for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP- 2 (Tyr(1009)), Or PLC-gamma 1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H2O2 production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the bindin g of PI3K mere replaced by Phe. In contrast, PDGF-induced H2O2, production in cells expressing a receptor in which the binding sites for GAP, SHP-2, a nd PLC-gamma 1 were all mutated was slightly greater than that in cells exp ressing the wildtype receptor. Only the PI3K binding site was alone suffici ent for PDGF-induced H2O2 production. The effect of PDGF on H2O2 generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpre ssion of a dominant negative mutant of Rad. These results suggest that a pr oduct of PI3K is required for PDGF-induced production of H2O2 in nonphagocy tic cells, and that Rad mediates signaling between the PI3K product and the putative NADPH oxidase.