Autophosphorylated platelet-derived growth factor receptor triggers intracellular signaling cascades by recruiting SH2 domain-containing enzymes that include phosphatidylinositol 3-kinase (PI3K), the GTPase- activating protein of Ras (GAP), the protein tyrosine phosphatase (SHP- 2), and phospholipase C-gamma1 (PLCgamma1). PDGF-dependent H2O2 production was measured in HepG2 cells expressing various PDGF receptor mutants in which the Lys635 essential for kinase activity is changed to Arg or the Tyr residues for the binding of PI3K (Tyr740 and Tyr751), GAP (Tyr 771), SHP-2 (Tyr 1009), and PLCgamma1 (Tyr 1021) were mutated to Phe in various combinations. The kinase deficient receptor failed to produce H2O2. Elimination of PI3K binding site also abrogated H2O2 production, whereas H2O2 production was slightly enhanced when the binding sites for GAP, SHP-2, and PLC-gamma1 were removed. Among the binding sites for the four effector enzymes, the presence of PI3K binding site alone was sufficient for H2O2 production. The PDGF-induced H2O2 production in cells expressing wild-type receptor or the receptor containing only the PI3K binding site was completely blocked by a PI3K inhibitor, LY294002 or by overexpression of a dominant negative form of Rac1, N17Rac1. These results suggest that a product of PI3-kinase is necessary for the activation of putative NADPH oxidase in nonphagocytice cells stimulated with PDGF and that Rac1 provides the linkage between the PI3K product and NADPH oxidase. - H2O2, PDGF, platelet-derived growth factor; phosphoinositide 3-kinase, Rac, NADPH oxidase
