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

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidyl-inositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein- tyrosine phosphatase SHP-2, and phospholipase C-γ1 (PLC-γ1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF- induced generation of H₂O₂ have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase- deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr⁷⁴⁰ and Tyr⁷⁵¹), GAP (Tyr⁷⁷¹), SHP-2 (Tyr¹⁰⁰⁹), or PLC-γ1 (Tyr¹⁰²¹) 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 binding of PI3K were re- placed by Phe. In contrast, PDGF- induced H₂O₂ production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-γ1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H₂O₂ production. The effect of PDGF on H₂O₂ generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H₂O₂ in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.