Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47 phox , and we systematically evaluated the signal relay mechanisms by which the ␣PDGF receptor (␣PDGFR) induces ROS liberation. Stimulation of the

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... led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. ␣PDGFR activation is rapidly followed by the translocation of p47 phox and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47 phox (؊/؊) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGFdependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant ␣PDGFRs that lack specific binding sites for ␣PDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase C␥, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase C␥, or SHP-2) completely abolished PDGF-dependent p47 phox and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant ␣PDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110␣ (but not p110␤) was identified as the crucial isoform that elicits ␣PDGFRmediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110␣ mediates growth factor-dependent ROS production by recruiting p47 phox and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.