Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47phoxIs Critical for α-Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species

Anselm T. Bäumer, Henrik ten Freyhaus, Heinrich Sauer, Maria Wartenberg, Kai Kappert, Petra Schnabel, Christian Konkol, Jürgen Hescheler, Marius Vantler, Stephan Rosenkranz
2007 Journal of Biological Chemistry  
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
more » ... lation of the ␣PDGFR 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.
doi:10.1074/jbc.m704997200 pmid:18070887 fatcat:mn74pkdzm5cx7lwcxzfgyo3pp4