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J. Biol. Chem., Vol. 283, Issue 12, 7864-7876, March 21, 2008

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Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47^phox Is Critical for -Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species^*

Anselm T. Bäumer¹, Henrik ten Freyhaus¹, Heinrich Sauer, Maria Wartenberg^¶, Kai Kappert, Petra Schnabel, Christian Konkol, Jürgen Hescheler^||, Marius Vantler^**, and Stephan Rosenkranz^**2

From the Klinik III für Innere Medizin, Universität zu Köln, 50937 Köln, Institut für Physiologie, Justus-Liebig-Universität Giessen, 35392 Giessen, the ^¶Department of Cell Biology, GKSS Research Institute, 14513 Teltow, ^||Institut für Neurophysiologie, Universität zu Köln, 50937 Köln, and ^**Center for Molecular Medicine Cologne, Universität zu Köln, 50937 Köln, Germany

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 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 PDGF-dependent 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 p110β) was identified as the crucial isoform that elicits PDGFR-mediated 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.

Received for publication, June 18, 2007 , and in revised form, November 7, 2007.

^* This work was supported by the Deutsche Forschungsgemeinschaft Grants Ro 1306/2-1 and 2-2 (to S. R.), by the Köln Fortune Program of the medical faculty of the University of Cologne (to A. T. B.), and by the Hans and Gertie Fischer-Stiftung (Essen, Germany). This work contains parts of a doctoral thesis by H. t. F. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Klinik III für Innere Medizin, Universität zu Köln, Kerpener Str. 62, 50937 Köln, Germany. Tel.: 49-221-478-32402; Fax: 49-221-478-32400; E-mail: stephan.rosenkranz{at}uk-koeln.de.

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