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Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91^phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67^phox, and p47^phox

Houssam Raad^*, Marie-Hélène Paclet, Tarek Boussetta^*, Yolande Kroviarski, Françoise Morel, Mark T. Quinn, Marie-Anne Gougerot-Pocidalo^*,, Pham My-Chan Dang^* and Jamel El-Benna^*,1

^* INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Université Paris 7 site Bichat, UMRS 773, Paris, France;

GREPI, TIMC-IMAG, UMR CNRS 5525, Centre Hospitalier Universitaire de Grenoble, Grenoble France;

AP-HP, Centre Hospitalier Universitaire Xavier Bichat, CIB Phenogen, Paris, France; and

Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA

¹Correspondence: INSERM U773, CRB3, Faculté de Medecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. E-mail: jamel.elbenna{at}inserm.fr

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47^phox, p67^phox, p40^phox, and Rac2) translocate to the membranes, where they associate with flavocytochrome b₅₅₈, which is composed of gp91^phox/NOX2 and p22^phox, to form the active system. During neutrophil stimulation, p47^phox, p67^phox, p40^phox, and p22^phox are phosphorylated; however, the phosphorylation of gp91^phox/NOX2 and its potential role have not been defined. In this study, we show that gp91^phox is phosphorylated in stimulated neutrophils. The gp91^phox phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91^phox, which confirms that this phosphoprotein is gp91^phox. The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91^phox, and protein kinase C (PKC) phosphorylated the recombinant gp91^phox- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91^phox-cytosolic tail on the same peptides that were phosphorylated on gp91^phox in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91^phox flavoprotein cytosolic domain and its binding to Rac2, p67^phox, and p47^phox. These results demonstrate that gp91^phox is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91^phox/NOX2 is a novel mechanism of NADPH oxidase regulation.—Raad, H., Paclet, M.-H., Boussetta, T., Kroviarski, Y., Morel, F., Quinn, M. T., Gougerot-Pocidalo, M.-A., Dang, P. M.-C., El-Benna, J. Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91^phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67^phox, and p47^phox.

Key Words: neutrophils • PMN • cytochrome b558 • inflammation • innate immunity

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