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

ABSTRACT 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 b 558 , 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 gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox. FASEB J . 23, 1011–1022 (2009)