Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

We investigated the mechanism of H2O2 activation of the Ca2+-regulated NADPH oxidase NOX5. H2O2 induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H2O2 is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H2O2. H2O2 NOX5 activation was inhibited by extracellular Ca2+ chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP–c-Abl colocalized with vesicular sites of superoxide production in a Ca2+-dependent manner. In contrast to H2O2, the Ca2+ ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP–c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H2O2 treatment. Furthermore, H2O2-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP–Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca2+-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.