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.