Tyrosine phosphorylation of IκBα activates NFκB through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation

NFkappaB is a critical transcription factor involved in modulating cellular responses to environmental injuries. Tyrosine 42 phosphorylation of IkappaBalpha has been shown to mediate NFkappaB activation following hypoxia/reoxygenation (HR) or pervanadate treatment. This pathway differs from the canonical proinflammatory pathways, which mediate NFkappaB activation through serine phosphorylation of IkappaBalpha by the IKK complex. In the present study, we investigated the involvement of c-Src in the redox activation of NFkappaB following H/R or pervanadate treatment. Our results demonstrate that pervanadate or H/R treatment leads to tyrosine phosphorylation of IkappaBalpha and NFkappaB transcriptional activation independent of the IKK pathway. In contrast, inhibition of c-Sre by pp2 treatment or in c-Sre (-/-) knockout cell lines, demonstrated a significant reduction in IkappaBalpha tyrosine phosphorylation and NFkappaB activation following pervanadate or H/R treatment. Overexpression of glutathione peroxidase-1 or catalase, but not Mn-SOD or Cu,Zn-SOD, significantly reduced both NFkappaB activation and tyrosine phosphorylation of IkappaBalpha. In vitro kinase assays further demonstrated that immunoprecipitated c-Src has the capacity to directly phosphorylate GST-IkappaBalpha and that this IkappaBalpha kinase activity is significantly reduced by Gpx-1 overexpression. These results suggest that c-Sre-dependent tyrosine phosphorylation of IkappaBalpha and subsequent activation of NFkappaB is controlled by intracellular H2O2 and defines an important redox-regulated pathway for NFkappaB activation following H/R injury that is independent of the IKK complex.