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

Abstract

NFκB is a critical transcription factor involved in modulating cellular responses to environmental injuries. Tyrosine 42 phosphorylation of IκBα has been shown to mediate NFκB activation following hypoxia/ reoxygenation (H/R) or pervanadate treatment. This pathway differs from the canonical proinflammatory pathways, which mediate NFκB activation through serine phosphorylation of IκBα by the IKK complex. In the present study, we investigated the involvement of c-Src in the redox activation of NFκB following H/R or pervanadate treatment. Our results demonstrate that pervanadate or H/R treatment leads to tyrosine phosphorylation of IκBα and NFκB transcriptional activation independent of the IKK pathway. In contrast, inhibition of c-Src by pp2 treatment or in c-Src (-/-) knockout cell lines, demonstrated a significant reduction in IκBα tyrosine phosphorylation and NFκB 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 NFκB activation and tyrosine phosphorylation of IκBα. In vitro kinase assays further demonstrated that immunoprecipitated c-Src has the capacity to directly phosphorylate GST-IκBα and that this IκBα kinase activity is significantly reduced by Gpx-1 overexpression. These results suggest that c-Src-dependent tyrosine phosphorylation of IκBδ and subsequent activation of NFκB is controlled by intracellular H202 and defines an important redox-regulated pathway for NFκB activation following H/R injury that is independent of the IKK complex.