Src glutathionylation and its role in oxidant and TGFβ1‐mediated Src activation (1095.13)

Abstract

Src is a critical enzyme involved in epithelial‐mesenchymal transition that contributes to metastasis and possibly fibrosis. Previous studies have demonstrated that oxidants are involved in Src activation by various stimuli. As glutathionylation of critical cysteine residues is an important mechanism of redox signaling, we studied the glutathionylation of Src and its role in Src activation in response to TGF‐β1, hydrogen peroxide (H2O2) and α,β‐unsaturated aldehydes. Human H358 lung carcinoma cells were transfected with FLAG‐tagged human c‐Src constructs, exposed to agents or control media. Then, FLAG‐Src was immunoprecipitated with anti‐FLAG antibody. Src protein was glutathionylated upon exposure to TGF‐β1 and H2O2, but not to aldehydes. Roles of individual cysteines in c‐Src activation were examined by mutating the cysteine (to alanine), transfecting cells with mutant c‐Src, and then examining oxidant‐stimulated auto‐phosphorylation of c‐Src at Tyr418. Mutation at C248 or C501 abolished Src activation by oxidants and TGF‐β1 and glutathionylation was absent in these mutants. The data suggests that glutathionylation plays a critical role in c‐Src activation by both TGF‐β1 and oxidative stimuli.Grant Funding Source: ES020942