Oxidative stress induces tyrosine phosphorylation of PDGF α-and β-receptors and pp60c−src in mesangial cells

Oxidative stress induces tyrosine phosphorylation of PDGF α- and β-receptors and pp60 c−src in mesangial cells. Reactive oxygen species are autocrine and paracrine modulators of cell behavior. Hydrogen peroxide, a cellular oxidant, has been shown to stimulate mesangial cell proliferation. In the present study we analyzed the H 2 O 2 -induced early signaling events. Immunofluorescence analysis revealed a H 2 O 2 induced dose-dependent increase in tyrosine phosphorylation. Short treatment (2 or 5 min) with 5mM H 2 O 2 induced a mitogenic response and a significant (P 2 O 2 (0.1 to 10 mM) treated cells were separated on SDS-PAGE and subjected to immunoblot analysis with anti-phosphotyrosine. A dose-dependent induction of tyrosine phosphorylation of 180kDa, 120kDa and 60kDa proteins was observed within 1 to 10 minutes. By sequentially using immunoprecipitation and immunoblotting the 180kDa tyrosine phosphorylated band was shown to represent both PDGF α -and β -receptors. The tyrosine phosphorylated 60kDa protein was identified as the cytoplasmic protein tyrosine kinase pp60 c−src . The c- src phosphorylation was associated with an inhibition of c- src kinase activity, suggesting phosphorylation of tyrosine 527 in the c- src regulatory domain. Pretreatment with catalase completely abrogated the H 2 O 2 -induced PDGF receptor and c- src tyrosine phosphorylation. These data support the notion that the activation of a signaling pathway involving the PDGF receptors and c- src contributes to the mitogenic effects of reactive oxygen species.