Activation of protein phosphorylation by oxidants in vascular endothelial cells: identification of tyrosine phosphorylation of caveolin.

Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H2O2), vanadate, and pervanadate (V(4+)-OOH) on [32Pi] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [32Pi] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 microM) induced maximum incorporation of [32Pi] into cells (two- to threefold over control) followed by H2O2 (1 mM) and vanadate (100 microM) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21-205 kDa, as evidenced by autoradiography. While the majority of the incorporated [32Pi] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 microM), while genistein showed only partial attenuation. Furthermore, H2O2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases.