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 throu gh modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular inju ry, we studied the effect of hydrogen peroxide (H2O2), vanadate, and p ervanadate (V4+-OOH) on [P-32(i)] uptake and protein phosphorylation i n bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [P-32(i)] into BPAEC was dependent on the concentration o f the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 mu M) induced maximum incorporation of [P-32(i)] into cells (two- to threefold over control) followed by H2O2 (1 mM) and va nadate (100 mu M) and clear differences in labeled protein profiles we re noticed between control and oxidant treated cells. The proteins, an alyzed by SDS-PAGE, showed distinct increases in labeling patterns ran ging from 21-205 kDa, as evidenced by autoradiography. While the major ity of the incorporated [P-32(i)] was in serine/threonine residues, im munoprecipitation and immunoblotting of cell lysates, using an antipho sphotyrosine antibody, revealed that oxidant treatment resulted in sig nificant increases in total protein tyrosine phosphorylation. Most sig nificantly, immunoprecipitation of cell lysates, from pervanadate trea tment showed distinct tyrosine phosphorylation of 22 kDa protein, whic h was identified as caveolin, a marker of caveolae. Pervanadate-mediat ed phosphorylation was effectively inhibited by staurosporine (5 mu M) , while genistein showed only partial attenuation. Furthermore, H2O2 t reatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhib ited increased tyrosine kinase activity and decreased phosphatase acti vity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases. Copyright (C) 1996 Elsevier Sci ence Inc.