PROTEIN-KINASE C-DEPENDENT PHOSPHORYLATION OF ANNEXINS-1 AND ANNEXINS-11 IN MESANGIAL CELLS

In this study we describe the phosphorylation of annexins from culture d rat mesangial cells by protein kinase C (PKC) both in vitro and in v ivo. Annexins I and II were detected either by Western-blot analysis o r by immunoprecipitation using specific antibodies. In the presence of [gamma-P-32]ATP, cytosolic annexin I and annexin II were phosphorylat ed in vitro only when Ca2+ and phospholipids were added, but not in th e presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on P-32-la belled mesangial cells, the addition of two well-known activators of P KC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), i ncreased preferentially the phosphorylation of annexin I. Annexin II w as phosphorylated to a much lesser extent after All treatment. Phospho amino acid analysis of annexins, either by two-dimensional chromatogra phy or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to me sangial cells increased serine phosphorylation of annexin I and annexi n II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylate d both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.