Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells.
In this study we describe the phosphorylation of annexins from cultured rat mesangial cells by protein kinase C (PKC) both in vitro and in vivo. Annexins I and II were detected either by Western-blot analysis or by immunoprecipitation using specific antibodies. In the presence of [gamma-32P]ATP, cytosolic annexin I and annexin II were phosphorylated in vitro only when Ca2+ and phospholipids were added, but not in the presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on 32P-labelled mesangial cells, the addition of two well-known activators of PKC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), increased preferentially the phosphorylation of annexin I. Annexin II was phosphorylated to a much lesser extent after AII treatment. Phosphoamino acid analysis of annexins, either by two-dimensional chromatography or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to mesangial cells increased serine phosphorylation of annexin I and annexin II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylated both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.
INSERM U64, Hôpital Tenon, Paris, France.
J P
JP
Oudinet
F
F
Russo-Marie
J C
JC
Cavadore
B
B
Rothhut
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D017305:Annexin A1 / Q000378:metabolism*; D017306:Annexin A2 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017305:Annexin A1; D017306:Annexin A2; D010743:Phospholipids; D000804:Angiotensin II; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium