Insulin receptor beta-subunit serine phosphorylation in permeabilized cultured fetal rat hepatocytes.

Regulation of cellular protein phosphorylation by insulin was investigated after short exposure at 37 degrees C prior to applying the permeabilization/phosphorylation step in the presence of digitonin and [gamma-32P]ATP for 30 min at 4 degrees C. The results revealed major 32P incorporation into a limited number of membrane polypeptides exhibiting a molecular mass of 95, 58 and 51 kDa. Phosphorylation of 95 kDa protein was selectively inhibited with Ca(2+)-free EGTA-containing permeabilization/phosphorylation buffer and became predominant in the presence of Ca2+. Considering in particular its immunoprecipitation by a monoclonal antibody directed against insulin receptor, the 32P-labeled 95 kDa protein represented the beta-subunit of the insulin receptor. Its phosphorylation was transiently stimulated after exposure to insulin (35% after 2 min), and concerned mostly serine residues under both basal and stimulated conditions. Vanadate had a similar effect and both agents favored glycogenesis, whereas heparin which inhibited 95 kDa protein phosphoseryl phosphorylation had an opposite effect on glycogenesis. These results suggest a biological role for the membrane-associated phosphoseryl-protein kinase(s) and phosphatase(s) acting on the insulin receptor beta-subunit in cultured fetal hepatocytes.