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-P-32 P]ATP for 30 min at 4-degrees-C. The results revealed major P-32 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 Ca2+-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 P-32-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.