Insulin stimulates phosphorylation of a 120-kDa glycoprotein substrate (pp120) for the receptor-associated protein kinase in intact H-35 hepatoma cells.
The insulin receptor possesses protein kinase activity, which may play a role in mediating insulin action. Recently, we have identified a glycoprotein (pp120) in rat liver plasma membranes that is phosphorylated by the solubilized insulin receptor in a cell-free system. We now report that insulin stimulates phosphorylation of pp120 in intact H-35 cells. H-35 cells were preloaded with [32P]orthophosphate to label the intracellular ATP pool. Insulin caused a 10-fold increase in the phosphorylation of its receptor and a 2-fold increase in phosphorylation of pp120 (P less than 0.001). The time course of insulin's stimulation of pp120 closely paralleled that of insulin receptor phosphorylation over the time period investigated (15-45 min). This effect had the specificity corresponding to the insulin receptor. Epidermal growth factor was inactive, and insulin-like growth factor I had approximately equal to 1% the potency of insulin in this regard. Insulin increased 32P incorporation into pp120 in a linkage that was stable to alkaline hydrolysis, as would be expected for tyrosine-specific phosphorylation. Direct phosphoamino acid analysis confirmed that insulin increased 32P incorporation into phosphotyrosine residues in pp120.
D000596:Amino Acids / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D002460:Cell Line; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008113:Liver Neoplasms / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*
D000596:Amino Acids; D007328:Insulin; D008565:Membrane Proteins; D010710:Phosphates; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human