Receptor-mediated phosphorylation of the hepatic insulin receptor: evidence that the Mr 95,000 receptor subunit is its own kinase.
Insulin stimulates the phosphorylation of its own receptor. In the work reported here, the kinase activity responsible for the insulin-stimulated phosphorylation of the insulin receptor was localized. In a first approach, partially purified insulin receptors derived from normal rat hepatocytes were immunoprecipitated with antibodies specific for the insulin receptor; thereafter, the immunoprecipitates were incubated with [gamma-(32)P]-ATP in the absence or presence of insulin (1 muM). NaDodSO(4)/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the beta subunit (M(r) 95,000) of the insulin receptor; the alpha subunit (M(r) 130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor beta subunit, indicating that anti-receptor antibodies precipitate a functional and insulin-stimulable protein kinase that appears to be independent of cyclic AMP and calcium. To localize more precisely the insulin receptor-related kinase activity, we searched for an ATP-binding site on solubilized insulin receptors. By using covalent labeling with oxidized [alpha-(32)P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the beta subunit of the insulin receptor (M(r) 95,000) was specifically immunoprecipitated with anti-receptor antibodies. Further, its appearance was prevented when the immunoprecipitation was preceded by incubation with unlabeled insulin. In conclusion, we have shown that an insulin-stimulated phosphorylation site and an ATP-binding site coexist on the beta subunit of the insulin receptor. The simultaneous presence of these two sites on the same receptor subunit indicates that the insulin receptor acts as its own protein kinase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011972:Receptor, Insulin