Receptor-mediated phosphorylation of the hepatic insulin receptor: Evidence that the M_r 95,000 receptor subunit is its own kinase

Abstract

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 [γ-³²P]-ATP in the absence or presence of insulin (1 μM). NaDodSO₄/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the β subunit (M _r 95,000) of the insulin receptor; the α subunit (M _r 130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor β 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 [α-³²P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the β 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 β 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.

• autoantibodies against insulin receptors
• covalent affinity labeling
• ATP