Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its β subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor β subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100 % higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor β subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated β subunit autophosphorylation activities when incubated with [γ-³²P]ATP. Similarly, the ability of insulin to stimulate the receptor β subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the β-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of ³²P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor β subunit. However, the tumor promoter has no effect on the insulin-stimulated tyrosine phosphorylation of the receptor. Consistent with this finding, prior exposure of cells to PMA has no effect on the ability of insulin to activate the receptor histone kinase activity under in vivo conditions. These results suggest that serine phosphorylation of the insulin receptor in response to phorbol ester action has little influence on the tyrosine kinase activity of the receptor in H-35 cells. This contrasts with the action of the cyclic AMP-mediated mechanism that appears to uncouple the insulin receptor kinase.