Insulin activation of insulin receptor tyrosine kinase in intact rat adipocytes. An in vitro system to measure histone kinase activity of insulin receptors activated in vivo.
We have studied the effect of incubation of intact cells with insulin on insulin receptor kinase activity. Following exposure of rat adipocytes to insulin, cells were solubilized and insulin receptors purified by specific immunoprecipitation or by insulin affinity chromatography. Kinase activity of the receptors, as measured by phosphorylation of histone 2B, was then determined. Insulin treatment of the cells resulted in a 10-20-fold increase in histone kinase activity of the subsequently isolated insulin receptors. The insulin effect was half-maximal at 3 s and maximal within 15 s of exposure, was dose-dependent (EC50 = 21 ng/ml), and was rapidly reversible following dissociation of insulin from the cells. The insulin effect in intact cells on insulin receptor kinase activity could be partially reversed in vitro by dephosphorylation of the isolated receptors by alkaline phosphatase. It is proposed that: in intact cells, insulin causes alterations in insulin receptors, such that their kinase activity toward non-receptor substrates increases; increased insulin receptor kinase activity following insulin stimulation in intact cells is, at least in part, the result of an increased phosphate content of the receptors; and effects of insulin on insulin receptors in intact cells can be preserved during receptor isolation and thus can be measured in a cell-free system.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D007328:Insulin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D006656:Protamine Kinase; D000469:Alkaline Phosphatase