Tumor-promoting phorbol esters increase the Km of the ATP-binding site of the insulin receptor kinase from rat adipocytes.

Abstract

Phorbol ester treatment of isolated rat adipocytes inhibits insulin stimulation of the glucose transport system. We studied whether this effect is related to a modification of the insulin receptor kinase. Insulin receptor of tetradecanoyl-beta-phorbol acetate (TPA)-treated adipocytes was solubilized and partially purified, and its kinase activity was studied in vitro. We found that insulin (10(-7) M) increased the tyrosine autophosphorylation of the insulin receptor kinase from TPA-treated cells only 3-fold in contrast to a 12-fold stimulation in control cells. The rate of insulin-stimulated 32P incorporation into the receptor of TPA-treated cells at insulin concentrations between 10(-10) M and 10(-7) M and at nonsaturating [32P]ATP levels (5 microM) was reduced to only 5-8% of the values found with receptor from control cells. 125I-insulin binding to the solubilized receptor of TPA-treated cells was reduced as well, apparently due to a decreased affinity of the binding site. Decreased binding was however, not sufficient to explain the difference of kinase activity. The inhibition of kinase activity of the receptor from TPA-treated cells decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis of receptor phosphorylation revealed that the Km for ATP of the receptor kinase from TPA-treated cells was increased to greater than 100 microM compared to 25 microM for the receptor of control cells. An analogous change of the Km for ATP was found when we studied the phosphorylation of a synthetic polymer of tyrosine and glutamic acid as a substrate of the receptor kinase. We conclude from the data that phorbol treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of a mechanism leading to insulin resistance in these cells.