| Abstract: | Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism. |
