Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations.
Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.
Sacks
D B
DB
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
McDonald
J M
JM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D006657:Histones / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011479:Protamines / Q000494:pharmacology; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*
D002147:Calmodulin; D006657:Histones; D007328:Insulin; D011479:Protamines; D011107:Polylysine; D008345:Manganese; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin; D008274:Magnesium