Calmodulin as substrate for insulin-receptor kinase. Phosphorylation by receptors from rat skeletal muscle.
Calmodulin is a substrate for insulin-receptor kinase obtained from rat adipocytes and hepatocytes and human placenta. In this study, we demonstrate that insulin stimulates the phosphorylation of calmodulin via insulin receptors partially purified from rat skeletal muscle. Phosphorylation of calmodulin was maximal in the presence of Mg2+ and insulin and the absence of Ca2+. Free-Ca2+ concentrations greater than 0.1 microM progressively inhibited phosphorylation with almost total inhibition at 200 microM Ca2+. Insulin-stimulated phosphorylation of calmodulin was dose dependent and saturable with half-maximal effect obtained at approximately 5 x 10(-10) M insulin. There was an absolute requirement for certain basic proteins, e.g., polylysine or protamine sulfate, to obtain phosphate incorporation into calmodulin. Polylysine stimulated the phosphorylation of calmodulin independently of insulin, but this was increased up to sixfold by the addition of insulin. Phosphate incorporation into calmodulin increased with increasing concentration of the substrate up to a saturating concentration of 2.4 microM. The Km for calmodulin was approximately 0.2 microM. Up to 0.15 mol of phosphate was incorporated per mole of calmodulin with tyrosine the predominant amino acid phosphorylated. The observations that calmodulin is phosphorylated by insulin-receptor kinase from all three classic target organs for insulin confirm that calmodulin is a general substrate for this kinase and suggest that Ca2+ and calmodulin may be components of the insulin-signaling mechanism.
Sacks
D B
DB
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
McDonald
J M
JM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007328:Insulin / Q000494:pharmacology; D008297:Male; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin
D000596:Amino Acids; D002147:Calmodulin; D007328:Insulin; D011107:Polylysine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D002118:Calcium