Characteristics of calmodulin phosphorylation by the insulin receptor kinase.
Calmodulin is a substrate for the insulin receptor kinase. The time sequence of events resulting in insulin-stimulated phosphorylation of calmodulin was analyzed at a number of different insulin concentrations using partially purified solubilized insulin receptor preparations from rat adipocytes. The respective insulin concentrations needed to reach half-maximal binding, phosphorylation of the beta-subunit of the insulin receptor, and phosphorylation of calmodulin were 4.5 X 10(-10), 4.3 X 10(-10), and 3.9 X 10(-10) M, respectively. At all insulin concentrations, the time to reach 50% of the maximum (defined as the value obtained at 60 min) occurred in the sequence: insulin binding less than beta-subunit phosphorylation less than calmodulin phosphorylation. Insulin binding and beta-subunit phosphorylation occurred almost immediately, whereas there was a lag phase preceding calmodulin phosphorylation. Although stoichiometry was generally low under routine assay conditions (0.01-0.10 mol phosphate/mol calmodulin), it could be increased 4.3 +/- 0.5-fold (n = 5) by pretreating the calmodulin with 0.1 N NaOH. Insulin-stimulated phosphorylation of calmodulin was exclusively on tyrosine residues. The calmodulin molecule in animals contains only two tyrosine residues, located at positions 99 and 138. The amount of phosphate incorporation into a semisynthetic calmodulin (VU1) which contains only one of these tyrosine residues (tyrosine-138) was half that obtained with porcine or chicken calmodulin. Therefore, insulin, via its receptor kinase, stimulates the phosphorylation of calmodulin; calmodulin can be phosphorylated on both tyrosine residues 99 and 138.
Wong
E C
EC
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Sacks
D B
DB
Laurino
J P
JP
McDonald
J M
JM
D000273:Adipose Tissue / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D002147:Calmodulin; D046911:Macromolecular Substances; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin