The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.
Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.
Laurino
J P
JP
Metabolic Research, Upjohn Company, Kalamazoo, Michigan 49001.
Colca
J R
JR
Pearson
J D
JD
DeWald
D B
DB
McDonald
J M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000302:isolation & purification; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011479:Protamines / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000502:physiology*; D013917:Thrombin
D002147:Calmodulin; D006657:Histones; D009943:Organophosphorus Compounds; D011479:Protamines; D011107:Polylysine; D008345:Manganese; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D013917:Thrombin; D008274:Magnesium