Identification of an adipocyte protein that binds to calmodulin in the absence of Ca2+ and is phosphorylated in response to insulin and tumor-promoting phorbol esters.
The present experiments were performed to identify calmodulin-binding proteins phosphorylated in response to insulin. Homogenates were prepared from 32Pi-labeled rat adipocytes. After centrifugation, the supernatants (+/- Ca2+) were applied to calmodulin-Sepharose columns. The bound proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and phosphoproteins were visualized by autoradiography. Several proteins bound to the affinity resin in the presence of Ca2+, two bound +/- Ca2+, but only one protein, Mr = 170,000 (denoted pp170), bound in the absence of Ca2+. Binding of pp170 was inhibited by adding calmodulin (micromolar) or Ca2+ (nanomolar) to extracts prior to affinity chromatography. Physiological concentrations of insulin rapidly and reversibly increased (by as much as 4-fold) 32P-labeled pp170. Phorbol 12-myristate 13-acetate (PMA) increased (up to 3-fold) phosphorylation of pp170; but 4 alpha-phorbol 12,13-didecanoate was without effect. Phosphorylation of pp170 in response to insulin and PMA occurred predominantly on serine residues; no phosphotyrosine was detected. Protein kinase C inhibitors attenuated PMA-stimulated phosphorylation of pp170, but had no effect on insulin-stimulated phosphorylation. Peptide mapping indicated that pp170 was phosphorylated on multiple sites and that insulin stimulated the phosphorylation of at least one site not phosphorylated in response to PMA. The results indicate that insulin and PMA stimulate the phosphorylation of pp170 via different pathways, the latter presumably via protein kinase C.
McDonald
J R
JR
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Lawrence
J C
JC
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002148:Calmodulin-Binding Proteins; D007328:Insulin; D010750:Phosphoproteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium