Insulin and phorbol ester stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes.
Several substrates of endogenous Ca2+- and phospholipid-sensitive protein kinase have been identified in plasma membranes and cytosol from rat adipocytes. Specifically, Ca2+ stimulates phosphorylation of a 40-kDa protein in isolated plasma membranes, an effect which is further enhanced by the addition of the phorbol ester tetradecanoylphorbol acetate and phospholipase C. The 40-kDa phosphoprotein is also present in the cytosol, and its phosphorylation is stimulated in a Ca2+-dependent manner by phosphatidylserine, diacylglycerol, and phorbol ester. Direct addition of insulin to adipocyte plasma membranes stimulates phosphorylation of the 40-kDa protein in a concentration-dependent manner. Maximal stimulation was observed at 10(-8) M insulin. At 6.7 X 10(-8) M insulin, phosphorylation of the 40-kDa protein was stimulated by 68 +/- 9% (n = 6). Addition of phorbol ester (1, 10, and 100 ng/ml) plus insulin further enhanced the phosphorylation (286 +/- 39, n = 3; 350 +/- 65, n = 4; and 323 +/- 42%, n = 5, stimulation, respectively). Analysis of the 40-kDa phosphoprotein by two-dimensional polyacrylamide gel electrophoresis revealed that incubations containing no additions, insulin, and/or phorbol ester all resulted in the generation of a single and apparently identical phosphorylated 40-kDa species. These studies indicate that insulin and Ca2+- and phospholipid-dependent protein kinase stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes.
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010704:Phorbols / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; D007328:Insulin; D008565:Membrane Proteins; D010704:Phorbols; D010718:Phosphatidylserines; D010750:Phosphoproteins; D013755:Tetradecanoylphorbol Acetate