Action of insulin and catecholamines on the phosphorylation of proteins associated with the cytosol, membranes, and “fat cake” of rat fat cells.

Hormone-induced phosphoprotein phosphorylations were studied in isolated rat fat cells and in subcellular fractions derived from fat cells.Insulin action increased the phosphorylation of two phosphoproteins designated 2 and fib, the molecular weights of which are 130,000 and 62,000, respectively.Epinephrine and isoproterenol action decreased the phosphorylation of phosphoprotein-6b and markedly increased the phosphorylation of phosphoprotein-5, the molecular weight of which is approximately 65,000 to 70,000.Changes in the phosphorylations of phosphoproteins-5 and -6b and fat cell glycerol production varied as a function of epinephrine concentrations.Isoproterenol-induced phosphoprotein-6b dephosphorylation was studied in "cold chase" experiments.At each time point of the "cold chase" isoproterenol increased the dephosphorylation of phosphoprotein-6band increased the phosphorylation of phosphoprotein-5.N-Ethylmaleimide at 0.1 mM inhibited fat cell protein phosphorylation 35%.However, it inhibited specifically phosphoprotein-6b phosphorylation and increased phosphoprotein-5 phosphorylation relative to other protein phosphorylations.Periodic acid-Schiff stain of gel electrophoretograms containing fat cell proteins showed that radiolabeled phosphoproteins-3 and -4 were glycoproteins.Subcellular fractionation of :'2P-labeled fat cells demonstrated that phosphoprotein-2 was found with the cytosol fraction, while phosphoproteins-4 and -6a were present in the "crude membrane" fraction.Phosphoprotein-Gb was not extracted by standard buffers but 6b was found with the "fat cake."Nonionic detergent extraction of the "fat cake" released phosphoprotein-6b from the "fat cake."Phosphoprotein-6b was distributed both in a new pellet and supernatant fraction.Electron microscopy of the pellet demonstrated it to be composed of filaments and short membrane segments.Since the phosphorylation of phosphoprotein-6b