Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes.
The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology*; D007330:Insulin Antibodies; D008297:Male; D008345:Manganese / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013696:Temperature
D007328:Insulin; D007330:Insulin Antibodies; D009842:Oligopeptides; D008345:Manganese; C037642:kemptide; D006493:Heparin; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D004229:Dithiothreitol