Rat skeletal muscle glycogen synthase: phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases.
Glycogen synthase was purified to near homogeneity from rat skeletal muscle, and was found to resemble the rabbit skeletal muscle enzyme in several respects. An apparent molecular weight (Mapp) of 86,000 was estimated from the electrophoretic mobility of the subunit on polyacrylamide gels in the presence of sodium dodecyl sulfate. Limited proteolysis of the rat synthase with trypsin resulted in the formation of species with MappS equal to 75,000, 69,000, and 67,000. The enzyme could be phosphorylated by cAMP-dependent protein kinase, phosphorylase kinase, and the cAMP-independent protein kinases, PC0.7 and FA/GSK-3. Essentially all of the phosphorylation observed occurred on serines located in two cyanogen bromide fragments, denoted CB-1 (Mapp = 13,000) and CB-2 (Mapp = 22,000). FA/GSK-3 and cAMP-dependent protein kinase phosphorylated sites in both fragments. Phosphate introduced by phosphorylase kinase was located exclusively in CB-1, and that incorporated with PC0.7 was found in CB-2. Phosphorylation by FA/GSK-3 reduced the electrophoretic mobility of the subunit, introduced heterogeneity into CB-2, and was synergistic with phosphorylation by PC0.7. To separate phosphorylation sites more completely, samples of glycogen synthase were subjected to extensive proteolysis using trypsin, followed by reverse-phase liquid chromatography. When phosphorylated by the same kinases, the pattern of fragments obtained with rat and rabbit skeletal muscle glycogen synthase were almost identical. The results presented provide strong evidence that the subunit of rat skeletal muscle glycogen synthase has at least five phosphorylation sites that are very similar, if not identical, to sites present on the rabbit muscle enzyme.
Hiken
J F
JF
Lawrence
J C
JC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006006:Glycogen Synthase / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D000242:Cyclic AMP; D006006:Glycogen Synthase; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D014357:Trypsin