Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites.
In skeletal muscle, insulin activates glycogen synthase by reducing phosphorylation at both NH2- and COOH-terminal sites of the enzyme and by elevating the levels of glucose-6-phosphate, an allosteric activator of glycogen synthase. To study the mechanism of regulation of glycogen synthase by insulin and glucose-6-phosphate, we generated stable Rat-1 fibroblast clones expressing rabbit muscle glycogen synthase with Ser-->Ala substitutions at key phosphorylation sites. We found that 1) elimination of the phosphorylation of either NH2- or COOH-terminal sites did not abolish insulin stimulation of glycogen synthase; 2) mutations at both Ser-7 and Ser-640 were necessary to bypass insulin activation; 3) mutation at Ser-7, coupled with the disruption of the motif for recognition by glycogen synthase kinase-3 (GSK-3), did not eliminate the insulin effect; and 4) mutation of either Ser-7 or Ser-640 increased the sensitivity of glycogen synthase to glucose 6-phosphate >10-fold. We conclude that Ser-7 and Ser-640 are both involved in mediating the response of glycogen synthase to insulin and activation by glucose 6-phosphate. In Rat-1 fibroblasts, GSK-3 action is not essential for glycogen synthase activation by insulin, and GSK-3-independent mechanisms also operate.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202. USA. askurat@iupui.edu
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Skurat
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Dietrich
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PJ
Roach
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D019298:Glucose-6-Phosphate / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012694:Serine; D014162:Transfection
D007328:Insulin; D011994:Recombinant Proteins; D012694:Serine; D019298:Glucose-6-Phosphate; D006006:Glycogen Synthase; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000409:Alanine