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 phospho rylation at both NH2 - and COOH-terminal sites of the enzyme and by elevati ng the levels of glucose-6-phosphate, an allosteric activator of glycogen s ynthase. To study the mechanism of regulation of glycogen synthase by insul in and glucose-6-phosphate, we generated stable Rat-1 fibroblast clones exp ressing rabbit muscle glycogen synthase with Ser --> Ala substitutions at k ey phosphorylation sites. We found that 1) elimination of the phosphorylati on of either NH2 - or COOH-terminal sites did not abolish insulin stimulati on of glycogen synthase; 2) mutations at both Ser-7 and Ser-640 were necess ary to bypass insulin activation; 3) mutation at Ser-7, coupled with the di sruption 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-phospha te >10-fold. We conclude that Ser-7 and Ser-640 are both involved in mediat ing the response of glycogen synthase to insulin and activation by glucose 6-phosphate. In Rat-1 fibroblasts, GSK-3 action is not essential for glycog en synthase activation by insulin, and GSK-3-independent mechanisms also op erate.