Multiple phosphorylation of rabbit skeletal muscle glycogen synthase. Evidence for interactions among phosphorylation sites and the resolution of electrophoretically distinct forms of the subunit.

Phosphorylation of rabbit skeletal muscle glycogen synthase by a cyclic nucleotide and Ca2+-independent protein kinase, PC0.7, caused the enzyme to be a better substrate for phosphorylation by another cyclic nucleotide and Ca2+-independent protein kinase, FA/GSK-3. In contrast, phosphorylation by the combination of FA/GSK-3 and cyclic AMP-dependent protein kinase led to less phosphorylation than predicted from the individual actions of the protein kinases. These results are explained in part by the existence of cooperative interactions among the phosphorylation sites of glycogen synthase. Phosphorylation by FA/GSK-3 also correlated with a reduction in the electrophoretic mobility, in the presence of sodium dodecyl sulfate, of the glycogen synthase subunit from an apparent molecular weight of 85,000-86,000 to values of 88,000 and ultimately 90,000. The synergistic phosphorylation by PC0.7 and FA/GSK-3 was associated with an increased formation of the species of reduced electrophoretic mobility. The effects on subunit mobility were also reflected in the behavior of a larger phosphorylated CNBr fragment of glycogen synthase, CB-2, which gave apparent molecular weights of 22,000-27,000 depending on its phosphorylation state.