RABBIT SKELETAL-MUSCLE GLYCOGEN-SYNTHASE EXPRESSED IN COS CELLS - IDENTIFICATION OF REGULATORY PHOSPHORYLATION SITES

Rabbit skeletal muscle glycogen synthase contains multiple sites for p hosphorylation. To investigate the relative importance of these sites, the enzyme was overexpressed in COS M9 cells, and Ser --> Ala mutatio ns were introduced singly, or in combinations, at nine known phosphory lation sites. Overexpressed wild-type enzyme had a very low -/+ glucos e-6-P activity ratio of similar to 0.01, indicative that the glycogen synthase is in a highly phosphorylated state. No single Ser --> Ala mu tation was able to cause a substantial increase in activity ratio; rat her, simultaneous mutation at both NH2- and COOH-terminal sites was ne eded. The most effective combinations were mutations at site 3a (Ser-6 40) or site 3b (Ser-644) together with site 2 (Ser-7). The results wer e consistent with site 2 phosphorylation being a prerequisite for phos phorylation of site 2a (Ser-10). Mutation of site 5 (Ser-656) perturbe d COOH-terminal phosphorylation but did not prevent inactivation. Expr ession of the most active mutants correlated with increased glycogen a ccumulation in the COS M9 cells. In summary, we con elude that (i) the sites most important for activating the enzyme are sites 2, 2a, 3a, a nd 3b; (ii) removal of phosphate from both NH2- and COOH-terminal site s is required for activation; and (iii) sites 3a and/or 3b can be phos phorylated in COS cells by mechanisms that do not depend on phosphoryl ation of site 5.