RATE-DETERMINING STEPS IN THE BIOSYNTHESIS OF GLYCOGEN IN COS CELLS

Consistent with previous results, overexpression of rabbit skeletal mu scle glycogen synthase in COS cells did not lead to overaccumulation o f glycogen unless activating Ser --> Ala mutations were present at key regulatory phosphorylation sites 2 (Ser(7)) and 3a (Ser(644)) in the enzyme. In addition, we found that expression of glycogenin, glycogen branching enzyme, or UDP-glucose pyrophosphorylase alone in COS cells had no effect on the glycogen level. However, coexpression of the hype ractive 2,3a glycogen synthase mutant with either glycogenin or UDP-gl ucose pyrophosphorylase led to higher glycogen accumulation than that obtained from the expression of glycogen synthase alone, Coexpression of glycogenin with the 2,3a mutant of glycogen synthase led to the app earance of glycogenin with a lower molecular weight suggestive of redu ced glucosylation. Increased glycogen synthesis may lead to competitio n between glycogenin and glycogen synthase for their common substrate UDP-glucose. In summary, we conclude that (i) glycogen synthase is a p rimary rate-limiting enzyme of glycogen biosynthesis in COS cells, (ii ) that phosphorylation of glycogen synthase is regulatory for glycogen accumulation, and (iii) once glycogen synthase is activated, the reac tion mediated by UDP-glucose pyrophosphorylase can become rate-determi ning. (C) 1996 Academic Press, Inc.