PHOSPHORYLATION OF SITES 3A AND 3B (SER(640) ANS SER(644)) IN THE CONTROL OF RABBIT MUSCLE GLYCOGEN-SYNTHASE

Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser(652) (site 4), Ser(648) (site 3c), Ser(644) (site 3b), and Ser(640) (site 3a). Effective recognition of glycogen synthase by glycogen synthase k inase-3 occurs only after the phosphorylation of Ser(656) (site 5) cat alyzed by casein kinase II. The present study addresses specifically t he role of sites 3a and 3b in the regulation of glycogen synthase expr essed in COS cells, Simultaneous Ser --> Ala substitutions at sites 3 a, b and c, 4, and 5 in the same protein molecule eliminated P-32 labe ling in the proteolytic fragment Arg(634)-Lys(682), which contains the se sites, This mutant enzyme (which also had a Ser --> Ala substitutio n at site 2 in the NH2 terminus) had a -/+ glucose-6-P activity ratio of similar to 0.8, similar to that of totally dephosphorylated enzyme, Reinstating serine residues at either site 3a or site 3b restored lab eling in the Arg(634)-Lys(682) peptide and caused a decrease in the ac tivity ratio to 0.4-0.6, When both sites 3a and 3b were reintroduced, there was complete inactivation of the enzyme. Thus, sites 3a and 3b a re sufficient for the inactivation of glycogen synthase and act synerg istically to control activity. This investigation demonstrates the exi stence of an alternate mechanism for the phosphorylation of sites 3a a nd 3b that does not depend on prior phosphorylation of site 5.