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 kinase-3 occurs only after the phosphorylation of Ser(656) (site 5) catalyzed by casein kinase II. The present study addresses specifically the role of sites 3a and 3b in the regulation of glycogen synthase expressed 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 labeling in the proteolytic fragment Arg(634)-Lys(682), which contains these sites, This mutant enzyme (which also had a Ser --> Ala substitution 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 labeling in the Arg(634)-Lys(682) peptide and caused a decrease in the activity 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 are sufficient for the inactivation of glycogen synthase and act synergistically to control activity. This investigation demonstrates the existence of an alternate mechanism for the phosphorylation of sites 3a and 3b that does not depend on prior phosphorylation of site 5.