MULTIPLE MECHANISMS FOR THE PHOSPHORYLATION OF C-TERMINAL REGULATORY SITES IN RABBIT MUSCLE GLYCOGEN-SYNTHASE EXPRESSED IN COS CELLS

Glycogen synthase can be inactivated by sequential phosphorylation at the C-terminal residues Ser(652) (site 4), Ser(648) (site 3c), Ser(644 ) (site 3b) and Ser(640) (site 3a) catalysed by glycogen synthase kina se-3. In vitro, glycogen synthase kinase-3 action requires that glycog en synthase has first been phosphorylated at Ser(656) (site 5) by case in kinase II. Recently we demonstrated that inactivation is linked onl y to phosphorylation at site 3a and site 3b, and that, in COS cells, m odification of these sites can occur by alternative mechanisms indepen dent of any C-terminal phosphorylations [Skurat and Roach (1995) J. Bi ol. Chem. 270, 12491-12497]. To address these mechanisms multiple Ser --> Ala mutations were introduced in glycogen synthase such that only site 3a or site 3b remained intact. Additional mutation of Arg(637) -- > Gin eliminated phosphorylation of site 3a, indicating that Arg(637) may be important for recognition of site 3a by its corresponding prote in kinase(s). Similarly, additional mutation of pro(645) --> Ala elimi nated phosphorylation of site 3b, indicating a possible involvement of 'proline-directed' protein kinase(s). Mutation of Arg(637) alone did not activate glycogen synthase as expected from the loss of phosphoryl ation at site 3a. Rather, mutation of both Arg(637) and the Ser --> Al a substitution at site 3b was required for substantial activation. The results suggest that sites 3a and 3b can be phosphorylated independen tly of one another by distinct protein kinases. However, phosphorylati on of site 3b can potentiate phosphorylation of site 3a, by an enzyme such as glycogen synthase kinase-3.