Abstract
Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b), and Ser640 (site 3a). Effective recognition of glycogen synthase by glycogen synthase kinase-3 occurs only after the phosphorylation of Ser656 (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 32P labeling in the proteolytic fragment Arg634-Lys682, 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 approximately 0.8, similar to that of totally dephosphorylated enzyme. Reinstating serine residues at either site 3a or site 3b restored labeling in the Arg634-Lys682 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.
Highlights
Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser[652], Ser[648], Ser[644], and Ser[640]
The present study addresses the role of sites 3a and 3b in the regulation of glycogen synthase expressed in COS cells
Phosphorylation tends to inactivate glycogen synthase full activity can be restored in the presence of glucose6-P, leading to the use of the -/+ glucose-6-P activity ratio as an index of the activation state of the enzyme (5)
Summary
Vol 270, No 21, Issue of May 26, pp. 12491-12497, 1995 Printed in U.S.A. Phosphorylation of Sites 3a and 3b (Ser[640] and Ser644 ) in the Control of Rabbit Muscle Glycogen Synthase*. Reinstating serine residues at either site 3a or site 3b restored labeling in the Arg634_Ly s682peptide 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. The activity of rabbit muscle glycogen synthase (EC 2.4.1.11) is under hormonal control mediated through multisite phosphorylation of the enzyme by multiple protein kinases (1-4). The costs of publication of this article were defrayed in part by the payment of page charges. We recently demonstrated that, for glycogen synthase expressed in COS cells, disruption of the recognition sequence for glycogen synthase kinase-3 by Ser ~ Ala substitution at site 3c, 4, or 5 did not preclude inactivation ofthe enzyme (15). We show that glycogen synthase expressed in COS cells could be phosphorylated directly at sites 3a or 3b when all adjacent phosphorylation sites were eliminated by mutagenesis
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