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Rabbit skeletal muscle glycogen synthase expressed in COS cells. Identification of regulatory phosphorylation sites
A V Skurat1, Y Wang, P J Roach
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Abstract:
Rabbit skeletal muscle glycogen synthase contains multiple sites for phosphorylation. To investigate the relative importance of these sites, the enzyme was overexpressed in COS M9 cells, and Ser-->Ala mutations were introduced singly, or in combinations, at nine known phosphorylation sites. Overexpressed wild-type enzyme had a very low +/- glucose-6-P activity ratio of approximately 0.01, indicative that the glycogen synthase is in a highly phosphorylated state. No single Ser-->Ala mutation was able to cause a substantial increase in activity ratio; rather, simultaneous mutation at both NH2- and COOH-terminal sites was needed. The most effective combinations were mutations at site 3a (Ser-640) or site 3b (Ser-644) together with site 2 (Ser-7). The results were consistent with site 2 phosphorylation being a prerequisite for phosphorylation of site 2a (Ser-10). Mutation of site 5 (Ser-656) perturbed COOH-terminal phosphorylation but did not prevent inactivation. Expression of the most active mutants correlated with increased glycogen accumulation in the COS M9 cells. In summary, we conclude that (i) the sites most important for activating the enzyme are sites 2, 2a, 3a, and 3b; (ii) removal of phosphate from both NH2- and COOH-terminal sites is required for activation; and (iii) sites 3a and/or 3b can be phosphorylated in COS cells by mechanisms that do not depend on phosphorylation of site 5.
Insights
Rabbit skeletal muscle glycogen synthase requires phosphorylation at multiple sites for full activation. Key sites (2, 2a, 3a, 3b) at both ends of the enzyme are crucial for regulating its activity and glycogen storage.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Rabbit skeletal muscle glycogen synthase (GS) is a key regulator of glycogen synthesis.
- GS activity is modulated by phosphorylation at multiple sites.
- Understanding these phosphorylation sites is crucial for comprehending glycogen metabolism regulation.
Purpose of the Study:
- To investigate the relative importance of different phosphorylation sites on rabbit skeletal muscle glycogen synthase.
- To determine which phosphorylation sites are critical for enzyme activation and glycogen accumulation.
Main Methods:
- Overexpression of wild-type and mutant glycogen synthase in COS M9 cells.
- Introduction of Ser-->Ala mutations at nine known phosphorylation sites, singly and in combination.
- Assay of enzyme activity using the +/- glucose-6-P activity ratio.
- Measurement of glycogen accumulation in transfected cells.
Main Results:
- Wild-type glycogen synthase exhibited a low activity ratio, indicating a highly phosphorylated and inactive state.
- No single mutation significantly increased the activity ratio; simultaneous mutations at NH2- and COOH-terminal sites were required.
- Mutations at sites 3a/3b combined with site 2 were most effective in increasing enzyme activity.
- Increased enzyme activity correlated with enhanced glycogen accumulation in COS M9 cells.
Conclusions:
- Sites 2, 2a, 3a, and 3b are the most important for activating glycogen synthase.
- Activation requires dephosphorylation at both NH2- and COOH-terminal sites.
- Sites 3a/3b can be phosphorylated independently of site 5, suggesting complex regulatory mechanisms.