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Mechanisms of multisite phosphorylation and inactivation of rabbit muscle glycogen synthase
W Zhang1, A A DePaoli-Roach, P J Roach
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Abstract:
Glycogen synthase, a rate-determining enzyme for glycogen biosynthesis, is regulated by complex multisite phosphorylation of its subunit. Previous work has suggested that phosphorylation by some protein kinases, casein kinase II and cyclic AMP-dependent protein kinase, potentiates the ability of other protein kinases, glycogen synthase kinase 3 and casein kinase I, respectively, to modify the enzyme. In the present study, active glycogen synthase was expressed in Escherichia coli using a pET vector. The purified recombinant glycogen synthase had specific activity and subunit M(r) similar to enzyme isolated from rabbit muscle. Prior phosphorylation by casein kinase II was found to be an obligate requirement for phosphorylation by glycogen synthase kinase 3, which introduced 4 mol phosphate/mol subunit. Casein kinase II action did not affect activity, whereas the phosphorylation catalyzed by glycogen synthase kinase 3 caused a potent inactivation, reducing the +/- glucose 6-phosphate activity ratio from 0.7 to 0.10. Casein kinase I alone phosphorylated the recombinant glycogen synthase, indicating that substrate phosphorylation was not an absolute requirement. However, the prior action of cyclic AMP-dependent protein kinase significantly potentiated the ability of casein kinase I to phosphorylate and inactivate glycogen synthase. All previous analyses of glycogen synthase phosphorylation have used enzyme purified from mammalian sources and containing residual covalent phosphate. By using recombinant substrate, the present study represents a rigorous assessment of the role of prior phosphorylation in the recognition of mammalian glycogen synthase by glycogen synthase kinase 3 and casein kinase I. The conclusion is that phosphorylation of glycogen synthase can involve the concerted action of multiple protein kinases.
Insights
This study reveals how protein kinases regulate glycogen synthase. Prior phosphorylation by casein kinase II is essential for glycogen synthase kinase 3 activity, while cyclic AMP-dependent protein kinase potentiates casein kinase I.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Glycogen synthase is a key enzyme in glycogen biosynthesis, regulated by multisite phosphorylation.
- Previous research suggested that certain protein kinases potentiate the phosphorylation activity of others on glycogen synthase.
Purpose of the Study:
- To investigate the role of prior phosphorylation in the modification of recombinant glycogen synthase by specific protein kinases.
- To rigorously assess the concerted action of multiple protein kinases on glycogen synthase using a purified recombinant enzyme.
Main Methods:
- Active glycogen synthase was expressed in Escherichia coli.
- Purified recombinant glycogen synthase was subjected to phosphorylation by casein kinase II, glycogen synthase kinase 3, cyclic AMP-dependent protein kinase, and casein kinase I, individually and sequentially.
- Enzyme activity and phosphorylation levels were analyzed.
Main Results:
- Prior phosphorylation by casein kinase II was an obligate requirement for glycogen synthase kinase 3 to phosphorylate the enzyme, leading to potent inactivation.
- Casein kinase I alone could phosphorylate glycogen synthase, but prior phosphorylation by cyclic AMP-dependent protein kinase significantly enhanced this activity and inactivation.
- The recombinant enzyme exhibited properties similar to native rabbit muscle glycogen synthase.
Conclusions:
- Phosphorylation of glycogen synthase can involve the coordinated action of multiple protein kinases.
- This study provides a rigorous assessment of kinase interactions using recombinant glycogen synthase, clarifying previous findings based on mammalian-derived enzymes.