Mechanisms of multisite phosphorylation and inactivation of rabbit muscle glycogen synthase.
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.
Zhang
W
W
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
DePaoli-Roach
A A
AA
Roach
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047388:Casein Kinases; D003001:Cloning, Molecular; D006006:Glycogen Synthase / Q000378:metabolism*; D038341:Glycogen Synthase Kinases; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006006:Glycogen Synthase; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases