Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a).
Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.
Wang
Y
Y
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Roach
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D047388:Casein Kinases; D018076:DNA, Complementary; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D012694:Serine / Q000378:metabolism*
D018076:DNA, Complementary; D008869:Microtubule-Associated Proteins; D012694:Serine; D006006:Glycogen Synthase; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3