Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a).

Y Wang,P.J Roach

doi:10.1016/s0021-9258(20)80467-0

Abstract

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.

Highlights

Rabbit skeletal muscleglycogen synthase, a rate- have been cloned and corresponding primary sequences have limitingenzyme for glycogen biosynthesis, is regulated beendeduced
Howthe -I+ glucose-6-Pactivity ratio to0.1 and increasing ever, in all previous analyses, it was impossibleto assign the the glucose-6-P concentration needed for half-maxi- relative contributions of sites 3a, 3b, 3c, and 4 to the inactimal activation by an orderof magnitude
Phorylation of site 5 by casein kinase I1 was an absolute requirement for GSK-3 to phospho~latseites 4, 3c, 3b, and

Summary

Ncol Asull I cDNA

Sac I EcoRl I and 1 mg of glycogen/ml. The cell suspension was passed through a French pressure cell twice at 12000 p.s.i. Enzyme solution (30 pl) was incubated 10 min at 30 'C, with 60 pl of assay mixture containing 50 mM Tris-HCl, pH 7.8,25 mM synthase purified from rabbit muscle, it did not permit dis- KF, 20 mM EDTA, 1% glycogen, and 6.67 mM UDP-['4C]glucose section of the relative roles of sites 3a, 3b, 3c, and 4 in the (specific activity 200 cpm/nmol), with or without 7.2 mM glucose-6-. After of activity is defined as theamount of enzyme that transfers 1 nmol digestion with SacII/EcoRI, the PCR product was purified by agarose of phosphate/min into a phosphorylated peptide gel electrophoresis and was used to replace the corresponding wild (KRREILSRRPS(P)YR) based on the sequence of the CAMP retype sequence in the pTRC-GSexpression vector. Glycogen Synthuse-Expression and purification of the recombinant glycogen synthase was by a modification of the method of Zhang et al [18].E. coli cells, strain RR1, were transformed with the pTRC-

Analysis of Wild Type andMutated Forms of Glycogen

Wild type

Phosphorylation of the Wild Type andMutant Glycogen

DISCUSSION

Glycogen synthase