Abstract
Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC0.7, FA/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, +/- glucose-6-P activity ratio reduced from approximately 1 to 0.6) or FA/GSK-3 (to approximately 1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination FA/GSK-3 plus PC0.7 was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reductions in the Vmax of the glycogen synthase with little effect on the S0.5 (substrate concentration corresponding to Vmax/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC0.7 acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent Mr = 12,300 (CB-I) and 16,000-17,000 (CB-II). PC0.7 and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-I. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.
Highlights
Purified rabbit livegrlycogen synthase wasfound to glycogen synthase (EC 2.4.1.11)
Phosphorylation by the combination FA/GSK-3 been studied invarious contexts besides glycogen synthase plus PC,., was synergistic, and more extensive inacti- phosphorylation,and most likely correspond to the casein vation was achieved
Activity-phosphorylation relationships for liver synthase was complicated by the inhibitoryeffect of glycogen glycogen synthase
Summary
The phosphorylation reactions kinase I and casein kinase I1 categories as reviewed recently just described caused significant reductions in tVh,e,, by Hathaway and Traugh (20) Anotheenrzyme of this group, of the glycogen synthase with little effect on theSo.[5] designated here FA/GSK-3, was purified by Hemmings et al. Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. To determine the dependence of muscle enzyme diluted in the above buffer were used for studies of activity on UDP-glucose concentration, in the absence of glucose-6- casein kinase-1 action (see under "Results"). Tris-HC1, mM EDTA, 0.6 mM EGTA, mM KF,[15] mM P- After standingfor 30 min, the precipitate was recovered by centrifumercaptoethanol, 10.5 mg/ml of rabbit liver glycogen, 0.0375 to 75 gation and dissolved in 70% (v/v) formic acid.Excess CNBr was mM UDP-[U-'4C]glucose (specific activity, 120-8,500 cpm/nmol, in- added, normally for 3 h, and the solution was evaporated to dryness creasing withlower concentrations). One was precipitated with trichloroacetic acidto besubjected to CNBrcleavage as described above
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