1. A glycogen synthase kinase was partially purified from rabbit skeletal muscle by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and chromatography on hydroxyapatite.2. The enzyme was highly specific for glycogen synthase. In the standard assay, the relative rates of phosphorylation were : glycogen synthase (loo), phosvitin (2.5), phosphorylase kinase ( < l), casein (0.3), protein phosphatase inhibitor-I (< ().I), phosphorylase (< 0.01), mixed histones (< 0.01). The enzyme was separated from virtually all phosvitin kinase and casein kinase activity by the chromatography on DEAE-cellulose.3. The K , values for ATP and GTP were 0.02 mM and 0.5 mM respectively, and a similar maximum reaction velocity was obtained with each nucleoside triphosphate. The activity of the enzyme was unaffected by cyclic AMP, cyclic GMP, calcium ions, calcium ions plus calmodulin, and the specific protein inhibitor of cyclic-AMP-dependent protein kinase.4. The properties of the enzyme demonstrated that it was distinct from both cyclic-AMPdependent protein kinase and phosphorylase kinase, the two well characterized glycogen synthase kinases in skeletal muscle. This enzyme was therefore termed glycogen synthase kinase-3.5. The phosphorylation of glycogen synthase by glycogen synthase kinase-3 reached a pleateau near 1.5 molecule phosphate incorporated per subunit under optimal conditions. The activity of glycogen synthase measured in the absence of glucose 6-phosphate was decreased fivefold and the apparent K, for glucose 6-phosphate was increased 15-fold, when 1.2 molecule phosphate per subunit had been introduced into the enzyme. Phosphorylation to a similar extent with either cyclic-AMP-dependent protein kinase or phosphorylase kinase produced smaller changes in activity.6. Glycogen synthase was phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase and glycogen synthase kinase-3, using conditions where the phosphorylation by any one protein kinase reached a plateau near one molecule of phosphate incorporated per subunit. The different protein kinases were used separately and in combination to generate seven different phosphorylated species of glycogen synthase. The phosphorylation of glycogen synthase approached two molecules per subunit when any two protein kinases werc combined, and three molecules per subunit when all three protein kinases were combined, and the inactivation produced by the different protein kinases was essentially additive. The results imply that each protein kinase preferentially phosphorylates a different site(s) on glycogen synthase, and this is confirmed by the amino acid sequence analysis described in the following paper in this journal.

Glycogen synthase has been shown to be phosphorylated in vitro by five protein kinases, cyclic-AMPdependent protein kinase (sites l a , I b and 2). phosphorylase kinase (site 2), glycogen synthase kinase 3 (sites 3a, 3b and 3c), glycogen synthase kinase 4 (site 2) and glycogen synthase kinase 5 (site 5 ) Eur. J. Biochem. 124,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]; Picton, C. et al. (1982) Eur. J. Biocliem. 124,[37][38][39][40][41][42][43][44][45]. All seven serine residues have now been found to be phosphorylated in vivo.Glycogen synthase purified from rabbits that had been injected with L-propranolol contained 0.33 mol of phosphate/mol subunit in site la, 0.38 in site Ib, 0.39 in site 2, 1.27 in sites 3a + 3b + 3c and 0.65 in site 5, totalling 3.0 mol/mol subunit. The activity ratio (i glucose-6 P) for these preparations was 0.21 and their K, for glucose-6-P was 1.4 mM. Glycogen synthase purified from animals that had been injected with I-adrenaline contained 0.59 niol phosphate/mol subunit in site la, 0.65 in site lb, 1.03 in site 2, 2.43 in sites 3a + 3b + 3c and 0.65 in site 5 , totalling 5.3 mol/mol subunit. The activity ratio of these preparations was 0.04 and their Ka for glucose-6-P was 6.0 mM. The results show that cyclic-AMP-dependent protein kinase, glycogen synthase kinase 3 and glycogen synthase kinase 5 act as glycogen synthase kinases in vivo. Glycogen synthase kinase 4 may be largely responsible for the phosphorylation of site 2 in propranolol-treated animals, and cyclic-AMP-dependent protein kinase and phosphorylase kinase for the increased phosphorylation of site 2 in response to adrenaline.The activity of glycogen synthase in vivo in propranolol-treated animals is largely determined by the state of phosphorylation of sites 3a, 3b and 3c, while the inactivation in response to adrenaline is due to increased phosphorylation at site 2 as well as at sites 3a, 3b and 3c. The finding that the phosphorylation of sites 3a, 3b and 3c is increased from 1.27 mol/mol subunit to 2.43 mol/mol subunit by adrenaline was unexpected, since this enzyme is not regulated by cyclic AMP. Possible explanations for this result are discussed.Phosphofructokinase was isolated as a byproduct of the purification of glycogen synthase. The phosphate content of this enzyme did not differ significantly between L-propranolol-treated (0.15 moljmol subunit) and adrenaline-treated (0.18 mol/mol subunit) animals. These results indicate that muscle phosphofructokinase is not a substrate for cyclic-AMP-dependent protein kinase in vivo.Glycogen synthase, the rate-limiting enzyme in glycogen synthesis is under hormonal control; its activity in muscle decreases in response to adrenaline [1,2] and increases in response to insulin [1,2]. These hormones alter the activity in the absence of glucose-6-P, but not in the presence of this allosteric effector. These changes in the activity ratio ( glucose-6-P) are consistent with the idea that adrenaline increases and insulin decreases the extent of phosphorylation of glyc...

Homogeneous preparations of phosphorylase kinase from rabbit skeletal muscle catalyse a calcium-dependent phosphorylation of glycogen synthase a isolated from the same tissue. The calcium-dependent glycogen synthase kinase activity copurifies with phosphorylase kinase throughout the standard procedure for the isolation of the latter enzyme. At the final step of the purification, gel filtration on Sepharose 4B, the elution profiles for glycogen synthase kinase and phosphorylase kinase activities are identical. ICR/IAn mice, which lack muscle phosphorylase kinase activity, do not contain detectable calcium-dependent glycogen synthase kinase activity. These results indicate that the calcium-dependent phosphorylation of glycogen synthase is catalysed by phosphorylase kinase and not by another calcium-dependent protein kinase that might be contaminating the preparation.The phosphorylation of glycogen synthase a by phosphorylase kinase reaches a plateau in the range 0.73 0.1 molecules of phosphate incorporated per subunit and is accompanied by a 2-fold decrease in the activity in the absence of glucose 6-phosphate. The phosphorylation takes place on a unique serine residue located seven amino acids from the N-terminus of the polypeptide chain. The amino acid sequence surrounding serine-7 is similar to the amino acid sequence surrounding the phosphoserine in phosphorylase a.Glycogen synthase kinase-2 is the name given to a protein kinase present as a trace contaminant in highly purified preparations of glycogen synthase [Nimmo, H. G. and Cohen, P. (1974) FEBS Lett. 47, 162 -1671. Glycogen synthase kinase-2 also phosphorylates serine-7 exclusively, and evidence is presented which demonstrates that glycogen synthase kinase-2 is merely a modified form of phosphorylase kinase which has lost its ability to be regulated by calcium ions at pH 6.8.The rate of phosphorylation of glycogen synthase a by phosphorylase kinase is 2 -3-fold slower than the rate of phosphorylation of phosphorylase h when identical concentrations of the two protein substrates are used (6 pM). At physiological concentrations of glycogen synthase (0.3 mg/ml) and phosphorylase (8.0 mgiml), the time required for half-maximal phosphorylation of each enzyme by phosphorylase kinase is similar. These results suggest that the phosphorylation of glycogen synthase by phosphorylase kinase may be physiologically significant, and the implications of these findings are considered.

Glycogen synthase preparations which were free of endogenous phosphorylase kinase were phosphorylated to different extents with cyclic-AMP-dependent protein kinase and the phosphopeptides obtained by tryptic digestion were analysed. Limited tryptic digestion of the native enzyme quantitatively released two phosphopeptides (site 1 a and site 1 b) which were soluble in trichloroacetic acid, The trichloroacetic-acid pellet contained a distinct phosphorylation site, which was shown to be identical to the serine residue which is phosphorylated by phosphorylase kinase (site 2).Sites l a , 1 b and 2 accounted for virtually all of the phosphorylation of glycogen synthase up to 2.5 molecules phosphate incorporated per subunit. Site 1 a was initially phosphorylated 7-10-fold faster than site 2, and 15-20-fold faster than site l b . The phosphorylation of site l a became much slower after about 0.5 molecules of phosphate had been incorporated per subunit.Cyclic-GMP-dependent protein kinase also phosphorylated glycogen synthase at sites 1 a, 1 b and 2. The order of phosphorylation was the same as that observed with cyclic-AMP-dependent protein kinase (site 1 a > site 2 > site 1 b).All three sites phosphorylated by cyclic-AMP-dependent protein kinase could be dephosphorylated by protein phosphatase 1. Site 2 was initially dephosphorylated 5 -10-fold faster than site 1 a and more than 100-fold faster than site 1 b. The MgATP-dependent protein phosphatase dephosphorylated site 2, site 1 a and site 1 b in a very similar manner to protein phosphatase 1.The different rates of phosphorylation and dephosphorylation of the three sites allowed the demonstration to be made that the activity of glycogen synthase depended on the state of phosphorylation of site 2 and site 1 a. The phosphorylation of site 1 b did not influence the activity under the conditions used.Incubation of glycogen synthase with high levels of phosphorylase kinase did not lead to the phosphorylation of either site 1 a or 1 b, or of the three sites phosphorylated by glycogen synthase kinase 3 (sites 3a, 3b and 3c). Similarly no phosphorylation of sites 3a, 3b or 3c by cyclic-AMP-dependent protein kinase was detected. It is concluded that site 2 is the only site at which overlapping substrate specificity occurs between these three glycogen synthase kinases.Glycogen synthase can exist as a dephosphorylated form of high activity, or as less active phosphorylated forms which require glucose 6-phosphate for activity [l, 21. Over the past few years several protein kinases have been identified which can decrease the activity of glycogen synthase in vitro [3 -191; which of these enzymes are important in regulating the activity of glycogen synthase in vivo has not yet been established.The first glycogen synthase kinase to be identified was cyclic-AMP-dependent protein kinase [20,21]. At first it was reported that this enzyme phosphorylated glycogen synthase to the extent of about 1 molecule per subunit, and that the activity ratio of the enzyme (defined as the activity in...

Curcumin, a bioactive compound in Curcuma longa, exhibits various pharmacological activities, including antimalarial effects. In silico docking simulation studies suggest that curcumin possesses glycogen synthase kinase-3β (GSK3β)-inhibitory properties. The involvement of GSK3 in the antimalarial effects in vivo is yet to be demonstrated. In this study, we aimed to evaluate whether the antimalarial effects of curcumin involve phosphorylation of host GSK3β. Intraperitoneal administration of curcumin into Plasmodium berghei NK65-infected mice resulted in dose-dependent chemosuppression of parasitemia development. At the highest dose tested (30 mg/kg body weight), both therapeutic and prophylactic administrations of curcumin resulted in suppression exceeding 50% and improved median survival time of infected mice compared to control. Western analysis revealed a 5.5-fold (therapeutic group) and 1.8-fold (prophylactic group) increase in phosphorylation of Ser 9 GSK3β and 1.6-fold (therapeutic group) and 1.7-fold (prophylactic group) increase in Ser 473 Akt in liver of curcumin-treated infected animals. Following P. berghei infection, levels of pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4 were elevated by 7.5-, 35.0-, 33.0-, and 2.2-fold, respectively. Curcumin treatment (therapeutic) caused a significant decrease (by 6.0- and 2.0-fold, respectively) in serum TNF-α and IFN-γ level, while IL-10 and IL-4 were elevated (by 1.4- and 1.8-fold). Findings from the present study demonstrate for the first time that the antimalarial action of curcumin involved inhibition of GSK3β.