Multiple phosphorylation of rabbit skeletal muscle glycogen synthase. Evidence for interactions among phosphorylation sites and the resolution of electrophoretically distinct forms of the subunit

Phosphorylation of rabbit skeletal muscle glycogen synthase by a cyclic nucleotide and Ca2+-independent protein kinase, PC0.7, caused the enzyme to be a better substrate for phosphorylation by another cyclic nucleotide and Ca2+-independent protein kinase, FA/GSK-3. In contrast, phosphorylation by the combination of FA/GSK-3 and cyclic AMP-dependent protein kinase led to less phosphorylation than predicted from the individual actions of the protein kinases. These results are explained in part by the existence of cooperative interactions among the phosphorylation sites of glycogen synthase. Phosphorylation by FA/GSK-3 also correlated with a reduction in the electrophoretic mobility, in the presence of sodium dodecyl sulfate, of the glycogen synthase subunit from an apparent molecular weight of 85,000-86,000 to values of 88,000 and ultimately 90,000. The synergistic phosphorylation by PC0.7 and FA/GSK-3 was associated with an increased formation of the species of reduced electrophoretic mobility. The effects on subunit mobility were also reflected in the behavior of a larger phosphorylated CNBr fragment of glycogen synthase, CB-2, which gave apparent molecular weights of 22,000-27,000 depending on its phosphorylation state.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol³²P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,³²P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

Role of protein kinase C in the regulation of rat liver glycogen synthase

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca2+- and phospholipid-dependent fashion to 1-1.4 mol PO4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3

The glycogen-binding (G) subunit of protein phosphatase-1G is phosphorylated stoichiometrically by glycogen synthase kinase-3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP-dependent protein kinase (A-kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS-, while the C-terminal serine (site 1) is one of the two residues phosphorylated by A-kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C-terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase-2.     

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.     

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium/phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - PDPK proline-dependent protein kinase     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.     

Ca2+, calmodulin-dependent phosphorylation, and inactivation of glycogen synthase by a brain protein kinase

Glycogen synthase from skeletal muscle was phosphorylated by a Ca2+, calmodulin-dependent protein kinase from brain, with concomitant inactivation. About 0.7 mol phosphate/mol subunit was sufficient for a maximal inactivation of glycogen synthase. Further phosphorylation of the enzyme had no effect on the activity. The concentrations required to give half-maximal phosphorylation and inactivation of glycogen synthase were 1.1 and 0.5 microM for Ca2+, and 22 and 11 nM for calmodulin, respectively. The molar ratio of the subunit of the protein kinase to calmodulin was 2-3:1 for half-maximal phosphorylation and inactivation of glycogen synthase. The Km values for glycogen synthase and ATP were 3.6 and 114 microM, respectively, for phosphorylation. Phosphate was incorporated into sites Ia, Ib, and 2 on glycogen synthase, and site 2 was the most rapidly phosphorylated. These results indicate that the brain Ca2+, calmodulin-dependent protein kinase is probably involved in glycogen metabolism in the brain as a glycogen synthase kinase.     

Cyclic AMP-independent casein/glycogen synthase kinases from pig polymorphonuclear leucocytes

1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein-Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the K(m) for casein (0.46mg/ml) and K(a) for Mg(2+) (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, (32)P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of (32)P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of (32)P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10-15 with casein kinase 1 and to 35-45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca(2+) and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca(2+). 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50 degrees C.     

Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.     

Purification and properties of cyclic AMP-independent glycogen synthase kinase 1 from rabbit skeletal muscle.

A cyclic AMP-independent casein (phosvitin) kinase eluted from a phosphocellulose column with 0.35 M KCl also possesses glycogen synthase kinase activity. This kinase, designated synthase kinase 1, is separable from other cyclic AMP-independent protein kinases, which also contain glycogen synthase kinase activity, by chromatography on a phosphocellulose column. This kinase was purified 15,000-fold from the crude extract. Synthase kinase activity co-purifies with casein and phosvitin kinase activities. Heat inactivation of these three kinase activities follow similar kinetics. It is suggested that these three kinase activities reside in a single protein. This kinase has a molecular weight of approximately 34,000 as determined by glycerol density gradient centrifugation and by gel filtration. The Km values for the synthase kinase-catalyzed reaction are 0.12 mg/ml (0.35 micronM) for synthase, 12 micronM for ATP, and 0.15 mM for Mg2+. The phosphorylation of glycogen synthase by the kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however, only two of the phosphate sites predominantly determine the glucose-6-P dependency of the synthase. Synthase kinase activity is sensitive to inhibition by NaCl or KCl at concentrations encountered during purification. Synthase kinase activity is insensitive to the allosteric effector (glucose-6-P) or substrate (UDP-glucose) of glycogen synthase at concentrations usually found under physiological condition.     

Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.     

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.     

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with ³²P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [γ-³²P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of M_r 85,000 and 80,000 were present in varying proportions. The ³²P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the homogeneous synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.     

Total conversion of glycogen synthase from the I- to the D-form by a cyclic AMP-independent protein kinase from rabbit skeletal muscle.

A newly discovered cyclic AMP-independent protein kinase, which catalyzes the total conversion of glycogen synthase from the I- to the D-form, has been isolated from rabbit skeletal muscle. This enzyme, designated glycogen synthase kinase, is separable from cyclic AMP-dependent protein kinase by column chromatography on phosphocellulose. Synthase kinase and cyclic AMP-dependent protein kinase are distinct in their specificity for protein substrates, the effects of cyclic AMP and the inhibitor of cyclic AMP-dependent protein kinase on their activities, and the extent to which they phosphorylate I-form glycogen synthase. The phosphorylation of I-form enzyme by synthase kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however only two of the phosphate sites seem predominantly to determine glucose-6-P dependence. The resulting multiply phosphorylated enzyme, which is highly dependent on glucose-6 P for activity, has a phosphate content comparable to the D-form enzyme isolated from rabbit muscle.