Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Effect of limited proteolysis on activity and phosphorylation of rabbit muscle glycogen synthetase.

Purified rabbit skeletal muscle glycogen synthetase, in both the glucose-6-phosphate (P)-dependent (phosphorylated) and the glucose-6-P-independent (dephosphorylated) forms, was subjected to limited proteolysis by trypsin. Both forms could be degraded from their original subunit molecular weight of 85,000 to 76,000 and subsequently to 68,000, as determined with acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Degradation of the glucose-6-P-dependent form of the enzyme resulted in essentially no change in the activity when measured either in the presence or in the absence of glucose-6-P. Degradation of the glucose-6-P-independent form was associated with a progressive increase in glucose-6-P dependency. Phosphorylation of the glucose-6-P-independent form with the adenosine 3′,5′-monophosphate-dependent protein kinase and subsequent digestion of the ³²P-labeled enzyme showed that the phosphate group was retained on these subunits. The protein kinase phosphorylated both the original subunit with molecular weight 85,000 and the partially digested subunit with molecular weight 76,000. Upon further digestion of the enzyme into a form having a subunit molecular weight of 68,000, the enzyme was unable to accept a phosphate group from ATP. By contrast with the phosphorylation reaction, the dephosphorylation reaction catalyzed by partially purified glycogen synthetase phosphatase is not stringent in terms of structural integrity of the synthetase. The phosphatase dephosphorylated the glucose-6-P-dependent form of glycogen synthetase equally well at various degrees of degradation.     

Characteristics of glycogen synthetase I from rabbit skeletal muscles]

Electrophoretic heterogeneity of glycosynthetase I from rabbit skeletal muscles is observed. Multiple glycosynthetase forms are separated in sucrose density gradient, their molecular weights are estimated. The existence of the enzyme as an equilibrium system of oligomeric forms, capable of reversible association-dissociation, is demonstrated. Dissociating effect of ATP, high pH values (11--12) and high ionic strength (2 M KCl) on oligomers of glycogen synthetase I is found to take place. Different activity of oligomers of different association degree is observed.     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes

Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase.     

Regulation of glycogen synthetase. Specificity and stoichiometry of phosphorylation of the skeletal muscle enzyme by cyclic 3':5'-AMP-dependent protein kinase.

Complete conversion of skeletal muscle glycogen synthetase from the I form to the D form requires incorporation of 2 mol of phosphate per enzyme subunit (90,000 g). Incubation of sythetase I with low concentrations of adenosine 3':5'-monophosphate(cAMP)-dependent protein kinase (10 units/ml) and ATP (0.1 to 0.3 mM) plus magnesium acetate (10 mM) results in incorporation within 1/2 hour of 1 mol of phosphate persubunit concomitant with a decrease in the synthetase activity ratio (minus glucose-6-P/plus glucose-6-P) from 0.85 to 0.25. Further incubation for 6 hours does not greatly increase the phosphate content of the synthetase or promote conversion to the D form. This level of phosphorylation is not increased by raising the concentration of protein kinase to 150 units/ml and is not influenced by the presence of glucose-6-P, UDP-glucose, or glycogen. However, at protein kinase concentrations of 10,000 to 30,000 units/ml a second mol of phosphate is incorporated per subunit, and the sythetase activity ratio decreases to 0.05 or less. In addition to the 2 mol of phosphate persubunit which are required for formation of sythetase D, further phosphorylation can be observed which is not associated with changes in synthetase activity. This phosphorylation occurs at a slow rate, is increased by raising the ATP concentration to 2 to 4mM, and is not blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. These data indicate that skeletal muscle glycogen synthetase contains multiple phosphorylation sites only two of which are involved in the synthetase I to D conversion.     

Comparative purification and characterization of invertebrate muscle glycogen synthase from the porcine parasite Ascaris suum

Glycogen synthase has been purified from the obliquely striated muscle of the swine parasite Ascaris suum. The muscle contains a concentration of glycogen synthase and glycogen which is 20-fold and 15-fold, respectively, greater than rabbit skeletal muscle. The enzyme could not be solubilized with salivary amylase, but partial solubilization was achieved by activation of endogenous phosphorylase. The enzyme was purified to 85-90% homogeneity (specific activity = 4.3 units/mg) by DEAE-cellulose, Sepharose 4B, and glucosamine 6-phosphate chromatography. The purified glycogen synthase was substantially similar to rabbit skeletal muscle enzyme with respect to Mr (gel electrophoresis and gel filtration), pH dependence, aggregation properties, temperature dependence, and kinetic constants for substrates and activators. Glycogen synthase I was converted to glycogen synthase D by the cyclic AMP-dependent protein kinase. The cyclic AMP-dependent protein kinase catalyzed the incorporation of 1.3 mol of phosphate into each glycogen synthase I subunit and the concomitant interconversion to glycogen synthase D. Since glycogen is the sole fuel utilized by this organism during nonfeeding periods of the host, the characterization of this enzyme provides further insight into the regulatory mechanisms which determine glycogen turnover.     

Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin

Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38-kDa component was separated from the 86-kDa catalytic subunit by gel filtration in the presence of 2 M LiBr, and a number of chymotryptic peptides were sequenced. This demonstrated that the 38-kDa subunit was glycogenin, the protein that is bound covalently to glycogen and believed to be the 'primer' involved in the initiation of de novo glycogen synthesis.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase

The phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase I is markedly enhanced if the enzyme has previously been phosphorylated by cAMP-dependent protein kinase. The presence of phosphate in the primary cAMP-dependent protein kinase sites, sites 1a, 1b, and 2 (serine 7), increases the activity of casein kinase I toward residues in the vicinity of these sites. This synergistic phosphorylation correlates with potent inactivation of the glycogen synthase. Analysis of the NH2 terminus of the enzyme subunit indicated that phosphorylation at serine 7 caused serine 10 to become a preferred casein kinase I site and that phosphoserine can be an important recognition determinant for casein kinase I. This finding can also explain how epinephrine stimulation of skeletal muscle provokes significant increases in the phosphorylation state of serine residues, in particular serine 10, not recognized by cAMP-dependent protein kinase.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

The role of phosphorylase kinase subunits in interaction with glycogen

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

Identity of long-chain acyl-coenzyme A synthetase of microsomes, mitochondria, and peroxisomes in rat liver

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.     

Variation of phosphorylase distribution in skeletal muscles

Summary Glycogen phosphorylase, glycogen alpha-4 UDP-glucosyl transferase, glycogen, and some enzymes were histochemically examined in rat skeletal muscles. Phosphorylase activity was abundantly demonstrated not only in large fibers of the white muscle, but also in small red fibers of soleus muscle and those in the deep fascicles of gastrocunemius and quadriceps femoris muscles. Small fibers with high phosphorylase activity did not always revealed high LDH activity.Native glycogen was abundant mostly in small fibers or in middlesized fibers. Neither glycogen synthetase, nor glycogenolytic enzyme activity was directly proportionate to native glycogen content.On Leave from Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan.     

Antibodies and autoantibodies of glycogen phosphorylase b: inactivation of pig and rabbit enzymes.

Pig skeletal muscle glycogen phosphorylase b was purified using ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sephadex G-200 column chromatography. The purified enzyme was used to immunize rabbits in the presence or in the absence of complete Freund adjuvant. Antibodies against pig phosphorylase in pure form were isolated from rabbit antisera using insoluble immunoadsorbents of pig phosphorylase. Autoantibodies against the rabbit enzyme were obtained from the same antisera using insoluble immunoadsorbents of rabbit phosphorylase. Complete inactivation of pig phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 4 and autoantibody/enzyme molar ratio equal to 130. Complete inactivation of rabbit phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 250 and autoantibody/enzyme molar ratio equal to 160. Passive haemagglutination technique gave positive results with minimum amounts of 0.02 microng/ml and 0.8 microng/ml for pig and rabbit phosphorylase respectively. Kinetic experiments have shown that antibodies and autoantibodies act as noncompetitive inhibitors of both enzymes with respect to AMP and glucose 1-phosphate but exhibit a mixed type of inhibition with respect to glycogen. When glycogen hydrolysates were used as substrate in place of intact glycogen molecules a pronounced decrease in the inhibitory capacity of antienzyme on the enzyme was demonstrated.     

Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit

Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes.     

Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4

Glycogen synthase I (EC 2.4.1.11) from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37) to the extent of 0.8 phosphates/subunit. For both phosphorylated enzymes, the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was 0.8 when determined with low concentrations of glycogen synthase and/or short incubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4 is an increased sensitivity to UDP inhibition.