Stimulation of site-specific phosphorylation of acetyl coenzyme A carboxylase by insulin and epinephrine

32P-labeled acetyl-CoA carboxylase was isolated from 32P-labeled rat epididymal fat pads by avidin-Sepharose affinity chromatography after exposure to epinephrine and insulin. Epinephrine led to an inactivation of the isolated enzyme by a reduction of Vmax, while the insulin stimulation observed in crude extracts did not survive enzyme purification. Both insulin and epinephrine caused only small increases in total 32P content of the enzyme. However, mapping of tryptic 32P-phosphopeptides by high performance liquid chromatography revealed that epinephrine and insulin stimulated the phosphorylation of 32P-peptides specific for each hormone. The major 32P-peptide phosphorylated by epinephrine co-migrated with the major 32P-peptide phosphorylated in vitro by the cAMP-dependent protein kinase, while the 32P-peptide phosphorylated in response to insulin co-migrated with that phosphorylated by casein kinase-I and casein kinase-II. The effects of epinephrine on carboxylase activity and phosphorylation can thus be accounted for by the expected epinephrine-induced activation of the cAMP-dependent protein kinase. While the increase in site-specific phosphorylation caused by insulin cannot be directly linked to insulin-induced activation in crude extracts, these data suggest that casein kinase-I and/or casein kinase-II may mediate the insulin-stimulated phosphorylation of acetyl-CoA carboxylase.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Threonine phosphorylation of rat liver glycogen synthase

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.     

Phosphorylation site specificities of glycogen synthase kinases: determination by peptide mapping using high-performance liquid chromatography

A method is described which separates the various phosphorylation sites in glycogen synthase based on reverse phase high-performance liquid chromatography (HPLC) of tryptic 32P-peptides. Using this method we studied the phosphorylation site specificities of the kinases which act on glycogen synthase. The cAMP-dependent protein kinase phosphorylated sites 1a, 1b, and 2, whereas casein kinase II phosphorylated only site 5. Two calcium, calmodulin-dependent kinases, phosphorylase kinase and liver calmodulin-dependent synthase kinase, both phosphorylated site 2, and the latter enzyme also phosphorylated site 1b. A cAMP-independent kinase (kinase 4) purified from liver also specifically phosphorylated site 2. Synthase kinase 3 catalyzed the phosphorylation of only site 3. This HPLC method was also used to establish that all of these sites were subject to phosphorylation in vivo.     

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.     

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.     

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.     

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.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.     

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.     

Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.     

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.     

Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation

Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.     

Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.     

Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Cyclic AMP-dependent protein kinase-induced vimentin filament disassembly involves modification of the N-terminal domain of intermediate filament subunits

The intermediate filament protein vimentin was phosphorylated with cAMP-dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine-specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N-terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32P-labeled species representing amino acid position 14-72, and a minor 32P-labeled peptide representing amino acid positions 1-13. These results demonstrate that phosphorylation of sites within the N-terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.     

Hormonal control of glycogen synthase in rat hemidiaphragms. Effects of insulin and epinephrine on the distribution of phosphate between two cyanogen bromide fragments

The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (Mapp = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 microM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The larger fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P] phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the 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.     

Insulin resistance in denervated skeletal muscle. Inability of insulin to stimulate dephosphorylation of glycogen synthase in denervated rat epitrochlearis muscles

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.