Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].     

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle: Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3

Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase     

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When ³²P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.     

Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II

Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.     

Identification of the C-terminus of rabbit skeletal muscle glycogen synthase

The primary structure of a tryptic peptide containing one of the phosphorylation sites on rabbit skeletal muscle glycogen synthase (site 1b) has been redetermined and shown to correspond to the C-terminus of the protein. The sequence is: -SNSVDTSSLSTPSEPLSSAPSLGEERN.     

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.     

Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase

Glycogen synthase kinase-3 phosphorylates three serine residues on glycogen synthase (sites 3a, 3b and 3c) which are all located in the same nine-amino-acid segment of the polypeptide chain. The sequence in this region is: Arg-Tyr-Pro-Arg-Pro-Ala-Ser(P)-Val-Pro-Pro-Ser(P)-Pro-Ser-Leu-Ser(P)-Arg-. These serine residues are distinct from the sites phosphorylated preferentially by cyclic-AMP-dependent protein kinase (sites 1a and 1b) and phosphorylase kinase (site 2). The N-terminal sequence of glycogen synthase containing the serine residue phosphorylated by phosphorylase kinase has been extended. The sequence in this region is: Pro-Leu-Ser-Arg-Thr-Leu-Ser(P)-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu-Asp-Trp-Glu-Asp- Glu-Phe-Asp-Leu-Glu-Asn-Ser-Val-Leu-Phe-(Asx2,Glx2,Ala2,Val2,Lys)-. The similarity to the N-terminal sequence of phosphorylase is confined to the immediate vicinity of the phosphorylation site (residues 4--15). The relationship of glycogen synthase kinase-3 to glycogen synthase kinases that have been described by other laboratories is discussed.     

Dephosphorylation of rabbit skeletal muscle glycogen synthase (phosphorylated by cyclic AMP-independent synthase kinase 1) by phosphatases

Phosphorylation of rabbit skeletal muscle glycogen synthase by cyclic AMP-independent synthase kinase 1 results in the incorporation of 4 mol of PO4/subunit. Incubation of the phosphorylated synthase with rabbit muscle phosphoprotein phosphatase brings about the hydrolysis of phosphates from all four major tryptic peptides and an increase in the synthase activity ratio from 0.01 to 0.85. Incubation of the phosphorylated synthase with calf intestinal alkaline phosphatase brings about the preferential hydrolysis of phosphates from three of the four major tryptic peptides and a slight increase in the four major tryptic peptides and a slight increase in the synthase activity ratio from 0.01 to 0.1. The phosphorylation site which is resistant to hydrolysis by calf intestinal alkaline phosphatase can be dephosphorylated by subsequent incubation with rabbit muscle phosphoprotein phosphatase. This dephosphorylation is accompanied by an increase in the synthase activity ratio to approximately 0.9. Measurements of the changes in the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase reveal that the phosphorylation sites susceptible to hydrolysis by alkaline phosphatase mainly affect the binding of glucose-6-P to the synthase. Comparison of the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase and by phosphoprotein phosphatase we find that the phosphorylation site resistant to hydrolysis by alkaline phosphatase affects both the binding of UDP-glucose and glucose-6-P to the synthase.     

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.     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

Identification of the phosphorylated sites of phosphofructokinase from skeletal muscle after in vivo and in vitro phosphorylation.

Phosphofructokinase from mice muscle was radioactively labelled either in vivo by the injection of [³²P]-phosphate or in vitro by the incubation with cAMP-dependent protein kinase and [γ-³²P]-ATP. Two labelled peptides were obtained after tryptic digestion in either case showing that at least two sites were phosphorylated. Independent of the labelling method, the labelled peptides showed an analogous pattern on the peptide maps, indicating that both methods led to the phosphorylation of the same sites.     

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.     

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.     

Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry

The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.