Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycogen synthase was purified to near homogeneity from rat skeletal muscle, and was found to resemble the rabbit skeletal muscle enzyme in several respects. An apparent molecular weight (Mapp) of 86,000 was estimated from the electrophoretic mobility of the subunit on polyacrylamide gels in the presence of sodium dodecyl sulfate. Limited proteolysis of the rat synthase with trypsin resulted in the formation of species with MappS equal to 75,000, 69,000, and 67,000. The enzyme could be phosphorylated by cAMP-dependent protein kinase, phosphorylase kinase, and the cAMP-independent protein kinases, PC0.7 and FA/GSK-3. Essentially all of the phosphorylation observed occurred on serines located in two cyanogen bromide fragments, denoted CB-1 (Mapp = 13,000) and CB-2 (Mapp = 22,000). FA/GSK-3 and cAMP-dependent protein kinase phosphorylated sites in both fragments. Phosphate introduced by phosphorylase kinase was located exclusively in CB-1, and that incorporated with PC0.7 was found in CB-2. Phosphorylation by FA/GSK-3 reduced the electrophoretic mobility of the subunit, introduced heterogeneity into CB-2, and was synergistic with phosphorylation by PC0.7. To separate phosphorylation sites more completely, samples of glycogen synthase were subjected to extensive proteolysis using trypsin, followed by reverse-phase liquid chromatography. When phosphorylated by the same kinases, the pattern of fragments obtained with rat and rabbit skeletal muscle glycogen synthase were almost identical. The results presented provide strong evidence that the subunit of rat skeletal muscle glycogen synthase has at least five phosphorylation sites that are very similar, if not identical, to sites present on the rabbit muscle enzyme.
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PMID:Rat skeletal muscle glycogen synthase: phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases. 298 12

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.
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PMID:Threonine phosphorylation of rat liver glycogen synthase. 299 12

Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK-3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F-II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport-independent pathway seems to involve a redistribution of phosphate in the synthase subunit.
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PMID:Control of glycogen synthase by insulin and isoproterenol in rat adipocytes. Changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation. 300 Oct 75

The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose-6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2-e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide.
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PMID:Phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1). Stoichiometry and distribution of the phosphorylation sites on the glycogen synthase subunit. 301 47

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.
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PMID:Multiple phosphorylation sites of rat liver glycogen synthase. 309 Oct 84

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 reverse-phase 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.
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PMID:Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: evidence of phosphorylation sites specific for casein kinase 1. 309 10

A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.
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PMID:Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I. 392 73

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speed gel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (CB1, Mr = 32,800 and CB2, Mr = 20,400). Sequence studies showed that the alignment of these two peptides was CB1 - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by cAMP-dependent protein kinase was found to be located on the CB1 peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenous or, at least, very similar in their primary structure.
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PMID:Studies on the primary structure of rat liver phenylalanine hydroxylase. 397 94

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.
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PMID:Multiple phosphorylation of rabbit skeletal muscle glycogen synthase. Evidence for interactions among phosphorylation sites and the resolution of electrophoretically distinct forms of the subunit. 630 12

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.
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PMID:Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme. 632 24


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