Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.
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PMID:Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene. 212 85

Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.
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PMID:Molecular cloning and expression of glycogen synthase kinase-3/factor A. 216 70

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.
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PMID:Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control. 249 21

Protein phosphorylation is the most prevalent covalent reversible modification used for the regulation of many biological functions. The dynamic phosphorylation state of cellular proteins results from the balance between the activities of protein kinases and phosphatases. Even autophosphorylation, shared by practically all protein kinases, is also considered as a mode of enzyme regulation. Phosphodephosphorylation reactions seem also to be regulated at the substrate level, and the structure of a target protein can be strongly altered through phosphorylation in a way which may either promote or depress the further action of a protein kinase or phosphatase. Second-site phosphorylation may have various consequences on the kinetic properties of the enzyme-substrate. Glycogen synthase is possibly one of the most complex examples of enzyme regulation through interactions between phosphorylation sites. An interesting example of regulation at the substrate level in a network protein phosphorylations, is provided by the effects of casein kinase-1 (CK-1), casein kinase-2 (CK-2) and protein kinase FA (PKFA) on the modulator protein of the ATP, Mg-dependent phosphatase. The crucial step in the activation of the enzyme is the transient phosphorylation of the modulator at the Thr-72 residue catalyzed by PKFA. The CK-2 mediated phosphorylation occurs at three Ser residues: Ser-86, Ser-120 and Ser-121. Although by itself CK-2 does not elicit any phosphatase activity, it potentiates the subsequent activation brought about by PKFA. The phosphorylation mediated by CK-1, probably directed toward Ser-86, blocks the activation of the ATP,Mg-dependent phosphatase mediated by PKFA.
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PMID:Metabolic regulation through second-site phosphorylation. 255 56

To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.
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PMID:Activation of rat liver and rabbit skeletal muscle glycogen synthases by rat liver cytosolic protein phosphatases. 282 Sep 52

Glycogen synthase a was purified from rabbit skeletal muscle by a procedure involving heparin-Sepharose chromatography. Glycogen synthase a was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to give synthase b1. Dephosphorylation and activation of synthase b1 was investigated using the catalytic subunits of protein phosphatase-1 and 2A. The dephosphorylation and activation of synthase b1 was biphasic with a larger rate constant for the initial phase. Analysis of tryptic phosphopeptides of glycogen synthase during the course of dephosphorylation revealed a faster initial phosphate release from site-2 by both phosphatases comparing to sites-1a and 1b. Ligand effects on synthase phosphatase reactions were also studied. Spermine was found to inhibit protein phosphatase-1 activity and to stimulate type-2A phosphatase using synthase b1 as substrate.
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PMID:Activation/dephosphorylation of rabbit muscle glycogen synthase by the catalytic subunits of protein phosphatase-1 and 2A. 285 91

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

Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine-serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96-144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody-enzyme complexes were immobilized on heat/formaldehyde-inactivated Staphylococcus aureus. The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase alpha and histones were phosphorylated at rates not higher than 1-4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation. The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten-times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki = 2.8 microM), and non-competitive towards the protein substrate (Ki = 15 microM).
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PMID:Catalytic properties of a human cytomegalovirus-induced protein kinase. 298 75

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.
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PMID:Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes. 299 30

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.
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PMID:Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4. 300 85


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