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:2.7.11.26 (GSK)
6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

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.
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PMID:Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon. 643 31

The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals. In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) EMBO J. 12, 803-808). A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates. Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies. The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity. GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues. In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+. Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues. The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity. The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity. A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction. We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes. Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity.
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PMID:Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. 751 73

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
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PMID:A novel tau-tubulin kinase from bovine brain. 755 43

Three genes TPK1, TPK2 and TPK3 encode in Saccharomyces cerevisiae distinct catalytic subunits of cAMP-dependent protein kinase (cAPK). We have measured cAPK activity in vitro and, indirectly, in vivo in yeast strains carrying only one of the three TPK genes. The strain containing TPK3 as the only intact TPK gene showed nearly undetectable phosphorylating activity and no TPK3 mRNA could be detected, although the cells grow normally. Overexpression of TPK3 in a high copy vector or under the control of the inducible GAL1 promoter did not by itself result in a corresponding increase in activity but coexpression of BCY1, the gene coding for the regulatory subunit, was necessary in both cases to achieve high levels of phosphorylating activity. Moreover, BCY1 overexpression not only increased Tpk3 catalytic activity but also increased the amount of TPK3 mRNA detected in Northern blots.
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PMID:Low activity of the yeast cAMP-dependent protein kinase catalytic subunit Tpk3 is due to the poor expression of the TPK3 gene. 838 30

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an "unregulated" mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.
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PMID:Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. 927 79

Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus.
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PMID:Nuclear glycogen and glycogen synthase kinase 3. 971 12

Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.
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PMID:Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle. 1021 53

Glycogen synthase kinase 3beta (GSK-3beta) is a proline-directed kinase that forms part of the wingless signaling pathway. Recent studies have shown that GSK-3beta phosphorylates the microtubule-associated protein tau in vitro and in cell culture. Tau is the principal component of the paired helical filaments (PHFs) found in the brains of patients with Alzheimer disease, and PHF-tau is hyperphosphorylated. GSK-3beta is therefore one of the candidate kinases for phosphorylating tau in Alzheimer disease. GSK-3beta activity is negatively regulated by phosphorylation on serine 9 and positively regulated by phosphorylation on tyrosine 216. However, since overexpression of GSK-3beta by transfection leads to increased activity in the absence of any stimuli, GSK-3beta activity may also be regulated at the transcriptional level. Indeed, increased GSK-3beta protein levels are found in Alzheimer disease brains, and GSK-3beta is found associated with PHFs in Alzheimer disease. To understand how GSK-3beta activity may be regulated at the transcriptional level, we have isolated the human GSK-3beta promoter. The GSK-3beta promoter does not contain a conventional TATA box although several other transcription factor binding sites were identified. A putative transcription start site was mapped by 5' RACE. Transfection of various GSK-3beta promoter CAT reporter genes into both COS-7 cells and SHSY5Y neuronal cells revealed that the GSK-3beta promoter is more active in neuronal cells. Such transfection studies involving promoter deletion mutants revealed that a negative transcriptional response element may be present at position -1421 to -1363 and an activator sequence at position -427 to -384. CP2 binding sites were also present within the promoter. CP2 has recently been shown to interact with the Alzheimer disease amyloid precursor protein binding protein Fe65. The significance of these results with respect to Alzheimer disease pathogenesis are discussed.
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PMID:Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter. 1048 3

The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1-20 microM Abeta on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against Abeta-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.
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PMID:Inhibition of tau phosphorylating protein kinase cdk5 prevents beta-amyloid-induced neuronal death. 1052 77


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