Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.
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PMID:Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody. 1172 83

We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.
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PMID:PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites. 1179 24

Experimental data support a role for FAK, an important component of the integrin signaling pathway, in insulin action. To test the hypothesis that FAK plays a regulatory role in hepatic insulin action, we overexpressed wild type (WT), a kinase inactive (KR), or a COOH-terminal focal adhesion targeting (FAT) sequence-truncated mutant of FAK in HepG2 hepatoma cells. In control untransfected (NON) and vector (CMV2)- and WT-transfected cells, insulin stimulated an expected 54 +/- 13, 37 +/- 4, and 47 +/- 12 increase in [U-(14)C]glucose incorporation into glycogen, respectively. This was entirely abolished in the presence of either KR (-1 +/- 7%) or FAT mutants (0 +/- 8%, n = 5, p < 0.05 for KR or FAT versus other groups), and this was associated with a significant attenuation of incremental insulin-stimulated glycogen synthase (GS) activity. Insulin-stimulated serine phosphorylation of Akt/protein kinase B was significantly impaired in mutant-transfected cells. Moreover, the ability of insulin to inactivate GS kinase-3beta (GSK-3beta), the regulatory enzyme immediately upstream of GS, by serine phosphorylation (308 +/- 16, 321 +/- 41, and 458 +/- 34 optical densitometric units (odu) in NON, CMV2, and WT, respectively, p < 0.02 for WT versus CMV2) was attenuated in the presence of either FAT (205 +/- 14, p < 0.01) or KR (189 +/- 4, p < 0.005) mutants. FAK co-immunoprecipitated with GSK-3beta, but only in cells overexpressing the KR (374 +/- 254 odu) and FAT (555 +/- 308) mutants was this association stimulated by insulin compared with NON (-209 +/- 92), CMV2 (-47 +/- 70), and WT (-39 +/- 31 odu). This suggests that FAK and GSK-3beta form both a constitutive association and a transient complex upon insulin stimulation, the dissociation of which requires normal function and localization of FAK. We conclude that FAK regulates the activity of Akt/protein kinase B and GSK-3beta and the association of GSK-3beta with FAK to influence insulin-stimulated glycogen synthesis in hepatocytes. Insulin action may be subject to regulation by the integrin signaling pathway, ensuring that these growth and differentiation-promoting pathways act in a coordinated and/or complementary manner.
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PMID:Focal adhesion kinase (FAK) regulates insulin-stimulated glycogen synthesis in hepatocytes. 1180 46

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. A key component of the Wnt signaling pathway is the cytosolic protein, beta-catenin. We have recently shown that the chondrogenic activity of BMP-2 in vitro involves the action of the cell-cell adhesion protein, N-cadherin, which functionally complexes with beta-catenin. The aim of this study is to test the hypothesis that Wnts may be involved in BMP-2 induced chondrogenesis, using an in vitro model of high-density micromass cultures of the murine multipotent mesenchymal cell line, C3H10T1/2. Expression of a number of Wnt members was detected in these cultures, including Wnt-3A and Wnt-7A, whose levels were up- and downregulated, respectively, by BMP-2. To assess the functional involvement of Wnt signaling in BMP-2 induced chondrogenesis, cultures were treated with lithium chloride, a Wnt-7A mimetic that acts by inhibiting the serine/threonine phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta). Lithium treatment significantly inhibited BMP-2 stimulation of chondrogenesis as well as GSK-3beta enzymatic activity, and decreased the levels of N-cadherin protein and mRNA. Furthermore, lithium decreased BMP-2 upregulation of total and nuclear levels of LEF-1 and beta-catenin as well as their interaction during later chondrogenesis; similarly, the interaction of beta-catenin with N-cadherin was also decreased. Interestingly, lithium treatment did not affect the ability of BMP-2 to decrease ubiquitination of beta-catenin, although it did reduce the interaction of beta-catenin with GSK-3beta during late chondrogenesis (days 9-13). We suggest that the chondro-inhibitory effect of lithium on BMP-2 induced chondrogenesis indicates antagonism between lithium-like Wnts and BMP-2 during mesenchymal condensation.
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PMID:Wnt signaling during BMP-2 stimulation of mesenchymal chondrogenesis. 1183 6

Although glycogen synthase kinase-3 (GSK-3) is but one of more than a thousand distinct serine/threonine kinases present in the mammalian genome, this enzyme has attracted attention for its role in a diverse range of cellular processes and its positioning at a nexus of several signaling pathways that are important in cancer and other human diseases. The association of GSK-3 with widely different functions, from glycogen metabolism to fruit fly segmentation and slime mold differentiation, was initially perplexing. However, as the context of the biological processes involving this enzyme has been clarified, unifying themes have emerged that begin to explain its pleiotropic nature. Unlike most protein kinases involved in signaling, GSK-3 is active in unstimulated, resting cells. Its activity is inactivated during cellular responses and its substrates therefore tend to be dephosphorylated. As more of these targets have been identified and the effects of their modification by GSK-3 determined, most have been found to be functionally inhibited by GSK-3. Hence, this kinase appears to act as a general repressor, keeping its targets switched off or inaccessible under resting conditions. The rarity of this form of regulation is perhaps related to the diversity of its targets. Over the past decade, the importance of GSK-3 has been established by three significant properties: its remarkable evolutionary conservation, allowing analysis in genetically tractable organisms; its involvement in the Wnt/wingless signaling pathway; and its inhibition by agonists of the prosurvival phosphatidylinositol 3' kinase (P13'K) pathway. This review covers recent advances in understanding the physiological roles of this enzyme, particularly in the context of cancer.
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PMID:Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways. 1188 28

The roles of glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation were examined in seven-day-old rats injected with the NMDA receptor antagonist (MK801) that is known to induce neuronal apoptosis. Immunoblot and immunohistochemical analysis of brain samples demonstrated a site-specific increase in tau phosphorylation associated with the relocalization of the protein to the nuclear/perinuclear region of apoptotic neurons. In addition, a tau 32-kDa fragment was detected, suggesting that tau was a target of intracellular proteolysis in MK801-treated brains. The proteolytically modified form of tau has reduced ability to bind to microtubules. GSK-3beta kinase assay and immunoblottings of active (tyrosine-216) and inactive (serine-9) forms of GSK-3beta revealed a rapid and transient increase in the kinase activity. Lithium chloride, a GSK-3beta inhibitor, prevented tau phosphorylation suggesting that tau phosphorylation is mediated by the activation of GSK-3beta. Confocal microscopy using double labelling of tau and GSK-3beta revealed that the activation of GSK-3beta in neurons was associated with early (2 h) nuclear translocation of tyrosine-216 GSK-3beta. The execution phase of neuronal apoptosis was accompanied by a selective phosphorylation of serine-9 and dephosphorylation of tyrosine-216 GSK-3beta. These findings demonstrate that in vivo, GSK-3beta kinase activation and nuclear translocation are early stress signals of neuronal apoptosis.
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PMID:In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3beta in neuronal apoptosis: links to tau phosphorylation. 1188 46

The serine-threonine kinase, Akt, is involved in the survival signaling pathways in many cell systems. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after traumatic brain injury (TBI) in mice. Immunohistochemistry showed phospho-Akt was decreased in the injured cortex 1 h after TBI, whereas it was temporally increased at 4 h in the perifocal damaged cortex. In the CA1 region of the hippocampus, phospho-Akt was increased after TBI. Western blot analysis showed that Akt was significantly decreased as early as 1 h after trauma; however, the phosphorylation was accelerated at 4 h. Double staining with phospho-Akt and phospho-BAD or phospho-GSK-3beta revealed the colocalization of phospho-Akt and downstream elements. Double staining with phospho-Akt and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling showed different cellular distributions after TBI. The present study implicates Akt phosphorylation in the signaling pathways that are involved in cell survival after TBI.
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PMID:Akt phosphorylation and neuronal survival after traumatic brain injury in mice. 1195 Feb 75

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.
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PMID:Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. 1195 36

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3beta and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.
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PMID:Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. 1200 Jul 90

Glycogen synthase kinase-3alpha and -3beta (GSK-3alpha and -3beta) are multi-substrate, serine/threonine-specific kinases that can phosphorylate microtubule-associated protein tau and other neuronal proteins. In this study, the expression level and mRNA distribution of two GSK-3 isoforms, GSK-3alpha and -3beta in mice were investigated. Northern blot analyses indicated that GSK-3alpha mRNA is encoded by a 2.5-kb transcript in adult tissues, whereas a 4.1-kb transcript was found in neonatal tissues. The GSK-3beta mRNA is encoded by a 1.6-kb transcript in the testis and a 7.6-kb transcript in the brain, and in many other adult tissues, but not neonatal tissues. Western blot analyses demonstrated that GSK-3beta protein was mainly expressed in the brain and heart, whereas GSK-3alpha was highly expressed in the brain, heart, and testis. A non-radioactive in situ hybridization study using specific digoxigenin-labeled RNA probes showed that GSK-3alpha and -3beta mRNAs were found in many brain regions, and were especially abundant in the hippocampus, cerebral cortex, and the Purkinje cells of the cerebellum. This implies the importance of GSK-3alpha and -3beta for brain function. The differential expression of GSK-3alpha and -3beta mRNAs as well as proteins in other tissues indicate that they play different roles in cellular functions and the developmental process.
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PMID:Expression of glycogen synthase kinase-3 isoforms in mouse tissues and their transcription in the brain. 1204 12


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