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)

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.
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PMID:Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK. 846 52

In Dictyostelium amoebae, cell-type differentiation, spatial patterning, and morphogenesis are controlled by a combination of cell-autonomous mechanisms and intercellular signaling. A chemotactic aggregation of approximately 10(5) cells leads to the formation of a multicellular organism. Cell-type differentiation and cell sorting result in a small number of defined cell types organized along an anteroposterior axis. Finally, a mature fruiting body is created by the terminal differentiation of stalk and spore cells. Analysis of the regulatory program demonstrates a role for several molecules, including GSK-3, signal transducers and activators of transcription (STAT) factors, and cAMP-dependent protein kinase (PKA), that control spatial patterning in metazoans. Unexpectedly, two component systems containing histidine kinases and response regulators also play essential roles in controlling Dictyostelium development. This review focuses on the role of cAMP, which functions intracellularly to mediate the activity of PKA, an essential component in aggregation, cell-type specification, and terminal differentiation. Cytoplasmic cAMP levels are controlled through both the regulated activation of adenylyl cyclases and the degradation by a phosphodiesterase containing a two-component system response regulator. Extracellular cAMP regulates G-protein-dependent and -independent pathways to control aggregation as well as the activity of GSK-3 and the transcription factors GBF and STATa during multicellular development. The integration of these pathways with others regulated by the morphogen DIF-1 to control cell fate decisions are discussed.
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PMID:Integration of signaling networks that regulate Dictyostelium differentiation. 1061 70

The physiological role of phosphodiesterase (PDE)11 is unknown and its biochemical characteristics are poorly understood. We have expressed human His-tagged PDE11A4 and purified the enzyme to apparent homogeneity. PDE11A4 displays K(m) values of 0.97 microM for cGMP and 2.4 microM for cAMP, and maximal velocities were 4- to 10-fold higher for cAMP than for cGMP. Given the homology between PDE11 and PDE5, we have compared the biochemical potencies of tadalafil (Cialis, Lilly-ICOS), vardenafil (Levitra, Bayer-GSK), and sildenafil (Viagra, Pfizer Inc.) for PDE11A4 and PDE5A1. PDE5A1/PDE11A4 selectivities are 40-, 9300-, and 1000-fold for tadalafil, vardenafil, and sildenafil, respectively. This suggests that none of these three compounds is likely to crossreact with PDE11A4 in patients.
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PMID:High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodiesterase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients. 1599 18

Cyclin D3 has been shown to play a major role in the regulation of cell cycle progression in lymphocytes. It is therefore important to understand the mechanisms involved in the regulation of this protein. We have previously shown that both basal and cAMP-induced degradation of cyclin D3 in Reh cells is dependent on Thr-283 phosphorylation by glycogen synthase kinase-3beta (GSK-3beta). We now provide evidence of an alternative mechanism being involved in the regulation of cyclin D3 degradation. Treatment of lymphoid cells with okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), induces rapid phosphorylation and proteasomal degradation of cyclin D3. This degradation is not inhibited by the GSK-3beta inhibitors lithium or Kenpaullone, or by substitution of Thr-283 with Ala on cyclin D3, indicating that cyclin D3 can be degraded independently of Thr-283 phosphorylation and GSK-3beta activity. Interestingly, in vitro experiments revealed that PP1, but not PP2A, was able to dephosphorylate cyclin D3 efficiently, and PP1 was found to associate with His-tagged cyclin D3. These results support the hypothesis that PP1 constitutively keeps cyclin D3 in a stable, dephosphorylated state, and that treatment of cells with OA leads to phosphorylation and degradation of cyclin D3 through inhibition of PP1.
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PMID:Degradation of cyclin D3 independent of Thr-283 phosphorylation. 1633 Dec 57

Several analogues of the 3-substituted-2-oxoindole chemotype were synthesized by condensing isatin or the appropriate haloisatin with some amino acids or histamine under neutral conditions. All the imino derivatives produced were tested for kinase inhibitory properties against three serine/threonine kinases, namely CDK1/cyclin B, CDK5/p25 and GSK3alpha/beta. Most of the histidine derivatives showed inhibitory properties to the three kinases in the low micromolar range. The histamine derivatives were less potent against CDK1/cyclin B and CDK5/p25 and totally inactive against GSK3alpha/beta. So, the management of the carboxyl function may be a tool to impart selectivity in such family of kinases. Docking of 2-[[-5-bromo-2-oxoindolin-3-ylidene]amino]-3-(1H-imidazol2-yl)propanoic acid 14 to CDK5/p25 indicates that this compound can interact with the enzyme through four hydrogen bonds; for GSK/3beta, the ligand poses itself in another orientation, also four hydrogen bonds can be formed between the ligand and the receptor, otherwise hydrophobic interactions seem to predominate. Also, all the final compounds were tested for their in vitro antitumor properties against MCF7 (breast), NCI-H460 (lung) and SF268 (CNS) cancer cell lines. None of the synthesized compounds was cytotoxic at 10(-4) molar concentration. Moreover, compounds 13 and 14 were tested for potential antiangiogenic properties by testing their ability to inhibit the proliferation of human umbilical vein endothelial cells (HUVECs), cord formation and migration in response to chemoattractant. Only compound 14 showed moderate inhibitory properties to HUVECs proliferation and cord formation while its non-brominated derivative 13 did not. Thus, the antiangiogenesis properties are not apparently caused by inhibition of any of the tested kinases.
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PMID:Synthesis of 3-substituted-2-oxoindole analogues and their evaluation as kinase inhibitors, anticancer and antiangiogenic agents. 1649 69

The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of many epithelial tumors including colorectal carcinoma (CRC). In the present study, a scattering and fibroblastic morphology with reduced intercellular contacts was found in the SW480 colon cancer cells overexpressing the gene encoding thymosin beta4 (Tbeta4), which was accompanied by a loss of E-cadherin as well as a cytosolic accumulation of beta-catenin, two most prominent markers of EMT. Whereas E-cadherin downregulation was likely to be accounted by a ZEB1-mediated transcriptional repression, the accumulation of beta-catenin was a result of glycogen synthase kinase-3beta inactivation mediated by integrin-linked kinase (ILK) and/or its downstream effector, Akt. Intriguingly, ILK upregulation in Tbeta4-overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with particularly interesting new Cys-His protein (PINCH) more efficiently. In the meantime, a strong correlation between the expression levels of Tbeta4, ILK and E-cadherin in CRC patients was also revealed by immunohistochemical analysis. Taken together, these data suggest a novel role of Tbeta4 in promoting CRC progression by inducing an EMT in tumor cells via upregulating ILK and consequentially its signal transduction.
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PMID:Thymosin beta4 triggers an epithelial-mesenchymal transition in colorectal carcinoma by upregulating integrin-linked kinase. 1707 45

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.
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PMID:Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis. 1708 64

Although GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins, here we describe N-terminal proteolysis as a novel way to regulate GSK-3. When brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of approximately 40 and 30 kDa, a proteolytic process that was inhibited by specific calpain inhibitors. Interestingly, instead of inhibiting this enzyme, GSK-3 truncation augmented its kinase activity. When we digested recombinant GSK-3 alpha and GSK-3beta protein with calpain, each isoform was cleaved differently, yet the truncated GSK-3 isoforms were still active kinases. We also found that lithium, a GSK-3 inhibitor, inhibits full-length and cleaved GSK-3 isoforms with the same IC(50) value. Calpain removed the N-terminal ends of His-tagged GSK-3 isoenzymes, and exposing cultured cortical neurons with ionomycin, glutamate, or N-methyl-d-aspartate led to the truncation of GSK-3. This truncation was blocked by the calpain inhibitor calpeptin, at the same concentration at which it inhibits calpain-mediated cleavage of NMDAR-2B and of p35 (the regulatory subunit of CDK5). Together, our data demonstrate that calpain activation produces a truncation of GSK-3 that removes an N-terminal inhibitory domain. Furthermore, we show that GSK-3 alpha and GSK-3beta isoenzymes have a different susceptibility to this cleavage, suggesting a means to specifically regulate these isoenzymes. These data provide the first direct evidence that calpain promotes GSK-3 truncation in a way that has implications in signal transduction, and probably in pathological disorders such as Alzheimer disease.
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PMID:N-terminal cleavage of GSK-3 by calpain: a new form of GSK-3 regulation. 1756 62

Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.
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PMID:A high-throughput screen for endothelial lipase using HDL as substrate. 1856 79

The receptor-binding domain of Plasmodium vivax Duffy-binding protein, region II (PvRII), is an attractive candidate for a vaccine against P. vivax malaria. Here, we have studied the safety and immunogenicity of recombinant PvRII in Macaca mulatta (rhesus monkeys). Recombinant PvRII with a C-terminal 6-histidine tag was expressed in E. coli, recovered from inclusion bodies, refolded into its functional conformation, purified to homogeneity and formulated with three adjuvants, namely, Alhydrogel, Montanide ISA 720 and the GSK proprietary Adjuvant System AS02A for use in immunogenicity studies. All the PvRII vaccine formulations tested were safe and highly immunogenic. The overall magnitude of the antibody response was significantly higher for both Montanide ISA 720 and AS02A formulations in comparison with Alhydrogel. Furthermore, there was a significant correlation between antibody recognition titers by ELISA and binding inhibition titers in in vitro binding assays. The PvRII vaccine formulations also induced IFN-gamma recall responses that were identified using ex vivo ELISPOT assays. These results provide support for further clinical development of a vaccine for P. vivax malaria based on recombinant PvRII.
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PMID:Preclinical assessment of the receptor-binding domain of Plasmodium vivax Duffy-binding protein as a vaccine candidate in rhesus macaques. 1857 99


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