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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.
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PMID:Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells. 1690 7

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

Activation and proliferation of human liver progenitor cells has been observed during acute and chronic liver diseases. Our goal was to investigate the presence of these putative progenitors in the liver of patients who underwent lobectomy for various reasons but did not show any hepatic insufficiency. Hepatic lesions were evaluated by histological analysis. Nonparenchymal epithelial (NPE) cells were isolated from samples of human liver resections located at a distance from the lesion that motivated the operation and were cultured and characterized. These cells exhibited a marked proliferative potential. They did not express the classic set of stem cell/progenitor markers (Oct-4, Rex-1, alpha-fetoprotein, CD90, c-kit, and CD34) and were faintly positive for albumin. When cultured at confluence in the presence of hepatocyte growth factor and either epidermal growth factor or fibroblast growth factor-4, they entered a differentiation process toward hepatocytes. Their phenotype was quantitatively compared with that of mature human hepatocytes in primary culture. Differentiated NPE cells expressed albumin; alpha1-antitrypsin; fibrinogen; hepatobiliary markers such as cytokeratins 7, 19, and 8/18; liver-enriched transcription factors; and genes characterized by either a fetal (cytochrome P4503A7 and glutathione S-transferase pi) or a mature (tyrosine aminotransferase, tryptophan 2,3-dioxygenase, glutathione S-transferase alpha, and cytochrome P4503A4) expression pattern. NPE cells could be isolated from the liver of several patients, irrespective of the absence or presence of lesions, and differentiated toward hepatocyte-like cells with an intermediate hepatobiliary and mature/immature phenotype. These cells are likely to represent a resident progenitor population of the adult human liver, even in the absence of hepatic failure. Disclosure of potential conflicts of interest is found at the end of this article.
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PMID:Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver. 1741 93

Evidence suggests that mitogen-activated protein kinase kinase (MEK) plays a role in cell transformation and tumor development and might be a significant target for chemoprevention. 3,5,4'-Trihydroxy-trans-stilbene (resveratrol), a non-flavonoid polyphenol found in various foods and beverages, including red wines, is reported to be a natural chemopreventive agent. However, the concentrations required to exert these effects might be difficult to achieve by drinking only one or two glasses of red wine a day. On the other hand, the flavonol content of red wine is approximately 30 times higher than that of resveratrol. Here we demonstrated that 3,3',4',5,5',7-hexahydroxyflavone (myricetin), one of the major flavonols in red wine, is a novel inhibitor of MEK1 activity and transformation of JB6 P+ mouse epidermal cells. Myricetin (10 microM) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF)-induced cell transformation by 76 or 72%, respectively, compared with respective reductions of 26 or 19% by resveratrol (20 microM). A combination of myricetin and resveratrol exerted additive but not synergistic effects on either TPA- or EGF-induced transformation. Myricetin, but not resveratrol, attenuated tumor promoter-induced activation of c-fos or activator protein-1. Myricetin strongly inhibited MEK1 kinase activity and suppressed TPA- or EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) or p90 ribosomal S6 kinase, downstream targets of MEK. Moreover, myricetin inhibited H-Ras-induced cell transformation more effectively than either PD098059, a MEK inhibitor, or resveratrol. Myricetin directly bound with glutathione S-transferase-MEK1 but did not compete with ATP. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines.
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PMID:Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1. 1769 61

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.
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PMID:Equol, a metabolite of the soybean isoflavone daidzein, inhibits neoplastic cell transformation by targeting the MEK/ERK/p90RSK/activator protein-1 pathway. 1772 30

Despite over a decade of research, only recently have the mechanisms governing transient receptor potential channel (TRPC) channel function begun to emerge, with an essential role for accessory proteins in this process. We previously identified a tyrosine phosphorylation event as critical in the plasma membrane translocation and activation of hTRPC4 channels following epidermal growth factor (EGF) receptor activation. To further characterize the signaling events underlying this process, a yeast-two hybrid screen was performed on the C terminus of hTRPC4. The intracellular C-terminal region from proline 686 to leucine 977 was used to screen a human brain cDNA library. Two members of the spectrin family, alphaII- and betaV-spectrin, were identified as binding partners. The interaction of hTRPC4 with alphaII-spectrin and betaV-spectrin was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. Deletion analysis identified amino acids 730-758 of hTRPC4 as critical for the interaction with this region located within a coiled-coil domain, juxtaposing the Ca(2+)/calmodulin- and IP(3)R-binding region (CIRB domain). This region is deleted in the proposed deltahTRPC4 splice variant form, which failed to undergo both EGF-induced membrane insertion and activation, providing a genetic mechanism for regulating channel activity. We also demonstrate that the exocytotic insertion and activation of hTRPC4 following EGF application is accompanied by dissociation from alphaII-spectrin. Furthermore, depletion of alphaII-spectrin by small interference RNA reduces the basal surface expression of alphahTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of alphaII-spectrin did not affect the expression of the delta variant. Taken together, these results demonstrate that a direct interaction between hTRPC4 and the spectrin cytoskeleton is involved in the regulation of hTRPC4 surface expression and activation.
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PMID:The spectrin cytoskeleton influences the surface expression and activation of human transient receptor potential channel 4 channels. 1804 48

Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.
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PMID:MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling. 1805 59

The cancer preventive action of (-)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The K(d) value for EGCG binding to the Fyn SH2 domain was 0.367 +/- 0.122 microM and B(max) was 1.35 +/- 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation.
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PMID:Fyn is a novel target of (-)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation. 1809 72

The Ser/Thr kinase ribosomal S6 kinase 2 (RSK2) has been demonstrated to phosphorylate transcription factor CREB (cyclic AMP-responsive-binding protein) and histone H3 in response to mitogenic stimulation by epidermal growth factor (EGF). EGF activates the MEK/ERK pathway to activate RSK2. We recently reported that receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) directly tyrosine phosphorylates RSK2 at Tyr-529, which consequently regulates RSK2 activation by facilitating inactive ERK binding to RSK2 that is required for ERK-dependent phosphorylation and activation of RSK2 (Kang, S., Dong, S., Gu, T. L., Guo, A., Cohen, M. S., Lonial, S., Khoury, H. J., Fabbro, D., Gilliland, D. G., Bergsagel, P. L., Taunton, J., Polakiewicz, R. D., and Chen, J. (2007) Cancer Cell 12, 201-214). Here we report that upon treatment of EGF, RSK2 was tyrosine-phosphorylated at Tyr-529 and activated in 293T and COS7 cells that do not express FGFR3. In contrast to FGFR3, the receptor tyrosine kinase EGF receptor did not directly phosphorylate RSK2 at Tyr-529 in an in vitro kinase assay using recombinant RSK2 and active EGF receptor or FGFR3. By mass spectroscopy-based studies, we identified Src tyrosine kinase family members Src and Fyn as upstream kinases of RSK2 Tyr-529. Treatment of Src inhibitor PP2 effectively attenuated EGF-dependent activation and Tyr-529 phosphorylation of RSK2, suggesting that Src family members are the kinases that phosphorylate RSK2 at Tyr-529 in response to EGF. Src and Fyn were able to directly phosphorylate RSK2 at Tyr-529 in the in vitro kinase assay. In vitro reconstitution of Tyr-529 phosphorylation by Src in glutathione S-transferase-tagged RSK2 enhanced inactive ERK binding to RSK2 wild type, but not the Y529F mutant. Together, our findings suggest that Src-dependent phosphorylation at Tyr-529 facilitates inactive ERK binding to RSK2, which might be a general requirement for RSK2 activation by EGF through the MEK/ERK pathway.
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PMID:Epidermal growth factor stimulates RSK2 activation through activation of the MEK/ERK pathway and src-dependent tyrosine phosphorylation of RSK2 at Tyr-529. 1815 74

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.
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PMID:Cell adhesion-dependent cofilin serine 3 phosphorylation by the integrin-linked kinase.c-Src complex. 1825 15


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