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)

Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.
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PMID:Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon. 1078 21

The 14-3-3 family of proteins is expressed in a broad range of organisms and tissues. Based on data essentially obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways, in particular those involving the activity of the Raf family protein kinases. The 14-3-3 theta mouse isoform is expressed almost exclusively in testis and brain. In an effort to understand the function of 14-3-3 theta in testis, we sought to identify endogenous proteins that interact with 14-3-3 theta in spermatogenic cells. A recombinant 14-3-3 theta fusion protein was used in Far Western and GST pulldown assays. Here we report that 14-3-3 theta interacts in vivo and in vitro with 93- to 95-kDa B-Raf, originally described as specific of neural tissues and never reported in male germ cells. Moreover, in mouse spermatids, i.e., the haploid cytodifferentiating cells, a so far unidentified protein complex formed by endogenous Rap1/B-Raf/14-3-3 theta can be coimmunoprecipitated. The intracellular localization of endogenous 14-3-3 theta, B-Raf, and Rap1 was analyzed in distinct spermatogenic cell types and a peculiar codistribution of the three proteins was immunorevealed in differentiating spermatids. Together, these data demonstrate that a protein complex formed by endogenous Rap1, 93- to 95-kDa B-Raf, and 14-3-3 theta exists in vivo and the finding that this has been detected in cytodifferentiating, not dividing cells, strengthens the hypothesis for a role of Rap1/B-Raf-mediated signaling in cell morphogenesis and differentiation.
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PMID:A novel rap1/B-Raf/14-3-3 theta protein complex is formed in vivo during the morphogenetic differentiation of postmeiotic male germ cells. 1085 65

The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.
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PMID:Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF). 1113 99

Na(+)/H(+) exchanger isoform-1 (NHE1), the ubiquitous form of the Na(+)/H(+) exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P)(703)) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P)(703) (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P)(703) exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 +/- 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 +/- 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and +2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P)(703). GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the base-line rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 +/- 0.02 versus 0.48 +/- 0.06 mmol of H(+)/min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3.
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PMID:14-3-3 Binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange. 1127 64

We have previously reported on the cloning of the 14-3-3 protein of Schistosoma mansoni. Here, we evaluate the potential use of this protein as a vaccine candidate against infection by S. mansoni. Sm14-3-3 was expressed and purified either as a free protein or as a fusion protein to SjGST or MBP. Sera from mice infected with S. mansoni recognized both SjGST and 14-3-3, indicating that antibodies against these two proteins are induced in the course of the natural infection. Furthermore, mice immunized with either 14-3-3, GST or 14-3-3-GST, reacted with cercaria lysate. A cellular immune response was also detected, particularly in mice immunized with 14-3-3-GST. With respect to the effect on biological functions, antibodies to 14-3-3 and 14-3-3-GST caused 23-32% complement-mediated cytotoxcity of S. mansoni schistosomula compared to only 10-11% induced by either normal mouse serum, or GST alone. In challenge infection with S. mansoni, immunization with 14-3-3, either as a fusion protein or as a free protein, led to protection ranging from 25-46%, as determined by reduction of adult worm burden, while SjGST alone elicited only 0-8% protection and MBP alone did not elicit any protection.
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PMID:The 14-3-3 protein as a vaccine candidate against schistosomiasis. 1129 98

It is well established that prolactin (PRL) sustains, while prostaglandin F(2 alpha) (PGF(2 alpha)) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20 alpha-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF(2 alpha). In addition to 20 alpha-HSD, PGF(2 alpha) stimulated and PRL inhibited PGF(2 alpha)-receptor, phospholipase C delta(1) and TGF beta(1) expression. In contrast PRL stimulated and PGF(2 alpha) inhibited the LH receptor, 11 beta-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GST mu(2), inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF(2 alpha). PGF(2 alpha) stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-beta, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF(2 alpha) also up-regulated the expression of the sodium channel beta(1), Na/K ATPase, annexin IV, GST7pi, and P450 reductase. In contrast PGF(2 alpha) inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450(AROM)) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3beta-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of beta(2)-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a "yin-yang" relationship between PRL and PGF(2 alpha) in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.
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PMID:Opposite effect of prolactin and prostaglandin F(2 alpha) on the expression of luteal genes as revealed by rat cDNA expression array. 1151 96

The plant plasma membrane H+ -ATPase is activated by the binding of 14-3-3 proteins to its extreme C-terminal amino acids (YTV) and phosphorylation of the penultimate threonine (YpTV) is necessary for this interaction in vivo. However, in the presence of the fungal toxin fusicoccin (FC), binding of 14-3-3 proteins occurs independently of phosphorylation but still involves the YTV motif. Since FC exclusively binds to the complex consisting of both 14-3-3 homologs and the C-terminal domain of the H+ -ATPase, the toxin was used as a tool to reveal potential protein-protein interaction sites in the enzyme's C terminus. We performed in vitro interaction studies by applying various C-terminal parts of the H+ -ATPase PMA2 from Nicotiana plumbaginifolia expressed as glutathione S-transferase fusion peptides in E. coli. Interestingly, the PMA2 region encompassing residues 905-922 is implicated in FC-dependent binding of 14-3-3 homologs. Recently, part of this region has been shown to contribute to the autoinhibitory action of the PMA2 C terminus. Site-directed mutagenesis of individual amino acids localized within this region resulted in a drastic decrease in FC-dependent binding of 14-3-3 proteins. Furthermore, by expressing the corresponding mutants of PMA2 in yeast, we observed a reduced capability of the mutant enzymes to functionally replace the endogenous H+ -ATPase. Notably, the decreased activity of the mutant enzymes was accompanied by a weakened binding of yeast 14-3-3 homologs to the plasma membrane of transformed cells. Taken together, our results suggest that a section of the autoinhibitory C-terminal PMA2 region contributes to binding of activatory 14-3-3 proteins in the absence of FC.
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PMID:Binding of regulatory 14-3-3 proteins to the C terminus of the plant plasma membrane H+ -ATPpase involves part of its autoinhibitory region. 1151 28

Promotion of apoptosis may potentiate the sensitivity of tumor cells to chemotherapeutic agents, thus improving the efficacy of cancer treatment. The transfection of the proapoptotic bax gene, which results in the overexpression of bax protein, augments the growth inhibition of A253 cells by BNP1350. Increased drug response was associated with the induction of DNA fragmentation in the size of 30-200 Kb, generating a cleaved fragment of 18 kDa from full-length 21 kDa bax and the cleavage of PARP. A253/vec cells treated with 0.07 microM(IC50) of BNP1350 accumulated in G2 phase at 24 h after drug removal. In contrast, A253/Bax cells treated with an equimolar concentration of BNP1350 primarily displayed a G1 phase accumulation with a concurrent decrease in G2 phase. Certain cell cycle regulatory protein expression and activities were altered following drug exposure in both cell lines under similar conditions. Cdk2- and cdc2-associated H1 kinase activities were markedly increased in the A253/Bax cell line with marginal increased activity in the A253/vec cell line. A chk1 activity assay was performed with GST-cdc25C (200-256) or GST-cdc25C(S216A) (200-256) fusion proteins as the substrate. Increased chk1 activity was observed in the A253/vec cell line, with little change in the A253/Bax cell line, when exposed to equimolar concentrations of BNP1350 (0.07 microM). A Western blot of immunoprecipitated chk1 indicated that increased chk1 phosphorylation following DNA damage induced by BNP1350 was accompanied by the observed G2 accumulation in the A253/vec cell line, while only a slight increase in chk1 phosphorylation was seen in the A253/Bax cell line. A decreased expression of cdc25C was observed in the BNP1350-treated A253/Bax cells, but not in the A253/vec cell line. Following exposure to BNP1350, increased binding of 14-3-3 proteins to chk1 occurred in both cell lines, with more being observed in the A253/vec cell line. The data have shown that inhibition of the chk1 pathway accompanied by the abrogation of G2 arrest is involved in sensitizing A253 cells to BNP1350 by bax gene transfer. These findings suggest that bax gene transfer sensitizes A253 cells to BNP1350 through apoptosis promoting and G2/M DNA damage checkpoint regulatory pathways.
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PMID:The Chk1-Cdc25C regulation is involved in sensitizing A253 cells to a novel topoisomerase I inhibitor BNP1350 by bax gene transfer. 1153 38

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.
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PMID:Identification of 14-3-3zeta as a protein kinase B/Akt substrate. 1195 22

The Protein 4.1 family contains at least two members that function as tumour suppressors, the neurofibromatosis 2 gene product merlin and the recently identified differentially expressed in adenocarcinoma of the lung (DAL-1)/Protein 4.1B molecule. DAL-1/Protein 4.1B loss is observed in a variety of tumours, including breast and lung cancers as well as meningiomas. We have previously demonstrated that DAL-1/Protein 4.1B interacts with some but not all merlin-binding proteins, raising the possibility that DAL-1/Protein 4.1B associates with additional unique proteins specific to its function as a negative growth regulator. Using yeast two-hybrid interaction cloning, we identified three 14-3-3 isoforms, beta, gamma and eta, to be DAL-1/Protein 4.1B-binding proteins. These interactions were verified by using glutathione S-transferase affinity chromatography in vitro and co-immunoprecipitation in vivo. The interaction of 14-3-3 with DAL-1/Protein 4.1B was specific, as 14-3-3 did not bind to the related Protein 4.1 family members merlin, ezrin or radixin. The DAL-1/Protein 4.1B domain that mediates 14-3-3 binding was mapped to residues Pro(244) and Leu(280) within the 4.1/ezrin/radixin/moesin domain. The identification of this novel DAL-1/Protein 4.1B-interacting protein represents the first step towards elucidating its potentially unique mechanism of action.
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PMID:The 4.1/ezrin/radixin/moesin domain of the DAL-1/Protein 4.1B tumour suppressor interacts with 14-3-3 proteins. 1199 70


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