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)

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.
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PMID:Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. 948 53

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-kappaB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAFs and NF-kappaB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-kappaB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-kappaB activation. These results indicate that the NF-kappaB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.
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PMID:Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation. 948 16

Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. We conclude that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB.
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PMID:Interaction of stat6 and NF-kappaB: direct association and synergistic activation of interleukin-4-induced transcription. 958 80

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.
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PMID:The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D. 1009 63

The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was not mimicked by 8-bromo-cAMP, was independent of Galpha(s), and was associated with activation of p21(ras). Accordingly, overexpression of the inactive S17N mutant of p21(ras) and of a dominant negative version of mSos (the exchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21(ras) and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21(ras) and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.
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PMID:Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway. 1046 24

c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.
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PMID:Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons. 1057 93

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.
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PMID:The RGG domain in hnRNP A2 affects subcellular localization. 1077 24

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca(2+)/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca(2+)/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525-529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. (32)P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca(2+)/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca(2+)/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2-3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.
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PMID:Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity. 1117 Oct 59

We investigated the interaction of the 12 kDa FK506-binding protein (FKBP12) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-inositol 1,4,5-trisphosphate (IP3) receptor isoforms (IP3R1 and IP3R3). Using glutathione S-transferase (GST)-FKBP12 affinity chromatography, we could efficiently extract RyR1 (42+/-7% of the solubilized RyR1) from terminal cisternae of skeletal muscle as well as RyR3 (32+/-4% of the solubilized RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20 microM) but were largely unaffected by RyR-channel modulators. In contrast, neither IP3R1 nor IP3R3 from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and IP3R-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-FKBP12 matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of FKBP12 with RyR1 but not with IP3R1. In order to determine the FKBP12-binding site, we fragmented both RyR1 and IP33R1 by limited proteolysis. We obtained a 45 kDa fragment of RyR1 that bound to the GST-FKBP12 matrix, indicating that it retained all requirements for FKBP12 binding. This fragment was identified by its interaction with antibody m34C and must therefore contain its epitope (amino acids 2756-2803). However, no fragment of IP3R1 was retained on the column. These molecular data are in agreement with the lack of correlation between FKBP12 and IP3R1 expression in various cell types. The observation that FKBP12 did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature IP3R1 and IP3R3, in contrast to RyR1 and RyR3, did not display a specific, high-affinity interaction with FKBP12.
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PMID:Characterization and mapping of the 12 kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor. 1117 Nov 21

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.
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PMID:Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases. 1152 19


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