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)

A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N. Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893). p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus. To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using [35S]guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA. Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021. This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region. These studies localized the minimum Rho binding region to amino acids 934-1015. To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations. This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding. The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560) and may define a distinct class of Rho effector motif.
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PMID:Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. 879 90

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.
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PMID:A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides. 931 76

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.
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PMID:Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. 972 79

Antiestrogens, including tamoxifen and raloxifene, block estrogen receptor (ER) action by blocking the interactions of an estrogen-dependent activation function (AF-2) with p160 coactivators. Although tamoxifen does show some agonist activity in the presence of ERalpha, this stems from a distinct constitutive activation function (AF-1) that lies within the ERalpha N terminus. Previous studies identified a naturally occurring mutation (D351Y) that allows ERalpha to perceive tamoxifen and raloxifene as estrogens. Here, we examine the contributions of ERalpha activation functions to the D351Y phenotype. We find that the AF-2 function of ERalpha D351Y lacks detectable tamoxifen-dependent activity when tested in isolation but does synergize with AF-1 to allow enhanced tamoxifen response. Weak tamoxifen-dependent interactions between the ERalpha D351Y AF-2 function and GRIP1, a representative p160, can be detected in glutathione S-transferase binding assays and mammalian two-hybrid assays. Furthermore, tamoxifen-dependent AF-2 activity can be detected in the presence of ERalpha D351Y and high levels of overexpressed GRIP1. We therefore propose that the D351Y mutation allows weak tamoxifen-dependent AF-2 activity but that this activity is only detectable when AF-1 is strong, and AF-1 and AF-2 synergize, or when p160s are overexpressed. We discuss the possible structural basis of this effect.
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PMID:An antiestrogen-responsive estrogen receptor-alpha mutant (D351Y) shows weak AF-2 activity in the presence of tamoxifen. 1098 90

Surfactant protein B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation and is developmentally regulated. Previous studies demonstrated that retinoic acid receptors (RARs) and thyroid transcription factor 1 (TTF-1) stimulated SP-B gene expression in respiratory epithelial cells. Clustered retinoic acid-responsive element and TTF-1 binding sites were identified in the enhancer region of the SP-B gene and were required for retinoic acid stimulation of the human SP-B (hSP-B) promoter. In addition, RAR and TTF-1 were colocalized in mouse bronchiolar and alveolar type II epithelial cells, the cellular site of SP-B synthesis. In the present studies, RAR and TTF-1 were colocalized in the nucleus of H441 cells. RAR and TTF-1 synergistically stimulated the hSP-B promoter in H441 cells. Direct protein-protein interactions between RAR and TTF-1 were demonstrated by the glutathione S-transferase pull-down assay and the mammalian cell two hybrid assay. Truncation/deletion studies showed that the RAR-TTF-1 interaction was mediated through the RAR DNA binding domain (DBD) and the TTF-1 homeodomain. RAR DBD greatly enhanced TTF-1 homeodomain DNA binding activity to a hSP-B enhancer oligonucleotide, in which retinoic acid-responsive element and TTF-1 DNA binding sites overlap. Chromatin immunoprecipitation assay demonstrated that retinoic acid treatment of H441 cells greatly stimulated both RAR and TTF-1 DNA binding to the hSP-B enhancer region in H441 cells. These findings support a model in which RAR/retinoid X receptor, TTF-1, and coactivators (p160 members and CBP) form an enhanceosome in the enhancer region of the hSP-B gene.
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PMID:Protein-protein interaction of retinoic acid receptor alpha and thyroid transcription factor-1 in respiratory epithelial cells. 1127 48

Activated estrogen receptor alpha (ERalpha) modulates transcription triggered by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demonstrated that the interference occurs without binding of ERalpha to DNA but probably results from protein.protein interactions. However, involvement of a direct interaction between ERalpha and AP-1 is still debated. Using glutathione S-transferase pull-down assays, we demonstrated that ERalpha bound directly to c-Jun and JunB but not to FOS family members, in a ligand-independent manner. The interaction could occur when c-Jun was bound onto DNA, as shown in a protein-protein-DNA assay. It implicated the C-terminal part of c-Jun and amino acids 259-302 present in the ERalpha hinge domain. ERalpha but not an ERalpha mutant deleted of amino acids 250-303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, as shown by two-hybrid and coimmunoprecipitation assays. We also show that ERalpha, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein complex in vitro and in intact cells and that the ERalpha.c-Jun interaction could be crucial for the stability of this complex. VP16-ERalpha and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induced transcription in the presence of estradiol, and this synergistic effect was not observed with the ERalpha mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ERalpha, was used instead of c-Jun. Finally, ER241G was inefficient for regulation of AP-1 activity, and an ERalpha truncation mutant encompassing the hinge domain had a dominant negative effect on ERalpha action. These results altogether demonstrate that ERalpha can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizing a multiprotein complex containing p160 coactivator, is likely to be involved in estradiol regulation of AP-1 responses.
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PMID:Characterization of the physical interaction between estrogen receptor alpha and JUN proteins. 1147 71

The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH(2) terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathione S-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.
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PMID:The FXXLF motif mediates androgen receptor-specific interactions with coregulators. 1177 76

The rat mineralocorticoid receptor (MR) has two activation functions in distinct regions of the A/B domain, designated activation function 1a (AF-1a; amino acids 1 to 169) and AF-1b (amino acids 451 to 600). Since the p160 family protein TIF2, a known component of the AF-2 coactivator complex, potentiates the transactivation function of AF-1b but not that of AF-1a, it is likely that some other, novel protein complex interacts with the AF-1a region. Therefore, we attempted to identify such coactivator complexes from HeLa nuclear extracts by biochemical purification using a glutathione S-transferase-MR AF-1a fusion protein. Purified AF-1a region-interacting proteins were found to contain RNA helicase A (RHA) and CBP. Further analysis showed that RHA interacted with the AF-1a region directly and then recruited a complex with histone acetyltransferase (HAT) activity that contained CBP. For full-length MR, aldosterone, but not hydrocortisone, was found to induce the binding of RHA/CBP complexes to the AF-1a region, as well as to allow the cooperative potentiation of MR transcriptional activity by RHA and CBP. In addition, a chromatin immunoprecipitation assay showed that aldosterone-bound MR, but not hydrocortisone-bound MR, recruited RHA/CBP complexes to native MR target gene promoters. Our results suggested that an altered conformation of the A/B region induced by aldosterone, but not hydrocortisone, might determine the accessibility of MR AF-1a to RHA/CBP complexes.
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PMID:Ligand-selective potentiation of rat mineralocorticoid receptor activation function 1 by a CBP-containing histone acetyltransferase complex. 2450 61

Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and activation of CAR.
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PMID:Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo. 1200 Jul 48

Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4)-induced tyrosine phosphorylation by direct interaction with coactivators. The CREB-binding protein and the nuclear coactivator 1 (NCoA-1), a member of the p160/steroid receptor coactivator family, bind independently to specific regions of STAT6 and act as coactivators. In this study we show that an LXXLL motif in the STAT6 transactivation domain mediates the interaction with NCoA-1. Peptides representing this motif as well as antibodies generated against this motif inhibited STAT6/NCoA-1 interaction in glutathione S-transferase pulldown assays. Peptides derived from the STAT6 transactivation domain adjacent to the LXXLL motif as well as antibodies against these peptides showed no inhibitory effect. Mutagenesis of the LXXLL motif eliminated the STAT6/NCoA-1 interaction in vitro and in vivo, supporting the specific role of this motif in NCoA-1 binding. Importantly, mutagenesis of the STAT-LXXLL motif strongly diminished the IL-4-regulated activation of the endogenous STAT6 target gene eotaxin-3. Taken together, these results indicate that the STAT6-LXXLL-binding motif mediates the interaction with NCoA-1 in transcriptional activation and represents a new potential drug target for the inhibition of the STAT6 transactivation function in allergic diseases.
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PMID:An LXXLL motif in the transactivation domain of STAT6 mediates recruitment of NCoA-1/SRC-1. 1213 96


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