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)

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.
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PMID:Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. 768 42

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.
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PMID:Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. 779 64

The mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein-protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N-terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S-transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S-radiolabeled MDA-MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2-DE, proteins of Mr 55,000, 31,500 and 34,000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2-DE and subjected to in-gel digestion and capillary or microbore reverse-phase high performance liquid chromatography (RP-HPLC). Resultant peptides were analyzed by peptide mass fingerprinting, N-terminal Edman degradation or tandem mass spectrometry. The 55,000 protein was identified as the cytoskeletal protein, beta-tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti-tubulin antibodies. The 31,500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.
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PMID:Two-dimensional electrophoretic analysis of mixed lineage kinase 2 N-terminal domain binding proteins. 962 20

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.
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PMID:Recruitment of the LIM protein hic-5 to focal contacts of human platelets. 966 39

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione-labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel.
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PMID:The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation. 969 34

Overexpression of 'tissue' transglutaminase (tTG) in the human neuroblastoma cells increases spontaneous apoptosis and renders these cells highly susceptible to death induced by various stimuli. We used immunoprecipitation to identify cellular proteins that interact specifically with tTG in SK-N-BE(2) -derived stable transfectants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that tTG binding proteins have molecular masses of 110, 50, 22, 14, and 12 kDa. Microsequencing and computer search analyses allowed us to identify these polypeptides as the beta-tubulin (50 kDa), the histone H2B (14 kDa), and two GST P1-1-truncated forms (22 and 12 kDa). The specificity of the interaction between tTG and these proteins was confirmed by competing tTG binding with purified enzyme and by detecting tTG in immunoprecipitates obtained using beta-tubulin or GST P1-1 mAb's. Here we demonstrate that the GST P1-1 acts as an efficient acyl donor as well as acceptor tTG substrate both in cells and in vitro. The tTG-catalyzed polymerization of GST P1-1 leads to its functional inactivation and is competitively inhibited by GSH. By contrast, the tTG-beta-tubulin interaction does not result in the cross-linking of this cytoskeletal protein, which suggests that microtubules act as the anchorage site for tTG and GST P1-1 interaction.
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PMID:Identification of 'tissue' transglutaminase binding proteins in neural cells committed to apoptosis. 997 24

In determining the role of Chk in T cell signaling, we have focused on its protein-protein interactions. We detected a tyrosine phosphoprotein that coimmunoprecipitated with Chk from pervanadate stimulated human blastic T cells. Subsequent Western blot analysis identified this tyrosine phosphoprotein as paxillin. Paxillin, a cytoskeletal protein involved in focal adhesions, was first identified as a v-Src substrate in transformed fibroblasts. Interestingly, Chk specifically bound tyrosine phosphorylated paxillin. Consistent with our in vivo data, Chk and paxillin were observed to localize in similar cellular regions prior to and following stimulation. Using GST fusion proteins, we determined that the Chk SH2 domain, not the SH3 domain, bound tyrosine phosphorylated paxillin. Specifically, paxillin bound to the FLVRES motif of the Chk SH2 domain. Using Far Western analysis, we revealed that the Chk SH2 domain directly associates with tyrosine phosphorylated paxillin. Finally, p52(Chk) expression in Csk-deficient mouse embryo fibroblasts decreased total phosphotyrosine levels of paxillin, implying a physiological role for Chk. These studies provide important insight into the role of Chk in tyrosine mediated signaling, as well as T cell physiology.
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PMID:The Csk homologous kinase, Chk, binds tyrosine phosphorylated paxillin in human blastic T cells. 1008 Sep 57

In response to the chemoattractants interleukin 8, C5a, N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.
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PMID:Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells. 1073 15

Erythroid protein 4.1 (4.1R) is an 80-kd cytoskeletal protein that stabilizes the membrane-skeletal network structure underlying the lipid bilayer. Using the carboxyl terminal domain (22/24 kd) of 4.1R as bait in a yeast 2-hybrid screen, we isolated cDNA clones encoding a polypeptide of eIF3-p44, which represents a subunit of a eukaryotic translation initiation factor 3 (eIF3) complex. The eIF3 complex consists of at least 10 subunits that play an essential role in the pathway of protein translation initiation. Northern blot analysis revealed that eIF3-p44 (approximately 1.35 kb) is constitutively expressed in many tissues. The essential sequence for this interaction was mapped to the carboxyl-terminus of 4.1R (residues 525-622) and a region (residues 54-321) of eIF3-p44. The direct association between 4.1R and eIF3-p44 was further confirmed by in vitro binding assays and coimmunoprecipitation studies. To characterize the functions of eIF3-p44, we depleted eIF3-p44 from rabbit reticulocyte lysates either by anti-eIF3-p44 antibody or by GST/4.1R-80 fusion protein. Our results show that the eIF3-p44 depleted cell-free translation system was unable to synthesize proteins efficiently. The direct association between 4.1R and elF3-p44 suggests that 4.1R may act as an anchor protein that links the cytoskeleton network to the translation apparatus. (Blood. 2000;96:747-753)
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PMID:Protein 4.1R binding to eIF3-p44 suggests an interaction between the cytoskeletal network and the translation apparatus. 1088 44

Metabotropic glutamate receptors (mGluRs) mediate a variety of responses to glutamate in the central nervous system. A primary role for group-III mGluRs is to inhibit neurotransmitter release from presynaptic terminals, but the molecular mechanisms that regulate presynaptic trafficking and activity of group-III mGluRs are not well understood. Here, we describe the interaction of mGluR7, a group-III mGluR and presynaptic autoreceptor, with the cytoskeletal protein, alpha tubulin. The mGluR7 carboxy terminal (CT) region was expressed as a GST fusion protein and incubated with rat brain extract to purify potential mGluR7-interacting proteins. These studies yielded a single prominent mGluR7 CT-associated protein of 55 kDa, which subsequent microsequencing analysis revealed to be alpha tubulin. Coimmunoprecipitation assays confirmed that full-length mGluR7 and alpha tubulin interact in rat brain as well as in BHK cells stably expressing mGluR7a, a splice variant of mGluR7. In addition, protein overlay experiments showed that the CT domain of mGluR7a binds specifically to purified tubulin and calmodulin, but not to bovine serum albumin. Further pull-down studies revealed that another splice variant mGluR7b also interacts with alpha tubulin, indicating that the binding region is not localized to the splice-variant regions of either mGluR7a (900-915) or mGluR7b (900-923). Indeed, deletion mutagenesis experiments revealed that the alpha tubulin-binding site is located within amino acids 873-892 of the mGluR7 CT domain, a region known to be important for regulation of mGluR7 trafficking. Interestingly, activation of mGluR7a in cells results in an immediate and significant decrease in alpha tubulin binding. These data suggest that the mGluR7/alpha tubulin interaction may provide a mechanism to control access of the CT domain to regulatory molecules, or alternatively, that this interaction may lead to morphological changes in the presynaptic membrane in response to receptor activation.
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PMID:Interaction between metabotropic glutamate receptor 7 and alpha tubulin. 1195 48


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