Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.
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PMID:Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors. Evidence for a role in mitogenic signaling. 879 17

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.
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PMID:Serine protein kinase activity associated with rotavirus phosphoprotein NSP5. 898 32

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The EHV-1 EICP22 protein, a homolog of ICP22 of herpes simplex virus, increased the in vitro DNA binding activity of the IE protein for sequences in the IE, early, and late promoters. The EICP22 protein affected the rate as well as the extent of the IE protein binding to promoter DNA sequences. To study the DNA binding activity of the IE protein, Trp493, Gln495, Asn496, and Lys498 of the WLQN region, which is directly involved in DNA binding, were replaced with Ser (IEW493S), Glu (IEQ495E), Ile (IEN496I), and Glu (IEK498E), respectively. Gel shift assays revealed that the glutathione S-transferase (GST)-IEQ495E(407-615) and GST-IEK498E(407-615) proteins failed to bind to the IE promoter, indicating that the Gln and Lys residues are important for the DNA binding activity. In the presence of the GST-EICP22 protein, DNA binding activity of the GST-IEQ495E(407-615) protein was restored, suggesting that the EICP22 protein cooperates with the IE protein to regulate EHV-1 gene expression. Transient-transfection assays also showed that the EICP22 protein allowed the IEQ495E mutant to be functional as a transactivator. These results are unique and may represent an important role for the EICP22 protein in EHV-1 gene regulation.
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PMID:The ICP22 protein of equine herpesvirus 1 cooperates with the IE protein to regulate viral gene expression. 899 19

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and alpha2A adrenergic receptors or overexpression of Gbeta1gamma2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gbeta1gamma2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gbetagamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.
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PMID:Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. 902 Jan 93

A cDNA expression clone of the human malarial parasite Plasmodium falciparum, lambdaPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone lambdaPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.
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PMID:Characterization of P0, a ribosomal phosphoprotein of Plasmodium falciparum. Antibody against amino-terminal domain inhibits parasite growth. 911 84

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.
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PMID:Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1. 914 93

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.
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PMID:Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase. 915 69

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the "subtractive probes" generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, a BLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na+, K+-ATPase alpha-subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a mu-glutathione S-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.
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PMID:Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening. 929 8

Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse alpha4 (malpha4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of alph4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/alpha4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the malpha4 cDNA into Jurkat cells or the increased association of PP2Ac/alpha4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-alpha4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that alpha4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.
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PMID:Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. 965 54

Large T-antigen, the major regulatory protein encoded by polyomaviruses, including Simian Virus 40 (SV40) and JC virus (JCV), is a multifunctional phosphoprotein that is involved in many viral and cellular events. In addition to its integral role in viral replication and cellular transformation, T-antigen also regulates transcription of both viral and cellular genes. In particular, the viral late promoter has been used as a model for the analysis of T-antigen-mediated transcriptional activation. Earlier studies have demonstrated that the cellular protein Puralpha is able to attenuate the transcriptional activity of JCV T-antigen. We investigated the mechanism whereby Puralpha affects T-antigen function. Co-immunoprecipitation studies demonstrated that Puralpha and JCV T-antigen associate in vivo, and glutathione S-transferase affinity binding assays revealed that these two proteins interact in vitro. Moreover, we localized the sequences of Puralpha that are important for the interaction between Puralpha and JCV T-antigen. In addition, we demonstrated that Puralpha interacts with the SV40 T-antigen. Transient transfection studies demonstrated that Puralpha and JCV T-antigen interact functionally as well. More specifically, Puralpha and a deletion mutant that interacts with T-antigen attenuated T-antigen-mediated transcriptional activation. A Puralpha deletion mutant that is unable to interact with JCV T-antigen, however, was found to be incapable of abrogating JCV T-antigen transactivation. Taken together, these data demonstrate that Puralpha and T-antigen interact both physically and functionally and that this interaction modulates T-antigen-mediated transcriptional activation. The implication of these findings with respect to the cellular role of Puralpha is discussed.
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PMID:Interaction of the single-stranded DNA-binding protein Puralpha with the human polyomavirus JC virus early protein T-antigen. 983 7


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