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)

Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.
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PMID:Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity. 850 24

The src family protein tyrosine kinases participate in signaling through cell surface receptors that lack intrinsic tyrosine kinase domains. One of the src family kinases, p59fyn (Fyn), plays an important role in the TCR-mediated signaling. Here we report that Fyn becomes associated with the zeta-associated tyrosine kinase, ZAP-70, in a T cell hybridoma upon stimulation. The association was transient; it occurred as early as 10 s after stimulation and disappeared after 10 min. The two proteins were also associated with each other when coexpressed in COS cells. Coexpression of the zeta-chain was not required for their interaction. Mutational analysis of Fyn and ZAP-70 revealed that their kinase activities were relevant to the association. Deletion of both the SH2 and SH3 domains of Fyn resulted in the decrease of the association with ZAP-70. Consistently, Fyn-SH2 and Fyn-SH3 fused to glutathione S-transferase were able to bind to ZAP-70. These data suggest that multiple sites of Fyn and ZAP-70 are involved in the association. Furthermore, coexpression of the wild-type of both kinases in COS cells enhanced tyrosine phosphorylation of the helix-turn-helix-containing protein, HS1. HS1 was also tyrosine phosphorylated upon TCR stimulation. Thus, we propose that Fyn phosphorylates and activates ZAP-70 and that both kinases cooperate in TCR signaling.
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PMID:Physical and functional interactions of protein tyrosine kinases, p59fyn and ZAP-70, in T cell signaling. 856 36

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.
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PMID:Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma. 861 Jan 51

The 2.3-kb BamHI-U DNA fragment (map units 0.319-0.335) of herpes simplex virus type 1 (HSV-1) genome contains the complete UL25 open reading frame (ORF). It specifies an essential viral protein reported previously to be involved in virus penetration and capsid assembly (C. Addison, F. J. Rixon, J. W. Palfreyman, M. O'Hara, and V. G. Preston, Virology 138, 246-259, 1984). To identify the protein encoded by the UL25 gene, the UL25 ORF was cloned in a eukaryotic expression vector (p91023) downstream of the adenovirus major late promoter to generate the expression plasmid p9-UL25. Synthesis of a 60-kDa protein was observed in COS-7 cells transfected with p9-UL25 plasmid DNA, but not in cells transfected with p91023 control plasmid DNA. To identify and characterize the UL25 protein from HSV-1-infected cells, we prepared a rabbit antiserum by using UL25-GST fusion protein expressed in Escherichia coli as immunogen. This rabbit antiserum readily immunoprecipitated the 60-kDa UL25 protein from HSV-1-infected cells. In HSV-1-infected cells, UL25 protein was expressed as a late (gamma) or a leaky late (gamma 1) viral protein. The rabbit antiserum raised against HSV-1 UL25 protein immunoprecipitated a UL25-homologue of identical size from HSV-2-infected cells. However, the reactivity of the antiserum with HSV-2 UL25-homologue was weaker than compared to the corresponding HSV-1 protein. Consistent with its classification as a virion component, the UL25 protein was found to be associated with purified HSV-1 virions.
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PMID:Characterization of an essential HSV-1 protein encoded by the UL25 gene reported to be involved in virus penetration and capsid assembly. 861 3

The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent Km = 0.8 microM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.
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PMID:The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells. 861 11

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.
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PMID:Interaction of Shc with adaptor protein adaptins. 861 12

Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.
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PMID:Physical and functional interactions between Lyn and p34cdc2 kinases in irradiated human B-cell precursors. 862 37

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD) in vitro. Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. We show here that a CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes the Km 0.4 microM to increase by 2 orders of magnitude. Direct binding of the CTD-ID to CTD and to RNAP II could be demonstrated in vitro. Phosphorylation of a recombinant glutathione S-transferase-CTD by c-Abl was observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylated nor associated with the glutathione S-transferase-CTD in vivo. Transient overexpression of c-Abl also led to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II could be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II was again dependent on the presence of the CTD-ID in Abl. Finally, the ability of c-Abl to phosphorylate and associate with RNAP II could be correlated with the enhancement of transcription by c-Abl in transient cotransfection assays. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo.
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PMID:Identification of a binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain of RNA polymerase II. 866 51

Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.
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PMID:Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene. 870 8

GH-induced activation of JAK2, a GH receptor (GHR)-associated tyrosine kinase, leads to tyrosine phosphorylation and activation of STATs (signal transducers and activators of transcription) 1, 3, and 5. The present study investigates the importance of the GHR cytoplasmic domain in the activation of STAT3 and STAT5b. As the perimembranous Box1 region of the GHR cytoplasmic domain is necessary for activation of wild-type (WT) JAK2 by GH, we examined this question using GHR/JAK2 chimeras that have an activatable JAK2 kinase domain replacing the GHR cytoplasmic domain. STAT5b and STAT3, when each was coexpressed in COS-7 cells with WT GHR and WT JAK2, were both strongly tyrosine phosphorylated in response to GH. Coexpression of STAT3 with GHR/ JAK2 chimeras resulted in a strong GH-independent tyrosine phosphorylation of STAT3 that was 40% as active as that seen with WT GHR plus WT JAK2, whereas STAT5b was more minimally phosphorylated (13% of WT GHR plus WT JAK2) when coexpressed with chimeras devoid of the GHR cytoplasmic domain. Transient coexpression of each STAT together with WT JAK2 and GHR COOH-terminal truncation mutants indicated that a GH-induced STAT3-DNA binding complex, but not a STAT5b-DNA binding complex, was detectable when a GHR devoid of 85% of the cytoplasmic domain COOH-terminus (but eliciting significant JAK2 tyrosine phosphorylation) was expressed. In vitro binding experiments using GST/GHR cytoplasmic domain fusions demonstrated that both STATs could interact at a low basal level with GHR regions distal to residue 317. Phosphorylation of tyrosine residues in those distal regions greatly enhanced the receptor's interaction with STAT5b, but not STAT3. We conclude that GH induces activation of STAT3 and STAT5b by two different pathways: one primarily dependent on activation of JAK2 (STAT3) and another that is additionally reliant on the presence of an intact and tyrosine-phosphorylated GHR cytoplasmic domain (STAT5b).
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PMID:Growth hormone receptor cytoplasmic domain differentially promotes tyrosine phosphorylation of signal transducers and activators of transcription 5b and 3 by activated JAK2 kinase. 892 68


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