Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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One of the major forms of glutathione S-transferase (designated as Ft transferase) has been identified and purified to near homogeneity from mouse testis. The purification was achieved by ammonium sulfate fractionation, DEAE cellulose chromatography, hydroxylapatite chromatography and the preparative isoelectric focusing. Purified Ft transferase has an isoelectric point of 4.9 +/- 0.3 and was shown to be a homodimer with a native molecular weight of about 50000. Immunologically, antisera to Ft transferase do not crossreact with F2 or F3 transferase. However, a weak cross reactivity was observed between the antisera to F3 transferase and FT transferase. Biochemical properties of purified Ft transferase are similar to those transferases isolated from mouse liver. Tissue distributions of the multiple forms of glutathione S-transferase were examined by column isoelectric focusing of various mouse tissue homogenates. It was found that mouse Ft transferase is present only in testis as a major form and in brain as a minor form, but not in other tissues that were examined.
Mol Cell Biochem 1982 Dec 10
PMID:Biochemical and immunological analysis of an abundant form of glutathione S-transferase, in mouse testis. 681 53

Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferase were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and Km's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.
Mol Cell Biochem 1982 Oct 18
PMID:Identity of microsomal glutathione S-transferases. 714 47

Maize glutathione S-transferase (GST) isozymes are encoded by a gene family comprising at least five genes, three of which (Gst I, II and III) have recently been isolated and sequenced. The enzymes are active as homo or heterodimers and exhibit intraspecific polymorphism including a "null" variant for the two major isoforms expressed in roots. Northern blot analyses performed on total root RNA from "null" and "plus" genotypes, using Gst I- and Gst II-specific probes, indicated that the Gst I gene controls the expression of the two major GST isoforms expressed in roots. Gst I and Gst II were mapped by RFLP analysis using an F2 population of 149 individuals previously characterized. Gst I was localized on the long arm of chromosome 8, while two putative Gst II loci were mapped to chromosome 8 (70 cM from Gst I) and 10, respectively.
Mol Gen Genet 1995 Sep 20
PMID:Molecular analysis and mapping of two genes encoding maize glutathione S-transferases (GST I and GST II). 747 52

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.
Mol Cell Biol 1993 Dec
PMID:Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides. 750 71

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.
Mol Cell Biol 1993 Dec
PMID:The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes. 750 74

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.
Mol Cell Biol 1994 Jan
PMID:Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. 750 91

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.
Mol Cell Biol 1994 Mar
PMID:Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways. 750 49

The antischistosomal agent oltipraz displays a unique ability to inhibit chemically induced carcinogenesis in a variety of animal models. Its apparent lack of carcinogen specificity and low toxicity make it an attractive candidate for further development as a chemopreventive agent. The mechanism by which oltipraz affords cellular protection is thought to involve the modulation of phase II detoxication enzymes. The present study examines the regulation of each class of glutathione S-transferase (EC 2.5.1.18) in mice after a single oral administration of oltipraz. Glutathione S-transferase activity in the liver increased in a dose-dependent manner after drug exposure. Oltipraz administration (1 g/kg, by gavage) elevated glutathione S-transferase activity to a maximum (4.5-fold) on day 4 after treatment. Western blot analyses demonstrated the induction of all three classes of glutathione S-transferase (alpha, mu, and pi) by oltipraz. Our murine studies suggest that the chemopreventive activity of oltipraz may be due in part to its ability to elevate glutathione S-transferase-mu activity. Consistent with this possibility, associations between the glutathione S-transferase-mu-null phenotype and increased risk for lung, larynx, and bladder cancer have been recently demonstrated in humans. Coordinate elevations in enzymatic activity were preceded by significant elevations in glutathione S-transferase alpha, mu, and pi RNA on day 2 after treatment. Although nuclear run-on assays confirmed the transcriptional induction of all three classes, the maintenance of elevations in enzymatic activity after RNA levels returned to base-line suggests that additional mechanisms are required to regulate glutathione S-transferase expression. Preclinical findings are presented that characterize the response of each class of glutathione S-transferase to oltipraz exposure and support the use of these enzymes as intermediate markers of the chemopreventive activity of oltipraz.
Mol Pharmacol 1994 Mar
PMID:Coordinate induction of glutathione S-transferase alpha, mu, and pi expression in murine liver after a single administration of oltipraz. 751 79

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration.
Mol Cell Biol 1994 May
PMID:The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck. 751 45

Cellular mechanisms for controlling membrane trafficking appear to involve small GTP-binding proteins such as the Rab proteins. Rab function is regulated by GDP dissociation inhibitor (GDI), which releases Rab proteins from membranes and inhibits GDP dissociation. Here we report the isolation of a full-length cDNA encoding a novel GDI isoform of 445 amino acids (GDI-2) with a deduced molecular weight of 50,649 from mouse skeletal muscle. Full-length and partial cDNA clones encoding a previously reported GDI protein (GDI-1) were also isolated from cDNA libraries prepared from rat brain and mouse skeletal muscle, respectively. The degree of deduced amino acid sequence identity between mouse GDI-2 and our mouse GDI-1 cDNA clone is 86%. Northern (RNA blot) analysis revealed that in human tissues, both GDI-1 and GDI-2 transcripts were abundant in brain, skeletal muscle, and pancreas but were weakly expressed in heart and liver. GDI-1 mRNA was expressed in kidney, whereas GDI-2 was almost absent, while in lung the relative amounts of these mRNA species were reversed. Specific antibodies against mouse GDI-1 and GDI-2 based on unique peptide sequences in the proteins were raised. Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes was accompanied by large increases in both mRNA and protein levels of GDI-1 and GDI-2. GDI-1 and GDI-2 expressed as glutathione S-transferase fusion proteins were both able to solubilize the membrane-bound forms of Rab4 and Rab5 in a GDP/GTP-dependent manner. Taken together, these data demonstrate that the protein products of at least two genes regulate the membrane dynamics of Rab proteins in mice.
Mol Cell Biol 1994 May
PMID:Cloning, characterization, and expression of a novel GDP dissociation inhibitor isoform from skeletal muscle. 751 52


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