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)

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.
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PMID:Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. 923 Jan 16

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.
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PMID:Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells. 924 37

To protect bone marrow cells from the toxicity of chemotherapy, a multidrug resistant gene or a dihydrofolate reductase gene has been introduced into stem cells. These genes, however, are not capable of conferring refractoriness to alkylating agents (AA), which are some of the most commonly used agents in chemotherapy regimens. In the present study, an attempt was made to endow human stem cell (CD34+ cells) with resistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase pi (GST-pi) gene whose product is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM. The gene transduction was carried out retrovirally with a virus titer of 1 x 10(5) FFU/ml, employing a recombinant fibronectin fragment; transduction efficiency was extremely low without the fragment. Incubation with interleukin-6 and stem cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34+ cells. This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34+ cell to the retroviral vector via fibronectin fragments and the subsequent transduction process. The GST-pi gene-transduced CD34+ cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34+ cells in the presence of 2.5 microg/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively. The transfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 microg/ml of 4-HC, 50 ng/ml of ADM) whereas neo gene-transduced or nontransduced CD34+ cells formed no colonies at all, indicating the possibility of selecting out the transfectants by exposing them to these anticancer drugs. Thus, we were able to demonstrate that transduction of the GST-pi gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach can be applied clinically for high-dose chemotherapy.
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PMID:Fibronectin fragment-facilitated retroviral transfer of the glutathione-S-transferase pi gene into CD34+ cells to protect them against alkylating agents. 938 56

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.
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PMID:Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater. 940 4

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.
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PMID:Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. 946 81

Leukocyte antigen-related protein (LAR) is a prototype for a family of transmembrane protein tyrosine phosphatases whose extracellular domain is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4-8 has been observed. The extracellular matrix laminin-nidogen complex was identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR- laminin-nidogen binding was regulated by alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence resulted in disruption of the laminin-nidogen-binding activity. Long cellular processes were observed when HeLa cells were plated on laminin-nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining revealed high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin-nidogen. Antibody-induced cross-linking of LAR inhibited formation of these cellular processes, and inhibition was correlated with changes in cellular actin cytoskeletal structure. Thus, LAR-laminin-nidogen binding may play a role in regulating cell signaling induced by laminin-nidogen, resulting in cell morphological changes.
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PMID:The laminin-nidogen complex is a ligand for a specific splice isoform of the transmembrane protein tyrosine phosphatase LAR. 964 58

Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, alpha5beta1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZ gene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G1. Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G1-to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.
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PMID:Cloning and characterization of a GC-box binding protein, G10BP-1, responsible for repression of the rat fibronectin gene. 967 87

Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg. AM but inhibited poorly by Eleg. PM and Kistrin. In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the alpha5 beta1 complex. In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an alpha5 beta1-dependent manner. Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the alpha5 beta1 complex by the PRGD molecules. These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.
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PMID:Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation. 976 21

The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes. The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments. Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1). The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components. The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs. The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain. The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay. The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels. Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2. In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified. Thus, the results indicate several previously undetected interactions of TSP1, which is known to be highly expressed during embryonic development, tissue remodeling and wound healing.
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PMID:Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1. 984 Apr 42

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.
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PMID:Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells. 985 37


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