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 aim of this study was to compare the antigenicity and the immunogenicity of five constructs of a peptide, including the peptide in single copy, a tandem repeat containing three copies, a copolymer with glutaraldehyde and two constructs based on the MAP (Multiple Antigenic Peptide) model, one containing two copies (MAP-2) and the other, eight copies of the peptide (MAP-8). The peptide used in this test was the 115-131 sequence derived from the rSm28-GST antigen of Schistosoma mansoni. All constructs were recognized by rSm28-GST specific antibodies in solid phase immunoassays. However, the binding was higher when the MAP-8 was used as antigen at least partly because of its better coating on the microtiter plates. In vitro lymphoproliferative assays showed that polymer was mitogenic, repeat and MAP-2 did not stimulate rSm28-GST specific T cells while MAP-8 induced a slight response. The injection of MAP-8 to rats led to important antibody and T cell responses higher than those obtained with the other constructs. The IgG2a (cytotoxic antibody in schistosomiasis)/IgG2c (blocking antibody) ratio was independent of the immunogen. Taken together these results demonstrate that both the antigenicity and the immunogenicity of a peptide containing T and B cell epitope(s) are strongly related to the molecular form whereby it is presented and that the MAP-8 construct can be useful in serodiagnosis or in vaccination trials using synthetic peptides.
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PMID:Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide. 160 96

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.
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PMID:Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. 841 57

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.
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PMID:Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level. 863 13

The function of the Xenopus c-mos proto-oncogene product (Mos(xe)) has been investigated during oocyte maturation. Experiments with a new antibody able to immunoblot Mos(xe) demonstrated the time course of MAP kinase (MAP K) activation in oocytes paralleled Mos(xe) accumulation, and in activated eggs the deactivation of MAP K paralleled the degradation of Mos(xe). Ablation of Mos synthesis by microinjection of antisense oligodeoxynucleotides abolished activation of MAP K by progesterone, but microinjection of GST-Mos fully restored both MAP K activation and germinal vesicle breakdown (GVBD). The Mos(xe) level at metaphase of Meiosis I (MI) was 2 - 3-fold less than that at metaphase of Meiosis II (MII), but MAP K activation was maximal at metaphase in both MI and MII. In the transition between MI and MII, both cyclin B and Mos(xe) levels rapidly declined in the presence of cycloheximide and injection of exogenous GST-Mos(xe) did not prevent degradation of either protein, although MAP K was activated. Microinjection of GST-Mos(xe) into oocytes was able to activate MAP K before GVBD and H1 kinase activation, and microinjection of constitutively-activated thiophosphorylated MAP K induced de novo synthesis of Mos(xe) before H1 kinase activation, suggesting the existence of a positive feedback loop between MAP K and Mos(xe) accumulation.
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PMID:Mos proto-oncogene function during oocyte maturation in Xenopus. 866 47

Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cerevisiae MAP kinases FUS3/KSS1, and a GST-Pmk1 fusion protein has kinase activity in vitro. pmk1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants. pmk1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmk1 acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GAL1 promoter in yeast, PMK1 can rescue the mating defect in a fus3 kss1 double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.
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PMID:MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. 894 11

STE20-homologous proteins have been implicated in mammalian MAP kinase pathways as important transducers of signals from p21 family GTPases. We have cloned a novel STE20 family member, which we call KHS for kinase homologous to SPS1/STE20, that encodes a kinase of 95 kD which is expressed in a variety of tissues. Transiently expressed fusion protein GST-KHS exhibits phosphotransferase activity toward a panel of test substrates, including myelin basic protein (MBP), which is phosphorylated by all known STE20 homologues. KHS is most closely related to another human STE20, GC kinase (74% similar in the catalytic domain), which has recently been placed upstream of the stress-activated MAP kinases (SAPKs/JNKs). KHS also activates JNK in transient coexpression experiments, suggesting a role for KHS in the stress response of fibroblasts. Characterization and comparison of the regulation of these two kinases will be important in elucidating MAP kinase signalling cascades.
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PMID:A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase. 903 72

Gamma-tubulin is localized at the microtubule organizing center and is thought to participate in the organizing of the microtubule network. In this study, we isolated a cDNA of rat gamma-tubulin. The rat gamma-tubulin cDNA encoded 451 amino acids, the same number as that of its counterpart in other vertebrates, and its structure was found to be highly conserved in vertebrates. In a previous work, we identified HP33 (hepatocarcinogenesis- and hepatocellular proliferation-related 33-kDa protein) that was localized at the centrosome of hepatic cells and that exhibited MAP-like activity. In vitro GST pull-down assay using highly purified recombinant HP33 and bacterially expressed gamma-tubulin demonstrated that HP33 bound to gamma-tubulin directly. These results suggest that HP33 is localized at the centrosome via association with both the microtubule and its minus end-specific component, gamma-tubulin.
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PMID:Structure of rat gamma-tubulin and its binding to HP33. 1047 Aug 52

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 + MKK7-activated JNK3 alpha 1 had Vmax 715-fold greater than nonactivated JNK3 alpha 1 and MKK7-activated JNK3 alpha 1 had Vmax 250-fold greater than nonactivated JNK3 alpha 1. In contrast, MKK4-activated JNK3 alpha 1 had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of mass spectrometry. These data suggest that MKK7 was largely responsible for JNK3 alpha 1 activation and that a single threonine phosphorylation may be all that is needed for JNK3 alpha 1 to be active. The steady-state rate constants kcat, Km(GST-ATF2++), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK3 alpha 1 were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylation does not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor, SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK3 alpha 1 compared to the bisphosphorylated JNK3 alpha 1, suggesting only a modest effect of tyrosine phosphorylation on inhibitor binding.
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PMID:Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1. 1071 36

The effects of pituitary and extrapituitary prolactin include cellular proliferation and differentiation. PC12 cells was used as a model to delineate respective signaling of prolactin. Prolactin acted as a mitogen for undifferentiated PC12 cells, as measured by significant increases in bromodeoxyuridine incorporation and in cell numbers, with an efficacy equal to epidermal growth factor. Both the long and short form of the prolactin receptor was expressed, yet only the long isoform was tyrosine-phosphorylated upon agonist binding. Functional prolactin receptor signaling was further demonstrated in the activation of JAK2 and phosphorylation activation of the transcription factors Stat1, -3, and -5a. Surprisingly, prolactin stimulated a sustained activation of Raf-B, without activation of the MAP kinases ERK1 or -2. Instead, in solid phase kinase assays using a glutathione S-transferase-c-Jun fusion protein (amino acids 1-79) as the substrate, a significant activation of the mitogen-activated protein Janus kinase (c-Jun N-terminal kinase; JNK) was observed. The prolactin-induced activation of JNK was prolonged and accompanied by a significant increase in c-Jun mRNA abundance and c-Jun protein synthesis. Moreover, analysis of bromodeoxyuridine incorporation at the single cell level revealed that epidermal growth factor-dependent incorporation was inhibited by PD98059 and independent of SB203580, whereas prolactin-induced incorporation was ERK and mitogen-activated protein kinase p38 independent but was abolished with JNK inhibition by 30 microm SB203580. Our studies suggest that prolactin may have a role in the growth of PC12 cells, where it stimulates concurrent mitogenic and differentiation-promoting signaling pathways.
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PMID:Prolactin-induced cell proliferation in PC12 cells depends on JNK but not ERK activation. 1080 11

Microtubule-associated protein 2 (MAP-2) isoforms are developmentally expressed in the nervous system and contain a number of functional domains. Adjacent to the first repeat of the microtubule-binding domain is an RTPPKSP motif for binding SH3 domains. To identify SH3-containing proteins that interact with MAP-2, transfections, filter overlay assays, glutathione S-transferase (GST)-mediated binding assays, co-immunoprecipitations and enzyme-linked immunosorbent assays were performed. Transfections of MAP-2a, MAP-2b, and MAP-2c constructs into COS7 cells, followed by incubation of the cell lysates with SH3-GST fusion proteins, determined that the strongest interaction was between MAP-2c and the non-receptor tyrosine kinase Fyn; however, MAP-2b and MAP-2c also bound to Grb2. Co-immunoprecipitation of Fyn and MAP-2c from human fetal homogenates confirmed the interaction in vivo. MAP-2 synthetic peptides spanning the RTPPKSP motif bound to Fyn, and the interaction was regulated by phosphorylation. Co-transfections with MAP-2c and the extracellular signal-regulated kinase 2 (ERK2) demonstrated that MAP-2c is threonine/serine-phosphorylated on its RTPPKSP motif and that threonine phosphorylation abolished the MAP-2c/Fyn binding. Kinase assays and co-transfection of MAP-2c and Fyn confirmed that Fyn tyrosine kinase phosphorylates MAP-2c. Thus, the activation of signaling pathways may regulate cytoskeletal dynamics by altering the state of phosphorylation of MAP-2 by both ERK2 and Fyn kinase.
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PMID:Binding of Fyn to MAP-2c through an SH3 binding domain. Regulation of the interaction by ERK2. 1154 90


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