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 21 kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. Less well understood are the downstream signal transduction cascades initiated by Ras in response to external stimuli. Only recently have many diverse studies in lower eukaryotes and vertebrates converged to demonstrate that Ras directly regulates multiple signaling pathways. In most eukaryotes, Ras functions as a positive regulator of an ERK/MAPK signal transduction cascade through the activation of a MEKK. In mammalian cells the primary Ras-responsive MEKK is the protein kinase Raf. Although Raf remains the most significant mediator of Ras signaling in most model systems, it does not explain all the biochemical responses observed in cells with activated Ras proteins. Yeast two hybrid and GST-fusion protein binding studies have identified new proteins distinct from Raf that could interact with Ras in other signaling pathways. In addition to Raf, other potential Ras target proteins include MEKK1, PI(3)K, p120GAP, ralGDS, and PKC zeta. This review will attempt to summarize the current literature of accepted and potential Ras-dependent signaling proteins in both lower eukaryotes and vertebrates.
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PMID:Ras target proteins in eukaryotic cells. 755 21

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

The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and JNK/SAPK MAP kinase cascades have been compared with their ability to elicit histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10T1/2 cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of c-fos and c-jun, showing that neither cascade is absolutely essential for these responses. We then analysed the relationship between ERKs, JNK/SAPKs and the transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and c-jun, respectively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, particularly ERK-2, to GST-Elk1(307-428); there is no cross-specificity of binding. Further, GST-Elk1(307-428) binds preferentially to active rather than inactive ERK-2. In vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1(307-428), whereas ERKs phosphorylate GST-Elk1(307-428) but not GST-cJun1-79. Thus, neither ERKs nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and c-jun induction. The data suggest either that activation of a single MAP kinase subtype is sufficient to elicit a complete nuclear response, or that other uncharacterised routes exist.
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PMID:Neither ERK nor JNK/SAPK MAP kinase subtypes are essential for histone H3/HMG-14 phosphorylation or c-fos and c-jun induction. 858 71

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.
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PMID:Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. 908 72

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
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PMID:Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. 956 38

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.
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PMID:A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors. 1007 22

The interaction of tumor necrosis factor-alpha (TNFalpha) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42(mapk/erk2) phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207-425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFalpha, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42(mapk/erk2) and by specific inhibition of p42(mapk/erk2) activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42(mapk/erk2), and constitutively active MEK-1 followed by metabolic labeling with [(32)P]orthophosphate indicated that p42(mapk/erk2) phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42(mapk/erk2)-dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.
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PMID:Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization. 1055 65

Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.
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PMID:Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK. 1080 94

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

A recent report that microinjection of the SH3 domain of PLC-gamma1 could induce DNA synthesis raised the functional importance of the SH3 domain of PLC-gamma1 in mitogenic signaling. In this report, we provide evidence that SOS1, a p21Ras-specific guanine nucleotide exchange factor, directly binds to the SH3 domain of PLC-gamma1, and that the SH3 domain of PLC-gamma1 is involved in SOS1-mediated p21Ras activation. SOS1 was coprecipitated with the GST-fused SH3 domain of PLC-gamma1 in vitro. The interaction between SOS1 and the PLC-gamma1 SH3 domain is mediated by direct physical interaction. The carboxyl-terminal proline-rich domain of SOS1 is involved in the interaction with the PLC-gamma1 SH3 domain. Moreover, PLC-gamma1 could be co-immunoprecipitated with SOS1 antibody in cell lysates. From transient expression studies, we could demonstrate that the SH3 domain of PLC-gamma1 is necessary for the association with SOS1 in vivo. Intriguingly, overexpression of the SH3 domain of PLC-gamma1, lipase-inactive PLC-gamma1, or wild-type PLC-gamma1 elevated p21Ras activity and ERK activity when compared with vector transfected cells. The PLC-gamma1 mutant lacking the SH3 domain could not activate p21Ras. p21Ras activities in cell lines overexpressing either PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 were elevated about 2-fold compared to vector transfected cells. This study is the first to demonstrate that the PLC-gamma1 SH3 domain enhances p21Ras activity, and that the SH3 domain of PLC-gamma1 may be involved in the SOS1-mediated signaling pathway.
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PMID:Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-gamma1. 1091 76


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