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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.
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PMID:Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases. 165 5

Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNFalpha and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production of NO and TNFalpha. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNFalpha, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNFalpha mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNFalpha gene expression in endotoxin-activated glial cells.
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PMID:Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. 946 88

Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA.
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PMID:Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils. 1035 7

Stimulating macrophages with bacterial endotoxin (LPS) activates numerous intracellular signaling pathways that lead to the production of TNF. In this study, we show that four mitogen-activated protein (MAP) kinase pathways are activated in LPS-stimulated macrophages: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation of a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full induction of the TNF gene. Interestingly, a dramatic induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases. Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expression. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases. The proximity of the MAP kinase-responsive element to the transcriptional initiation site suggested that MAP kinases regulate the transcriptional initiation complex. Utilizing alpha-amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats (Delta31) led to >90% reduction in MAP kinase-mediated TNF production. Thus, our data demonstrate coordination of multiple MAP kinase pathways in TNF production and suggest that the CTD of RNA polymerase II is required to execute MAP kinase signaling in TNF expression.
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PMID:Regulation of TNF expression by multiple mitogen-activated protein kinase pathways. 1084 89

Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore. in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mitogen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice. These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection.
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PMID:Trypanosoma cruzi infection (Chagas' disease) of mice causes activation of the mitogen-activated protein kinase cascade and expression of endothelin-1 in the myocardium. 1107 62

Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.
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PMID:Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. 1205 62

Conjugated linoleic acid (CLA) has chemoprotective properties in a variety of experimental cancer models. We have previously observed that dietary CLA inhibits colon tumorigenesis induced by 1,2-dimethylhydrazine in rats. In addition, our in vitro studies have shown that CLA inhibits DNA synthesis and induces apoptosis in HT-29 cells, the human colon adenocarcinoma cell line. The insulin-like growth factor (IGF) system regulates the growth of HT-29 cells by an autocrine mechanism. The present study examined whether the growth inhibitory effect of CLA is related to changes in the IGF system in HT-29 cells. To determine whether CLA inhibits IGF-II production, HT-29 cells were incubated in serum-free medium in the presence of various concentrations of CLA. CLA decreased protein levels of both mature and pro IGF-II and IGF-II transcripts. Whereas exogenous IGF-I and IGF-II produced an increase in cell number, neither IGF-I nor IGF-II counteracted the negative growth regulatory effect of CLA. Reverse transcriptase-polymerase chain reaction and Western blot analysis of total cell lysates revealed that CLA decreased IGF-I receptor (IGF-IR) transcript and protein levels in a dose-dependent manner. Immunoprecipitation/Western blot studies revealed that CLA inhibited IGF-I-induced phosphorylation of IGF-IR and insulin-receptor substrate (IRS)-1, recruitment of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to IGF-IR, IGF-IR-associated PI3K activity, and phosphorylated Akt and extracellular signal-regulated kinase (ERK)-1/2 levels. In conclusion, the inhibition of cell proliferation and induction of apoptosis by CLA in HT-29 cells may be mediated in part by its ability to decrease IGF-II synthesis and to downregulate IGF-IR signaling and the PI3K/Akt and ERK-1/2 pathways.
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PMID:Conjugated linoleic acid downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells. 1288 57

The TRAP/Mediator coactivator complex serves as a molecular bridge between gene-specific activators and RNA polymerase II. TRAP220/Med1 is a key component of TRAP/Mediator that targets the complex to nuclear hormone receptors and other types of activators. We show here that human TRAP220/Med1 is a specific substrate for extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. We demonstrate that ERK phosphorylates TRAP220/Med1 in vivo at two specific sites: threonine 1032 and threonine 1457. Importantly, we found that ERK phosphorylation significantly increases the stability and half-life of TRAP220/Med1 in vivo and correlates with increased thyroid hormone receptor-dependent transcription. Furthermore, ERK phosphorylates TRAP220/Med1 in a cell cycle-dependent manner, resulting in peak levels of expression during the G(2)/M phase of the cell cycle. ERK phosphorylation of ectopic TRAP220/Med1 also triggered shuttling into the nucleolus, thus suggesting that ERK may regulate TRAP220/Med1 subnuclear localization. Finally, we observed that ERK phosphorylation of TRAP220/Med1 stimulates its intrinsic transcriptional coactivation activity. We propose that ERK-mediated phosphorylation is a regulatory mechanism that controls TRAP220/Med1 expression levels and modulates its functional activity.
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PMID:Activation of TRAP/mediator subunit TRAP220/Med1 is regulated by mitogen-activated protein kinase-dependent phosphorylation. 1631 96

Legionella pneumophila causes community-acquired pneumonia with high mortality, but little is known about its interaction with the alveolar epithelium. The aim of this study was to investigate whether L. pneumophila infection of lung epithelial cells (A549) resulted in pro-inflammatory activation. L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-alpha, IL-1beta, interferon-gamma and granulocyte colony-stimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colony-stimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-kappaB subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-kappaB complex dose-dependently lowered IL-8 expression. Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase- and nuclear factor-kappaB/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.
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PMID:Legionella pneumophila-induced NF-kappaB- and MAPK-dependent cytokine release by lung epithelial cells. 1697 6

Lung caner cells have a striking tendency to metastasize to bone. The chemokine stromal cell-derived factor-1 (SDF-1) is constitutively secreted by osteoblasts and bone marrow stromal cells and plays a key role for homing of hematopoietic cells to the bone marrow. Reverse transcriptase-polymerase chain reaction and flow cytometry studies demonstrated SDF-1 receptor (CXCR4) messenger RNA (mRNA) and surface expression of CXCR4 in lung cancer cell lines, especially higher in those with high invasiveness (A549) as compared with lower level in H928 cells and H1299 cells. SDF-1, osteoblast-conditioned medium (OBCM) and stromal cell-conditioned medium all induced the invasiveness of lung cancer cells. Matrix metalloproteinase (MMP)-9 small interfering RNA inhibited the SDF-1alpha- or OBCM-induced MMP-9 expression and thereby significantly inhibited the SDF-1alpha- or OBCM-induced cell invasion. Furthermore, mitogen-activated protein kinase kinase inhibitor PD98059 suppressed SDF-1alpha-induced MMP-9 mRNA expression. Transient transfection with dominant-negative extracellular signal-regulated kinase (ERK) mutant also showed that the ERK-signaling pathway was involved in SDF-1alpha-induced MMP-9 expression. Moreover, nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide suppressed the MMP-9 promoter activity enhanced by SDF-1alpha. Both mitogen-activated protein kinase kinase inhibitor and ERK mutant also antagonized SDF-1alpha-induced NF-kappaB-driven luciferase promoter activity. Taken together, our results indicated that bone marrow-derived-SDF-1alpha enhances the invasiveness of lung cancer cells by increasing MMP-9 expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.
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PMID:Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis. 1791 7


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