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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated in IMR90 cells the effects of N-formyl-Met-Leu-Phe (N-fMLP) and WKYMVm (W peptide) on activation of the NADPH oxidase-like enzyme. In serum-deprived human fibroblasts, exposure to 100 microM N-fMLP or 10 microM peptide W for 1 min induced both p47phox translocation and NADPH-dependent superoxide generation. These effects were in large part mediated by prevention of the rapid activation of extracellular signal-regulated kinases (ERKs) by preincubation with the MEK1 inhibitor PD098059. Furthermore, responses to N-fMLP or W peptide were inhibited by pertussis toxin, suggesting the involvement of a seven-transmembrane G protein-coupled receptor(s) for peptides. RT-PCR experiments demonstrated the expression in these cells of the low-affinity receptor FPRL1, but not the high-affinity receptor FPR. Incubation with radiolabeled WKYMVm, which had a higher efficiency on FPRL1, revealed that human fibroblasts express binding sites for 125I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a Kd of 155.99 nM and a receptor density of about 16,200 molecules/cell. HEK293 cells, which express a NADPH oxidase-like enzyme but not formyl peptide receptors, transiently transfected with FPRL1 cDNA produced superoxide on stimulation with N-fMLP or W peptide, demonstrating that this receptor is biologically functional.
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PMID:Low-affinity receptor-mediated induction of superoxide by N-formyl-methionyl-leucyl-phenylalanine and WKYMVm in IMR90 human fibroblasts. 1474 31

We have previously reported that leukemia inhibitory factor (LIF) gradually increased cardiac L-type Ca2+ channel current (I(CaL)), which peaked at 15 minutes in both adult and neonatal rat cardiomyocytes, and this increase was blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. This study investigated the molecular basis of LIF-induced augmentation of I(CaL) in rodent cardiomyocytes. LIF induced phosphorylation of a serine residue in the alpha(1c) subunit (Ca(v)1.2) of L-type Ca2+ channels in cultured rat cardiomyocytes, and this phosphorylation was inhibited by PD98059. When constructs encoding either a wild-type or a carboxyl-terminal-truncated rabbit Ca(v)1.2 subunit were transfected into HEK293 cells, LIF induced phosphorylation of the resultant wild-type protein but not the mutant protein. Cotransfection of constitutively active mitogen-activated protein kinase kinase also resulted in phosphorylation of the Ca(v)1.2 subunit in the absence of LIF stimulation. In in-gel kinase assays, extracellular signal-regulated kinase phosphorylated a glutathione S-transferase fusion protein of the carboxyl-terminal region of Ca(v)1.2 (residues 1700 through 1923), which contains the consensus sequence Pro-Leu-Ser-Pro. A point mutation within this consensus sequence, which results in a substitution of alanine for serine at residue 1829 (S1829A), was sufficient to abolish the LIF-induced phosphorylation. LIF increased I(CaL) in HEK cells transfected with wild-type Ca(v)1.2 but not with the mutated version. These results provide direct evidence that LIF phosphorylates the serine residue at position 1829 of the Ca(v)1.2 subunit via the actions of extracellular signal-regulated kinase and that this phosphorylation increases I(CaL) in cardiomyocytes.
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PMID:Leukemia inhibitory factor activates cardiac L-Type Ca2+ channels via phosphorylation of serine 1829 in the rabbit Cav1.2 subunit. 1504 19

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-kappaB activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-kappaB activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-kappaB activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-kappaB activation. Further, wild-type LRR more effectively suppressed NF-kappaB activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.
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PMID:Reciprocal cross-talk between Nod2 and TAK1 signaling pathways. 1507 45

The tobacco N gene, a member of the Toll-interleukin 1 homology region/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class of resistance (R) genes, confers resistance to tobacco mosaic virus (TMV). We used a candidate gene approach to identify known defense genes that were also involved in N signaling. The requirement for these genes was determined by downregulating their expression using the well-established tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS). Silencing of genes encoding a mitogen-activated protein kinase (MAPK) NTF6/NRK1, or an MAPK kinase (MAPKK) MEK1/NQK1, attenuated N-mediated resistance to TMV. We also found that N resistance is compromised in plants in which expression of WRKY1-WRKY3 and MYB1 transcription factors were downregulated. In addition, suppression of jasmonic acid (JA) signaling component COI1 ortholog affected N function. However, downregulation of expression of CTR1 ortholog leads to more rapid hypersensitive response (HR). The involvement of these genes in N- and other R-gene-mediated defense provides further evidence for the convergence of downstream signaling pathways of different R genes.
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PMID:Involvement of MEK1 MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in N-mediated resistance to tobacco mosaic virus. 1514 81

We characterized the tracheal and bronchial relaxation caused by proteinase-activated receptor-2 (PAR-2) activation in ddY mice and/or in wild-type and PAR-2-knockout mice of C57BL/6 background. Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and Thr-Phe-Leu-Leu-Arg-amide, PAR-2- and PAR-1-activating peptides, respectively, caused relaxation in the isolated ddY mouse trachea and main bronchus. The relaxation was abolished by specific inhibitors of cyclooxygenase (COX)-1, COX-2, mitogen-activated protein kinase kinase (MEK), and p38 MAP kinase. The MEK and p38 MAP kinase inhibitors did not affect prostaglandin E(2)-induced relaxation. Inhibitors of cytosolic Ca(2+)-dependent phospholipase A(2) (PLA), Ca(2+)-independent PLA(2), diacylglycerol lipase, tyrosine kinase, and protein kinase C exhibited no or only minor inhibitory effects on the PAR-mediated relaxation. Trypsin, a PAR-2 activator, and 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide, a potent PAR-2-activating peptide, in addition to SLIGRL-NH(2), caused airway relaxation in wild-type C57BL/6 mice, as in ddY mice. In PAR-2-knockout mice, the peptide effects were absent and the potency of trypsin decreased. Desensitization of PAR-2 and/or PAR-1 greatly suppressed the relaxant effect of trypsin. The bronchial and tracheal tissues displayed distinct sensitivities toward trypsin and the PAR-2-activating peptides. Our data indicate an involvement of both COX-1 and COX-2, and the MEK-extracellular signal-regulated kinase and p38 MAP kinase signaling pathways in the PAR-2- and PAR-1-triggered relaxation of mouse airway tissue, and substantiate a role for PAR-2 in regulating both the trachea and bronchial responsiveness in the mouse lung.
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PMID:Proteinase-activated receptor-2-mediated relaxation in mouse tracheal and bronchial smooth muscle: signal transduction mechanisms and distinct agonist sensitivity. 1519 93

The development of specific inhibitors for the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) has been a recent research focus because of the association of JNK with cell death in conditions such as stroke and neurodegeneration. We have demonstrated previously the presence of critical inhibitory residues within an 11-mer peptide (TI-JIP) based on the sequence of JNK-interacting protein-1 (JIP-1). However, the corresponding region of JNK bound by this JIP-1-based peptide was unknown. To identify this region, we used a novel reverse two-hybrid approach with TI-JIP as bait. We screened a library of JNK1 mutants that had been generated by random PCR mutagenesis and found three mutants of JNK1 that failed to interact with TI-JIP. The mutations in JNK1 were L131R, R309W, and Y320H. Of these mutated residues, Leu-131 and Tyr-320 were located on a common face of the JNK protein close to other residues implicated previously in the interactions of MAPKs with substrates, phosphatases, and scaffolds. To test whether these JNK1 mutants were thus affected in their regulation, we evaluated their activation in mammalian cells in response to hyperosmolarity or cotransfection with a constitutively active upstream kinase or their direct phosphorylation by either MAPK kinase (MKK)4 or MKK7. In each situation, all three JNK mutants were not activated or phosphorylated to the same level as wild-type JNK. Therefore, the results of our unbiased reverse two-hybrid screening approach have identified residues of JNK responsible for binding JIP-1-based peptides as well as MKK4 or MKK7.
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PMID:Reverse two-hybrid screening identifies residues of JNK required for interaction with the kinase interaction motif of JNK-interacting protein-1. 1527 95

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [(3)H]thymidine and [(3)H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione-S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.
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PMID:Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway. 1529 55

Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.
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PMID:Inhibition of constitutively activated nuclear factor-kappaB radiosensitizes human melanoma cells. 1529 81

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.
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PMID:PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes. 1531 32

We investigated the effects of bradykinin (BK) on the production of interleukin (IL)-6 and prostaglandin PGE(2), whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as the signal transduction systems involved, in human osteoblasts (SaM-1 cells). BK receptors B1 (B1R) and B2 (B2R) were expressed in SaM-1 and osteosarcoma (SaOS-2, HOS, and MG-63) cells. Treatment of SaM-1 cells with BK increased the synthesis of both IL-6 and PGE(2) and the increase in both was blocked by HOE140 (B2R antagonist), but not by Des-Arg(9)-[Leu(8)]-BK (B1R antagonist). U-73122, a phospholipase C (PLC) inhibitor, suppressed BK-induced IL-6 and PGE(2) synthesis in SaM-1 cells. In addition, BK caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was inhibited by pretreatment with HOE140 or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) blocker. Furthermore, both SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]) and PD98059 (an inhibitor of MEK, upstream of ERK) attenuated the BK-induced IL-6 and PGE(2) synthesis. BK treatment resulted in the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK)1/2, and 2-APB could suppress BK-induced phosphorylation of ERK1/2. These findings suggest that BK increased both IL-6 and PGE(2) synthesis in osteoblastic cells via B2R and that PLC, IP(3)-induced [Ca(2+)]i, MEK, and MAPKs were involved in the signal transduction in these cells.
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PMID:Activation of osteoblastic functions by a mediator of pain, bradykinin. 1534 32


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