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)

Oocytes of Crassostrea gigas and Mytilus galloprovincialis are arrested in metaphase I when they are spawned and ready to be fertilized. To investigate the role of MAP kinase in maintaining metaphase I arrest, oocytes were exposed to the MEK inhibitor U0126, and the effects on chromosome behavior and MAPK activity were examined by bisbenzimide staining and in immunoblots with anti-phospho MAPK antibodies. Following treatment with 50 microM U0126, active MAPK was undetectable and oocytes resumed meiosis, forming enlarged polar bodies and undergoing chromosome decondensation. Prophase stage oyster oocytes maturing spontaneously in seawater completed germinal vesicle breakdown in the presence of U0126, but failed to arrest in metaphase I, and also formed polar bodies and underwent chromosome decondensation. Treatment of oyster oocytes with the protein synthesis inhibitor, emetine (500 microM), also caused them to resume meiosis, although substantial MAPK activity remained. Levels of phospho-MEK also decreased during emetine treatment. 35 S-methionine incorporation in emetine treated oocytes was reduced to only 5% of control values. These data show that, while active MAPK is necessary to maintain metaphase I arrest, other proteins are also required.
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PMID:MAPK is involved in metaphase I arrest in oyster and mussel oocytes. 1294 25

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

Activation of the hepatocyte growth factor receptor Met induces a morphogenic response and stimulates the formation of branching tubules by Madin-Darby canine kidney (MDCK) epithelial cells in three-dimensional cultures. A constitutively activated ErbB2/Neu receptor, NeuNT, promotes a similar invasive morphogenic program in MDCK cells. Because both receptors are expressed in breast epithelia, are associated with poor prognosis, and hepatocyte growth factor (HGF) is expressed in stroma, we examined the consequence of cooperation between these signals. We show that HGF disrupts NeuNT-induced epithelial morphogenesis, stimulating the breakdown of cell-cell junctions, dispersal, and invasion of single cells. This correlates with a decrease in junctional proteins claudin-1 and E-cadherin, in addition to the internalization of the tight junction protein ZO-1. HGF-induced invasion of NT-expressing cells is abrogated by pretreatment with a pharmacological inhibitor of the mitogen-activated protein kinase kinase (MEK) pathway, which restores E-cadherin and ZO-1 at cell-cell junctions, establishing the involvement of MEK-dependent pathways in this process. These results demonstrate that physiological signals downstream from the HGF/Met receptor synergize with ErbB2/Neu to enhance the malignant phenotype, promoting the breakdown of cell-cell junctions and enhanced cell invasion. This is particularly important for cancers where ErbB2/Neu is overexpressed and HGF is a physiological growth factor found in the stroma.
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PMID:HGF converts ErbB2/Neu epithelial morphogenesis to cell invasion. 1554 98

Our previous studies indicated that opioid-induced cardioprotection occurs via activation of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, other elements of the Met(5)-enkephalin (ME) cardioprotection pathway are not fully characterized. In the present study, we investigated the role of tyrosine kinase, MAPK, and phosphatidylinositol 3-kinase (PI3K) signaling in ME-induced protection. Ca(2+)-tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion and subjected to simulated ischemia for 180 min. ME was administered 15 min before the 180 min of simulated ischemia; blockers were administered 15 min before ME. Cell death was assessed by trypan blue as a function of time. The epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 (250 nM) blocked ME-induced protection, but the inactive analog AG-9 (100 microM) did not. Treatment with herbimycin (1 microM) completely eliminated ME-induced protection. To verify that ME activates EGFR and to determine the involvement of Src, Western blotting of EGFR was performed after ME administration with and without herbimycin A. ME resulted in herbimycin-sensitive robust phosphorylation of EGFR at Tyr(992) and Tyr(1068). Administration of the selective MAPK inhibitor PD-98059 (10 nM) and the specific MEK1/2 inhibitor U-0126 (10 microM) also inhibited ME-induced cardioprotection. ME-induced ERK1/2 phosphorylation was significantly reduced by PD-98059, the EGFR kinase inhibitor PD-153035 (10 microM), and chelerythrine (2 microM). The PI3K inhibitor LY-294002 (20 microM) abrogated ME-induced protection, and ME-induced Akt phosphorylation at Ser(473) was suppressed by LY-294002, PD-153035, and chelerythrine. We conclude that ME-induced cardioprotection is mediated via Src-dependent EGFR transactivation and activation of the PI3K and MAPK pathways.
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PMID:Met5-enkephalin-induced cardioprotection occurs via transactivation of EGFR and activation of PI3K. 1556 40

The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-microm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBalpha (Akt1), GSK3alpha, GSK3beta, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and beta1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, L-alpha-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation.
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PMID:Tumor cell pseudopodial protrusions. Localized signaling domains coordinating cytoskeleton remodeling, cell adhesion, glycolysis, RNA translocation, and protein translation. 1598 31

BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome c and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2 inactivation and caspase-independent down-regulation of Mcl-1. Inducible expression of a constitutively active MEK1 construct did not prevent Mcl-1 down-regulation, suggesting that this event is not related to MEK/ERK pathway inactivation. Furthermore, BAY 43-9006 did not induce major changes in Mcl-1 mRNA levels monitored by real-time PCR or Mcl-1 promoter activity demonstrated by luciferase reporter assays, but it did enhance Mcl-1 down-regulation in actinomycin D-treated cells. Inhibition of protein synthesis by cycloheximide or proteasome function with MG132 and pulse-chase studies with [35S]methionine demonstrated that BAY 43-9006 did not diminish Mcl-1 protein stability, nor did it enhance Mcl-1 ubiquitination, but instead markedly attenuated Mcl-1 translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor. Finally, ectopic expression of Mcl-1 in leukemic cells markedly inhibited BAY 43-9006-mediated cytochrome c cytosolic release, caspase-9, -7, and -3 activation, as well as cell death, indicating that Mcl-1 operates upstream of cytochrome c release and caspase activation. Together, these findings demonstrate that BAY 43-9006 mediates cell death in human leukemia cells, at least in part, through down-regulation of Mcl-1 via inhibition of translation.
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PMID:Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation. 1610 13

Inappropriate activation of MET, the receptor tyrosine kinase for hepatocyte growth factor (HGF), has been implicated in tumorigenesis. Although we have previously shown that HGF/MET signaling controls survival and proliferation of multiple myeloma (MM), its role in the pathogenesis of other B-cell malignancies has remained largely unexplored. Here, we have examined a panel of 110 B-cell malignancies for MET expression, which, apart from MM (48%), was found to be largely confined to diffuse large B-cell lymphomas (DLBCLs) (30%). No amplification of the MET gene was found; however, mutational analysis revealed 2 germ-line missense mutations: R1166Q in the tyrosine kinase domain in 1 patient, and R988C in the juxtamembrane domain in 4 patients. The R988C mutation has recently been shown to enhance tumorigenesis. In MET-positive DLBCL cells, HGF induces MEK-dependent activation of ERK and PI3K-dependent phosphorylation of PKB, GSK3, and FOXO3a. Furthermore, HGF induces PI3K-dependent alpha4beta1 integrin-mediated adhesion to VCAM-1 and fibronectin. Within the tumor microenvironment of DLBCL, HGF is provided by macrophages, whereas DLBCL cells themselves produce the serine protease HGF activator (HGFA), which autocatalyzes HGF activation. Taken together, these data indicate that HGF/MET signaling, and secretion of HGFA by DLBCL cells, contributes to lymphomagenesis in DLBCL.
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PMID:Functional analysis of HGF/MET signaling and aberrant HGF-activator expression in diffuse large B-cell lymphoma. 1618 74

The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.
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PMID:Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation. 1622 11

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals for monitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC50 of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.
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PMID:Microarrays for the functional analysis of the chemical-kinase interactome. 1631 6

Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell-cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal-binding partners and downstream signaling pathways in cell-cycle regulation. We show that cell-cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr-Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C-terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell-cycle progression and activation of MAPK and JNK, while the binding of phospholipase-Cgamma or phosphatidylinositol 3-kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3-kinase, phospholipase-Cgamma, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK-dependent and independent signaling contribute to Met receptor-mediated cell-cycle progression, but phospholipase-Cgamma or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3-kinase signaling are required for cell-cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors.
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PMID:Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling. 1633 88


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