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 studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08+/-0.008 (n=34) to 7.18+/-0.018 (n=33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29+/-0.022 pH U/min (n=14) to 0.50+/-0.024 pH U/min (n=14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.
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PMID:Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells. 1649 13

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.
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PMID:Caldesmon phosphorylation in actin cytoskeletal remodeling. 1654 74

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.
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PMID:A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis. 1671 Mar 59

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.
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PMID:Molecular mechanism of cofilin dephosphorylation by ouabain. 1671 81

The cardiotonic steroid, ouabain, a specific inhibitor of Na(+),K(+)-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of alpha(1)- and alpha(2)-subunits of Na(+),K(+)-ATPase and promoted interaction of alpha(1)- and alpha(2)-subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3alpha/beta, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nM ouabain increased phosphorylation of the alpha-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of alpha(2)-subunit, whereas abundance of the alpha(1)-subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nM concentration, similarly to 100 nM ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.
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PMID:Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism. 1671 87

Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to Baeyer-Villiger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.
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PMID:Degradation of alkyl methyl ketones by Pseudomonas veronii MEK700. 1735 Oct 32

We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.
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PMID:An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation. 1749 Jun

The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphorylation of PKC alpha/beta and delta isoforms, whereas phosphorylation of PKCzeta was unchanged. Ouabain exposure increased interaction of 1- and 2-subunits of Na-pump with c-Src, as assessed by co-immunoprecipitation with c-Src. Phosphorylation of ERK1/2, GSK 3 / and p90rsk activity was increased in response to ouabain, and these effects were prevented in the presence of PD98059 and PP2. In conclusion, the cardiac glycoside ouabain stimulates glycogen synthesis additively to insulin in rat skeletal muscle. This effect is mediated by activation of c-Src-, ERK1/2- p90rsk- and GSK3-dependent signaling pathway.
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PMID:Metabolic and signaling events mediated by cardiotonic steroid ouabain in rat skeletal muscle. 1753 36

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.
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PMID:Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump. 1761 54

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.
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PMID:Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes. 1772 97


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