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 have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.
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PMID:The acute and chronic stimulatory effects of endothelin-1 on glucose transport are mediated by distinct pathways in 3T3-L1 adipocytes. 1110 76

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.
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PMID:Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation. 1111 9

1. Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) in vivo. To determine the signal transduction cascade through which this is mediated, we measured the effect of inhibition of various signalling pathways on VEGF-mediated acute increases in L(p) in individually perfused frog mesenteric microvessels. 2. VEGF receptors have previously been shown to activate phospholipase C-gamma (PLCgamma), protein kinase C (PKC) and MEK, the mitogen-activated and extracellular signal-related kinase (ERK) kinase. To determine the role of these signalling pathways we measured the effects of inhibitors of each on the VEGF-mediated increase in L(p). 3. VEGF-mediated increases in L(p) were attenuated by pre-treatment with the PLC inhibitor U73122, but not affected by treatment with the inactive enantiomer U73343. The PLC inhibitor was also able to attenuate the increase in L(p) mediated by the inflammatory mediator ATP. 4. Inhibition of either PKC or MEK activation using the selective inhibitors bisindolylmaleimide (BIM, 1 microM) and PD98059 (30 microM), respectively, did not change the VEGF-mediated increase in L(p). However, PD98059, BIM and U73122 all reduced phosphorylation of ERK1/2 determined by Western blot analysis with anti-phospho-ERK1/2 antibodies. 5. Furthermore, inhibition of the conversion of diacyl glycerol (DAG) to arachidonic acid, by perfusion with the DAG lipase inhibitor RHC80267 (50 microM), did not attenuate the increase in L(p) brought about by VEGF. 6. These data suggest that VEGF acutely increases microvascular permeability in vivo through a mechanism that is dependent on PLC stimulation, but is independent of PKC or MEK activation or production of arachidonic acid from DAG. We therefore propose that VEGF acutely acts to increase L(p) through the direct actions of DAG, independently of PKC or arachidonic acid.
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PMID:In vivo mechanisms of vascular endothelial growth factor-mediated increased hydraulic conductivity of Rana capillaries. 1145 65

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.
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PMID:Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells. 1147 33

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.
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PMID:Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway. 1148 7

Nuclear phosphoinositide breakdown mediated by nuclear PLCbeta phosphorylation is a downstream effect of IL-2 stimulation in primary human NK cells that is involved in the proliferative response to IL-2. Here we investigated whether the nuclear phosphoinositide turnover in NK cells is a response confined to IL-2, or rather represents a more general mechanism of nuclear signalling linked to the proliferative response of NK cells to activatory cytokines. We therefore focused on IL-12 and IL-15-induced nuclear events in primary human NK cells. Our results show that IL-12 and IL-15 activate nuclear PLCbeta1 in NK cells, with delayed kinetics as compared to IL-2. The nuclear PLC activation induced by the cytokines could be blocked by the MEK-1 inhibitor PD 98059, suggesting its dependence upon MAPKinase activation. Our conclusion is that the three cytokines that activate NK cells and that bind partially similar (IL-2, IL-15) or different surface receptors (IL-12), all induce activation of PLCbeta1 in the nucleus of NK cells via MAPKinase. Thus, the activation of nuclear PLCbeta1 appears as a physiologically relevant general mechanism of response to cytokine stimulation in human natural killer cells.
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PMID:IL-12 and IL-15 induce activation of nuclear PLCbeta in human natural killer cells. 1174 56

Ecto-5'-nucleotidase (E5'N, CD73) is key enzyme responsible for formation of anti-inflammatory and immunosuppressive adenosine from extracellular nucleotides as well as an important surface molecule involved in cellular signalling. In this study we provide evidence that the pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha) may reduce the capacity of human endothelial cells to produce adenosine by a decrease in surface expression and in the activity of E5'N. Human umbilical vein endothelial cells incubated for 24 h with TNF-alpha lost 54% of the activity of E5'N while activities of the other enzymes involved in adenosine metabolism remained unaffected. Immunofluorescence staining with anti-E5'N (1E9) following exposure to TNF-alpha, showed reduced numbers of positive cells. TNF-alpha induced down-regulation of E5'N was prevented by addition of the PLC inhibitor neomycin, but not by inhibitors of MAPK-like pathways (MEK and p38). Therefore, we conclude that TNF-alpha through activation of endogenous PLC leads to cleavage of the GPI-linkage of E5'N resulting in loss of E5'N from the extracellular surface. This change may lead to decrease in formation of adenosine and could be an important mechanism of endothelial activation during inflammation.
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PMID:Regulation of ecto-5'-nucleotidase by TNF-alpha in human endothelial cells. 1203 Mar 67

Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca(2+)(o)) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca(2+)]. Ca(2+)(o), neomycin, and gentamicin also activated the extracellular signal-regulated kinases, ERK1 and ERK2. Neomycin-induced ERK activation was both dose- and time-dependent and was attenuated by inhibitors of phosphatidylinositol 3-kinase, phosphatidylinositol bisphosphate (PIP(2))-specific phospholipase C, and MEK1, but not of protein kinase C. Thus, proximal tubular OK cells express a CaR that mediates Ca(2+)(i) mobilization and PIP(2)-PLC-dependent ERK activation in response to AGAs and thus could play a role in AGA-induced nephrotoxicity.
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PMID:Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor. 1203 77

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.
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PMID:The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. 1218 53

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.
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PMID:Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-kappaB pathways in canine tracheal smooth muscle cells. 1222 Jun 16


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