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)

After a brain insult, ATP is released from injured cells and activates microglia. The microglia that are activated in this way then release a range of bioactive substances, one of which is tumor necrosis factor (TNF). The release of TNF appears to be dependent on the P2X7 receptor. The inhibitors 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene (U0126), anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580), which target MEK (mitogen-activated protein kinase kinase), JNK (c-Jun N-terminal kinase), and p38, respectively, all potently suppress the production of TNF in ATP-stimulated microglia, whereas the production of TNF mRNA is strongly inhibited by U0126 and SP600125. SB203580 did not affect the increased levels of TNF mRNA but did prevent TNF mRNA from accumulating in the cytoplasm. The ATP-provoked activation of JNK and p38 [but not extracellular signal-regulated kinase (ERK)] could be inhibited by brilliant blue G, a P2X7 receptor blocker, and by genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, which are general and src-family-specific tyrosine kinase inhibitors, respectively. Most important, we found that treatment of the microglia in neuron-microglia cocultures with the P2X7 agonist 2'-3'-O-(benzoyl-benzoyl) ATP led to significant reductions in glutamate-induced neuronal cell death, and that either TNF-alpha converting enzyme inhibitor or anti-TNF readily suppressed the protective effect implied by this result. Together, these findings indicate that both ERK and JNK are involved in the regulation of TNF mRNA expression, that p38 is involved in the nucleocytoplasmic transport of TNF mRNA, and that a PTK (protein tyrosine kinase), possibly a member of the src family, acts downstream of the P2X7 receptor to activate JNK and p38. Finally, our data suggest that P2X7 receptor-activated microglia protect neurons against glutamate toxicity primarily because they are able to release TNF.
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PMID:Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. 1471 32

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.
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PMID:Evidence for multiple P2Y receptors in trabecular meshwork cells. 1474 47

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca(2+)-dependent MLC kinase or Ca(2+)-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 microM) increased ERK2 phosphorylation from basal 17 +/- 3 to 53 +/- 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 microM) or U0126 (10 microM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca(2+) rise, because it was unaltered when this was suppressed by the Ca(2+) chelator BAPTA (10 microM) or xestospongin C (3 microM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 microM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 microM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca(2+)-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase.
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PMID:MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain. 1500 25

Kinase Suppressor of Ras1 (KSR1) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf-1, and pharmacologic inactivation of KSR1 may serve as a treatment for Rasdriven malignancies such as pancreatic cancer (Xing, H. R., Cordon-Cardo, C., Deng, X., Tong, W., Campodonico, L., Fuks, Z., and Kolesnick, R. (2003) Nat. Med. 9, 1266-1268). Although some studies demonstrated a requirement for KSR1 kinase activity for its action, others suggested KSR1 acts primarily as a scaffold facilitating assembly of the c-Raf-1/MEK module. We recently established a two-stage in vitro reconstitution assay to measure KSR1 kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). In this assay, KSR1, immunopurified to apparent homogeneity, never comes in contact with recombinant kinases other than c-Raf-1. In the first assay stage, activated KSR1 is incubated with recombinant c-Raf-1 and ATP. In the second stage, activated c-Raf-1 is separated from KSR1, and incubated with unactivated MEK1, unactivated MAPK, Elk-1, and ATP. Elk-1 phosphorylation serves as a specific readout for MAPK activation. However, because KSR1 constitutively associates with MEK1 and this interaction appears critical for KSR1 scaffolding function, it has been argued that the kinase activity detected is an artifact of KSR1-bound MEK1. To address these concerns, we depleted as much as 90% of KSR1-bound MEK1 by high salt washing without altering KSR1 kinase activity. Further, a complete inactivation of KSR1-bound MEK1 by pretreating with the MEK inhibitor PD 98059 prior to the first assay stage did not alter KSR1 kinase activity. In addition, the omission of exogenous recombinant GST-MEK1 from the reaction mixture during the second assay stage abolished Elk-1 phosphorylation confirming KSR1-bound MEK1 does not support MAPK activation in our in vitro assay. Moreover, a kinase-inactive mutant, FLAG-Ki-KSR1(D683A/D700A), which efficiently interacts with endogenous MEK1, lacks kinase activity. These results collectively support our contention that the kinase activity of KSR1 is an intrinsic property of this protein independent of KSR1-bound endogenous MEK.
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PMID:The kinase activity of kinase suppressor of Ras1 (KSR1) is independent of bound MEK. 2427 36

The extracellular-regulated kinases (ERK) modulate cell proliferation and survival in response to several different stimuli and are therefore important drug targets. ERKs are activated by the dual phosphorylation kinase MEK1 and MEK1 inhibitors PD98059, U0126 and CI-1040 are now widely used to inhibit ERKs in cell and animal studies. In an analysis of ERK functions in astrocytes we found that PD98059 (100microM) failed to inhibit ERK phosphorylation but U0126 (50microM) inhibited ERK phosphorylation to approximately 80%. Surprisingly, U0126 also caused profound depletion of ATP in glucose-deprived cells, leading to death by necrosis. Since glucose-deprived cells depend mainly on mitochondrial ATP-synthase for ATP production, we tested whether U0126 or PD184161, a derivative of CI-1040, might inhibit ATP synthase activity, using 143B(Rho0) cells (which lack a functional F0 subunit) to further parse this effect. We found that the F1F0ATPase activity extracted from U0126- or PD184161-treated parental 143B cells or astrocytes was indeed inhibited by >or=80% suggesting a covalent change in the enzyme. However, F1F0ATPase activity extracted from similarly treated 143B(Rho0) cells was spared. Because F1F0ATPase activity in isolated mitochondria was not inhibited directly, we propose that U0126 and PD184161 inhibit ATP-synthase via an indirect action on F0. The MEK1 inhibitors also induced necrosis of other glucose-deprived cell types including primary neurons at the same concentrations required for inhibition of ERK phosphorylation. Thus, the MEK1/ERK signalling pathway may modulate ATP synthase function, and its inhibition may cause cells unable to perform glycolysis to die by necrosis.
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PMID:Aggravation of necrotic death of glucose-deprived cells by the MEK1 inhibitors U0126 and PD184161 through depletion of ATP. 1519 7

A high-throughput screen for Ras-mitogen-activated protein kinase (MAPK) signaling inhibitors identified two series (class 1 and 2) of substituted 4-anilino-3-quinolinecarbonitriles as potent (IC(50)s <10 nmol/L) mitogen-activated protein/extracellular signal-regulated kinase 1 (MEK1) kinase inhibitors. These compounds had cyanoquinoline cores, but differed in their respective aniline groups [1a, 1b: 4-phenoxyphenylaniline; 2a, 2b: 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline]. These compounds were competitive inhibitors of ATP binding by MEK1 kinase, and they had minimal or no effect on Raf, epidermal growth factor receptor (EGFR), Akt, cyclin-dependent kinase 4 (CDK4), or MK2 kinases at concentrations >100-fold higher than those that inhibited MEK1 kinase. Both class 1 and 2 compounds inhibited in vitro growth of human tumor cell lines. A class 2 compound (2b) was the most potent inhibitor of human tumor cell growth in vitro, and this effect was linked to distinct suppression of MAPK phosphorylation in cells. Compound 2b did not affect phosphorylation status of other kinases, such as EGFR, Akt, and stress-activated protein (SAP)/c-jun-NH kinase (Jnk); nor did it affect overall tyrosine phosphorylation level in cells. However, compound 2b did inhibit MEK1 phosphorylation in cells. Inhibition of MEK1 phosphorylation by 2b was not due to a major effect on Raf kinase activity, because enzyme assays showed minimal Raf kinase inhibition. We believe compound 2b inhibits kinase activity upstream of Raf, and thereby affects MEK1 phosphorylation in cells. Even with the dual effect of 2b on MEK and MAPK phosphorylation, this compound was well tolerated and significantly inhibited growth of the human colon tumor cell line LoVo (at 50 and 100 mg/kg BID, i.p.) in a nude mouse xenograft model.
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PMID:Identification of 4-anilino-3-quinolinecarbonitrile inhibitors of mitogen-activated protein/extracellular signal-regulated kinase 1 kinase. 1521 Aug 62

Human CYP2J2 is abundant in heart and active in the biosynthesis of epoxyeicosatrienoic acids (EETs); however, the functional role of this P450 and its eicosanoid products in the heart remains unknown. Transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 were generated. CYP2J2 transgenic (Tr) mice have normal heart anatomy and basal contractile function. CYP2J2 Tr hearts have improved recovery of left ventricular developed pressure (LVDP) compared with wild-type (WT) hearts after 20 minutes ischemia and 40 minutes reperfusion. Perfusion with the selective P450 epoxygenase inhibitor N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH) for 20 minutes before ischemia results in reduced postischemic LVDP recovery in WT hearts and abolishes the improved postischemic LVDP recovery in CYP2J2 Tr hearts. Perfusion with the ATP-sensitive K(+) channel (K(ATP)) inhibitor glibenclamide (GLIB) or the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (5-HD) for 20 minutes before ischemia abolishes the cardioprotective effects of CYP2J2 overexpression. Flavoprotein fluorescence, a marker of mitoK(ATP) activity, is higher in cardiomyocytes from CYP2J2 Tr versus WT mice. Moreover, CYP2J2-derived EETs (1 to 5 micromol/L) increase flavoprotein fluorescence in WT cardiomyocytes. CYP2J2 Tr mice exhibit increased expression of phospho-p42/p44 mitogen-activated protein kinase (MAPK) after ischemia, and addition of the p42/p44 MAPK kinase (MEK) inhibitor PD98059 during reperfusion abolishes the cardioprotective effects of CYP2J2 overexpression. Together, these data suggest that CYP2J2-derived metabolites are cardioprotective after ischemia, and the mechanism for this cardioprotection involves activation of mitoK(ATP) and p42/p44 MAPK.
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PMID:Enhanced postischemic functional recovery in CYP2J2 transgenic hearts involves mitochondrial ATP-sensitive K+ channels and p42/p44 MAPK pathway. 1534 65

Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as alpha-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the alpha-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. alpha-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of alpha-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the alpha-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that alpha-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, alpha-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through alpha-enolase expression during ischemic hypoxia and reoxygenation.
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PMID:ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation. 1545 7

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

Recently we have reported that glucose deprivation induces the potentiated death and loss of ATP in immunostimulated astroglia via the production of NO and eventually peroxynitrite. This study examined the role of the ERK1/2 signaling pathways in the glucose deprivation-induced death of immunostimulated astroglia. Immunostimulation with LPS+IFN-gamma induced the sustained activation of ERK1/2 for up to 48 h. Glucose deprivation caused the loss of ATP and consequently cell death in immunostimulated astroglia, which was significantly blocked by the treatment with the ERK kinase (MEK1) inhibitor, PD98059 (10-40 microM), to inhibit the ERK1/2 pathways. The systems for generating NO (iNOS) or superoxide (NADPH oxidase) were regulated by the ERK1/2 signaling pathways because the addition of PD98059 reduced the level of both. Interestingly, glucose deprivation caused an approximately two-fold increase in the level of peroxynitrite formation in immunostimulated astroglia, which was significantly reduced by the PD98059 treatment. This demonstrates that the ERK1/2 signaling pathways play an important role in glucose deprivation-induced death in immunostimulated astroglia by regulating the generation of NO, superoxide and their reaction product, peroxynitrite.
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PMID:Role of MAPK/ERK1/2 in the glucose deprivation-induced death in immunostimulated astroglia. 1572 Dec 16


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