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)

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.
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PMID:Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. 1265 94

P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1-100 microm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-alpha. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis.
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PMID:The P2Y2 nucleotide receptor mediates UTP-induced vascular cell adhesion molecule-1 expression in coronary artery endothelial cells. 1271 97

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1-43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2-3 min and lasting for approximately 10-15 min. The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H. R., and Tilly, B. C. (1999) Biochem. J. 343, 579-586), was diminished after 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment. Uptake of TRITC-dextrans, however, was not affected. Treatment of the cells with the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-specific protease Clostridium botulinum toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis. Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans. Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.
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PMID:Increased vesicle recycling in response to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release. 1287 43

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.
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PMID:P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells. 1451 Nov 12

In mildly hyperosmotic medium, activation of the Na+ -K+ -2Cl- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (KATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a KATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of KATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of KATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions.
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PMID:ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle. 1459 11

Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mek1 kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase. We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.
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PMID:Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic double strand break repair in budding yeast. 1459 9

Hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) is a unique multifunctional protein, being not only the precursor of HCNP, which promotes the phenotype development of septo-hippocampal cholinergic neurons, but also the binding protein of phosphatidylethanolamine, ATP, Raf-1 kinase (known as "Raf-1 kinase inhibitory factor" in peripheral organs), and serine protease. We obtained a high-titer retroviral vector harboring HCNP-pp cDNA by the use of a modified packaging cell line and centrifugation, and by injecting it into embryonic mouse ventricles, we investigated the function of its gene product within the central nervous system (CNS). We found that efficient transduction into hippocampal pyramidal neurons can be achieved by injecting the vector into embryonic brain ventricles on embryonic day 14 (E14). Three days after receiving the intraventricular injection of the high-titer HCNP-pp retrovirus vector on E14, the tissues around the ventricles showed an overexpression of HCNP-pp. This was accompanied by a reduced amount of activated MEK and Erk (as analyzed by histochemical and Western blot methods), suggesting that HCNP-pp also regulates the MAP-kinase cascade within the CNS. Surprisingly, mouse brains that received the HCNP-pp retroviral vector showed massive malformation of the hippocampus and cerebellum when examined 30 days after birth. This shows that strictly regulated HCNP-pp gene expression is necessary for the normal development of the mouse brain, and that the moderate overexpression achieved by retroviral vector-mediated gene transfer is sufficient to cause severe abnormality of entire brain structures.
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PMID:Brain malformations caused by retroviral vector-mediated gene transfer of hippocampal cholinergic neurostimulating peptide precursor protein into the CNS via embryonic mice ventricles. 1461 62

In response to neurotransmitters, astrocytes show various types of calcium increase (transient, oscillatory, and complex), the physiological significance of which is still controversial. To explore this variability, we examined factors affecting the calcium increase pattern in cultured astrocytes and investigated the consequences of the astrocytic calcium response in slice preparations. We found that growth factors (GFs) (EGF plus basic FGF) promoted calcium oscillation in response to glutamate, ATP, or thimerosal (which directly activates the inositol-1,4,5 triphosphate receptor) and that this effect was suppressed by pro-inflammatory cytokines (interleukin-1beta or tumor necrosis factor-alpha), lipopolysaccharide, or a MEK (mitogen-activated protein kinase kinase) inhibitor, suggesting dual regulation of calcium oscillation in astrocytes by factors affecting brain function and pathology via the mitogen-activated protein kinase (MAPK) cascade. The calcium oscillation was accompanied by enlargement of the calcium store, cell proliferation, and the development of a hypertrophic morphology. The cytokines suppressed GF-induced MAPK-dependent immediate early gene promoter activation, but not phosphorylation of extracellular signal-regulated kinase (ERK), showing that they affected gene regulation by acting on the MAPK cascade downstream of ERK. In slice preparations, a metabotropic glutamate receptor agonist converted the spontaneous neuronal calcium increase, attributable to synaptic transmission, to an oscillatory response similar to that seen in astrocytes in culture, indicating that the calcium response in astrocytes acted as a feedback mechanism on the activity of neighboring neurons. This is the first evidence for a dual regulation of calcium oscillation by physiological factors and for the control of calcium dynamics actually being used in physiological processes.
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PMID:Dual regulation of calcium oscillation in astrocytes by growth factors and pro-inflammatory cytokines via the mitogen-activated protein kinase cascade. 1464 90

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappa B, we tested the hypothesis that LPS and nucleotides regulate NF-kappa B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X(7)-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-kappa B DNA-binding activity and a sustained attenuation of levels of the NF-kappa B inhibitory protein I kappa B alpha. Interestingly, a persistent reduction in I kappa B alpha levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating I kappa B alpha protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in I kappa B alpha levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-kappa B/I kappa B-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-kappa B and Ras/MEK/MAPK-associated pathways.
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PMID:Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages. 1468 87

One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, gamma-(32)P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras-->Raf-->MEK-->ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N(6)-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N(6)(2-phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with (32)P-N(6)(2-phenethyl) ATP in cell lysates.
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PMID:Engineering the serine/threonine protein kinase Raf-1 to utilise an orthogonal analogue of ATP substituted at the N6 position. 1470 20


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