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)

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.
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PMID:Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway. 1064 90

During wound healing, dermal fibroblasts switch from a migratory, repopulating phenotype to a contractile, matrix-reassembling phenotype. The mechanisms controlling this switch are unknown. A possible explanation is suggested by the finding that chemokines that appear late in wound repair prevent growth factor-induced cell-substratum de-adhesion by blocking calpain activation. In this study, we tested the specific hypothesis that fibroblast contraction of the matrix is promoted by a pro-repair growth factor, epidermal growth factor, and is modulated by calpain-mediated release of adhesions. We employed an isometric force transduction system designed to measure the contraction of a collagen matrix under tension by a population of NR6 fibroblasts transfected with the human epidermal growth factor receptor. By maintaining a fixed level of strain, we could monitor both the initial contraction and subsequent relaxation of the matrix. Epidermal growth factor stimulated a transient, dose-dependent increase in matrix contraction that peaked within 60 minutes and then decayed over the ensuing 3 to 6 hours. Calpain inhibitor I (ALLN) prevented epidermal growth factor-stimulated cell de-adhesion and resulted in a significantly slower decay of matrix contraction, with only a slight decrease of the peak magnitude of contraction. The mitogen-activated protein kinase kinase-1-selective inhibitor PD 98059 that blocks signaling through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, required for epidermal growth factor receptor-mediated activation of calpain and de-adhesion, does not significantly affect the magnitude of matrix contraction within minutes of epidermal growth factor addition, but slows the decay similarly to calpain inhibition. Epidermal growth factor receptor signaling thus stimulates the complementary mechanisms of intracellular contractile force generation and calpain-mediated de-adhesion, which are known to coordinately facilitate cell migration. These findings suggest that calpain can act as a functional switch for transmission of intracellular contractile force to the surrounding matrix, with calpain-mediated de-adhesion reducing this transmission and corresponding matrix contraction. Countervailing processes that down-regulate calpain activation can, accordingly, direct the transition of cell function from locomotion to matrix contraction.
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PMID:Epidermal growth factor induces acute matrix contraction and subsequent calpain-modulated relaxation. 1198 8

Estrogen influences the development and function of the nervous system through estrogen receptor-dependent changes in gene expression and by rapidly influencing diverse intracellular signaling pathways. We have investigated the influence of estradiol on developing neonatal rat cerebellar neurons in primary culture and found that low concentrations of 17beta-estradiol (17beta-E2), 17alpha-E2, 17beta-E2-BSA, and ICI182,780 stimulated phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK). Neither testosterone nor progesterone increased ERK1/2 phosphorylation. The effects of the estrogens were specific to the ERK1/2 MAPK pathway and were blocked by U0126, an inhibitor of the ERK1/2 MAPK kinase (MEK1/2). Compared with control cultures, significant MAPK-dependent decreases in viable granule cell numbers were observed in dissociated explant cultures of developing cerebellar neurons 24-96 hr after pulse treatment with 10 pm 17beta-E2 or 10 nm ICI182,780. In contrast, continuous exposure to 10 pm 17beta-E2 significantly increased granule cell numbers. Analysis of bromodeoxyuridine incorporation revealed that a 15 min pulsed treatment with 10 pm 17beta-E2 increased mitogenesis, whereas continuous exposure to the same concentration of 17beta-E2 was anti-mitotic. Estradiol did not increase caspase activity; however, significant increases in cellular permeability and lysis were observed. Cell lysis and death were independent of the pan-caspase inhibitor zVAD-fmk but were blocked fully by the irreversible calpain inhibitor PD150606. These results indicate that rapid activation of the ERK1/2 MAPK pathway by low concentrations of 17beta-E2 induces oncotic/necrotic, but not apoptotic, programmed cell death in a subpopulation of developing granule cells and increased mitogenesis of the granule cell neuroblasts refractory to estrogen-induced neurotoxicity.
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PMID:Estrogens and ICI182,780 (Faslodex) modulate mitosis and cell death in immature cerebellar neurons via rapid activation of p44/p42 mitogen-activated protein kinase. 1283 21

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.
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PMID:Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages. 1500 72

The intense host response to meningococcus reflects marked functional and morphological alterations in blood-brain barriers. We showed previously that mouse-derived cerebrovascular endothelium responded to meningococcal lysates with a robust nitric oxide (NO) response, resulting in the loss of cell viability. To understand how the NO synthase-2 gene in endothelium is activated by meningococcus, we investigated upstream roles for specific protein kinases. Using known kinase inhibitors, and measuring both mRNA expression and nitrite release, we found MAPK/ERK kinase (MEK)2, p38 kinase and phosphoinositide 3-kinase (but not MEK1 or phospholipase C) to be implicated in the NO synthase-2 response. Recruitment of these kinases by meningococcus did not depend on the prior release of the proinflammatory cytokines tumour necrosis factor alpha or interleukin-1beta from endothelium. These endothelial cells were found to express toll-like receptors (TLR) 2, 4 and 9 and antibodies directed against TLR 2 and 4 (but not TLR 9) blocked the NO synthase-2 response to meningococcus. Both meningococcus-induced translocation of nuclear factor-kB (NF-kB) and endothelial cell death were blocked by a known inhibitor of p38 kinase. Calpain inhibitor-1 blocked the NO synthase-2 response to meningococcus, which is further evidence of a role for NF-kB.
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PMID:Neisseria meningitidis-induced death of cerebrovascular endothelium: mechanisms triggering transcriptional activation of inducible nitric oxide synthase. 1514 9

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimer's disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.
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PMID:Calpain mediates calcium-induced activation of the erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons: relevance to Alzheimer's disease. 1533 4

Tumor necrosis-related apoptosis-inducing ligand (TRAIL) induces apoptosis of oligodendrocytes, target cells of immune attack in multiple sclerosis (MS). TRAIL-induced human oligodendrocyte (hOL) death depends on TRAIL ligation with its receptor 1 (TRAIL-R1). However, the intracellular signaling initiated with ligation of TRAIL-R1 in hOLs is unknown. We defined that intracellular transduction signaling involved in TRAIL-induced death of hOLs is associated with strong activation of c-jun NH2-terminal kinase (JNK) and a dominant negative mutant of MKK4/SEK1, MAP kinase upstream of JNK, inhibited TRAIL-induced apoptosis of hOLs. The immunoprecipitation experiments showed that JNK3 isoform was predominantly activated upon hOLs exposure to TRAIL and JNK-3 activation occurred before mitochondrial membrane dysfunction. The other mitogen-activated protein kinase p38 and ERK, as well as calpains and serine proteases, were not activated during TRAIL-induced hOL death. Accordingly, the calpain inhibitor, ZLLY.FMK, p38 kinase inhibitor, SB 203580, and serine proteases inhibitor, TPCK, did not protect hOLs from TRAIL-induced apoptosis. These results demonstrate that JNK pathway is critically involved in hOL death induced by TRAIL and might have significant importance in designing new molecules to protect immune-mediated hOLs demise.
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PMID:TRAIL-induced death of human adult oligodendrocytes is mediated by JNK pathway. 1620 63

Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MbetaCD (Methyl-beta-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbetaCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.
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PMID:Low substratum rigidity of collagen gel promotes ERK phosphorylation via lipid raft to augment cell migration. 1802 79

We studied the mechanisms underlying calpain inhibition-mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, c-Raf, and p21-activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signaling molecules (PAK, c-Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil-like HL-60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI-3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.
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PMID:Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils. 1844 89

17-Beta-estradiol (E2) is a steroid hormone involved in numerous brain functions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ERalpha and ERbeta receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes.
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PMID:17-Beta-estradiol increases neuronal excitability through MAP kinase-induced calpain activation. 1999 77


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