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)

Several proteases and their specific inhibitors modulate the interdependent processes of cell migration and matrix proteolysis as part of the global program of trauma repair. Expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor (SERPIN) important in the control of barrier proteolysis and cell-to-matrix adhesion, for example, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. PAI-1 mRNA/protein synthesis was induced early after epidermal monolayer scraping and restricted to keratinocytes comprising the motile cohort closely recapitulating, thereby, similar events during cutaneous healing. The time course of PAI-1 promoter-driven PAI-1-GFP fusion "reporter" expression in wound-juxtaposed cells approximated that of the endogenous PAI-1 gene confirming the location-specificity of gene regulation in this model. ERK activation was evident within 5 min after injury and particularly prominent in cells residing at the scrape-edge (suggesting a possible role in PAI-1 induction and/or the motile response) as was myosin light chain (MLC) phosphorylation. Indeed, MEK blockade with PD98059 or U0126 attenuated keratinocyte migration (by > or =60%), as did transient transfection of a dominant-negative ERK1 construct (40% decrease in monolayer repair), and completely inhibited PAI-1 transcript expression. Anti-sense down-regulation of PAI-1 synthesis (by 80-85%), or addition of PAI-1 neutralizing antibodies also inhibited injury site closure over a 24 h period establishing that PAI-1 was required for efficient long-term planar motility in this system. PAI-1 anti-sense transfection or actinomycin D transcriptional blockade, in contrast, did not affect the initial migratory response suggesting that residual PAI-1 protein levels (at least in transfectant cells and actinomycin D-treated cultures) may be sufficient to support early cell movement. Pharmacologic inhibition of keratinocyte MEK signaling effectively ablated scrape-induced PAI-1 mRNA expression but failed to attenuate wound-associated increases in cellular PAI-1 protein levels soon after monolayer injury. Collectively, these data suggest that basal PAI-1 transcripts may be mobilized for initial PAI-1 synthesis and, perhaps, the early motile response while maintenance of the normal rate of migration requires the prolonged PAI-1 expression that typically accompanies the repair response. To assess this possibility, scrape site closure studies were designed using keratinocytes isolated from PAI-1-/- mice. PAI-1-/- keratinocytes, in fact, had a significant wound healing defect evident even within the first 6 h following monolayer denudation injury. Addition of active PAI-1 protein to PAI-/- keratinocytes rescued the migratory phenotype that that approximating wild-type cells. These findings validate use of the present keratinocyte model to investigate injury-related controls on PAI-1 gene regulation and, collectively, implicate participation of PAI-1 in two distinct phases of epidermal wound repair.
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PMID:PAI-1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair. 1517

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.
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PMID:B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts. 1519 48

Alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that alpha2-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 microM) almost completely inhibited the contractile response to the alpha2-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 +/- 2.3% of 60 mM KCl response (mean +/- S.E.M.) in the presence of Y27632, compared with 64.9 +/- 7.1% in control tissues, n = 4]. However, Y27632 had no effect on alpha2-adrenoceptor-mediated ERK activation, as measured by Western blotting. Alpha2-adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 microM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 microM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.
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PMID:The role of Rho kinase and extracellular regulated kinase-mitogen-activated protein kinase in alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein. 1523 68

Little is known about the adaptation of uterine artery smooth muscle contractile mechanisms to pregnancy. The present study tested the hypothesis that pregnancy differentially regulates thick- and thin-filament regulatory pathways in uterine arteries. Isometric tension, intracellular free Ca(2+) concentration, and phosphorylation of 20-kDa myosin light chain (MLC(20)) were measured simultaneously in uterine arteries isolated from nonpregnant and near-term (140 days gestation) pregnant sheep. Phenylephrine-mediated intracellular free Ca(2+) concentration, MLC(20) phosphorylation, and contraction tension were significantly increased in uterine arteries of pregnant compared with nonpregnant animals. In contrast, phenylephrine-mediated Ca(2+) sensitivity of MLC(20) phosphorylation was decreased in the uterine arteries of pregnant sheep. Simultaneous measurement of phenylephrine-stimulated tension and MLC(20) phosphorylation in the same tissue indicated a decrease in MLC(20) phosphorylation-independent contractions in the uterine arteries of pregnant sheep. In addition, activation of PKC produced significantly lower sustained contractions in uterine arteries of pregnant compared with nonpregnant animals in the absence of changes in MLC(20) phosphorylation levels in either vessels. In uterine arteries of nonpregnant sheep, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitor PD-098059 significantly increased phenylephrine-mediated, MLC(20) phosphorylation-independent contractions. The results suggest that in uterine arteries, pregnancy upregulates alpha(1)-adrenoceptor-mediated Ca(2+) mobilization and MLC(20) phosphorylation. In contrast, pregnancy downregulates the Ca(2+) sensitivity of myofilaments, which is mediated by both thick- and thin-filament pathways.
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PMID:Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy. 1535 11

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.
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PMID:Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension. 1613 52

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.
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PMID:Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery. 1660 29

Infection with group B streptococcus (GBS) is the most common cause of early onset neonatal sepsis in many countries, leading to neonatal morbidity and mortality. There is much evidence for a direct involvement of platelets in the pathogenesis of inflammation and sepsis. Several bacteria are known to directly interact with platelets leading to activation and aggregation, a phenomenon also observed with GBS. Here, we demonstrate that GBS rapidly bound to platelets; however, only strains isolated from septic patients bound fibrinogen on their surface and induced platelet thromboxane synthesis, platelet aggregation, and P-selectin (CD62P) expression. In contrast, GBS strains isolated from healthy newborns or healthy pregnant women induced only shape change, but not platelet thromboxane synthesis, platelet aggregation, or CD62P expression. All GBS strains investigated were able to activate FcgammaRIIA receptor signaling pathways including phospholipase C gamma2 (PLCgamma2), as well as calcium/calmodulin-dependent myosin kinase II (CaMKII) and phosphorylation of myosin light chain (MLC). In contrast, protein kinase C (PKC) was exclusively activated by GBS strains isolated from septic patients, and p38 mitogen activated protein kinase (p38 MAP kinase) was preferentially activated by septic GBS strains. Furthermore, stress signaling kinase SEK1/MKK4 and focal adhesion kinase (FAK) were activated by all tested GBS strains in a FcgammaRIIA-independent way. This study demonstrates that septic, but not colonizing, GBS strains bind fibrinogen on their surface, and that septic GBS strains influence platelet function not only via the FcgammaRIIA receptor, but also via pathways distinct from IgG-mediated signalling. These mechanisms lead to platelet aggregation and secretion, thereby possibly modulating the pathophysiologic course of GBS infections.
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PMID:Group B streptococcus isolates from septic patients and healthy carriers differentially activate platelet signaling cascades. 1667 76

We previously showed that hydrogen peroxide (H2O2) induced resistance artery relaxation independent of endothelium. Thus, in this study we investigated the mechanism of relaxation induced by H2O2 on human renal vascular smooth muscle cell (HVSMC). HVSMC were stimulated with H2O2 and/or angiotensin II (Ang II), proline-rich-tyrosine-kinase-2 (PYK2), ERK1/2 MAP-Kinase, and myosin light chain 20 phosphorylation (Lc20) were assessed using Western blot analysis in the presence of potassium channel blockers, MAP-Kinase, and nitric oxide synthesis (NOS) inhibitors. H2O2 increased PYK2 and ERK1/2 phosphorylation, and at the same time decreased Lc20 phosphorylation. AngII increased phosphorylation of PYK2, ERK1/2 and Lc20, whereas, the pretreatment of HVSMC with H2O2 decreased Lc20 phosphorylation induced by AngII. MEK inhibition, decreased ERK1/2 phosphorylation, but had no effect on the inhibition of phosphorylation of Lc20 induced by H2O2. The inhibition of Ca2(+)-dependent K+ channels (BKCa) and NOS did not block the decrease of Lc20 phosphorylation in response to H2O2. On the other hand, pretreatment of HVSMC with 60 mM of KCl, increased rather than decreased Lc20 phosphorylation in response to H2O2. This study shows the evidence that H2O2 acts as a relaxing factor and as an activator of PYK2 and ERK1/2 in Human renal VSMC. The relaxation induced by H2O2 is independent of BKCa, ERK1/2 MAP-Kinase and NOS pathways. The relaxing effect to H2O2 changes to contracting effect when the potassium channels are compromised.
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PMID:Hydrogen peroxide acts as relaxing factor in human vascular smooth muscle cells independent of map-kinase and nitric oxide. 1672 Mar 30

The effect of granulocyte colony-stimulating factor (G-CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G-CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3-5 min after G-CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84.4 +/- 2.9 microm/5 min. A study using the Boyden chamber method revealed that G-CSF-stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G-CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G-CSF-induced redistribution of F-actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti-CD18 antibody, which prevented cell adherence. These findings indicate that G-CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.
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PMID:Enhanced neutrophil motility by granulocyte colony-stimulating factor: the role of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. 1690 68

Increased permeability of blood vessels is an important component of inflammation, but in some circumstances it contributes to tissue injury and organ failure. Previous work showed that p21-activated kinase (PAK) is a critical regulator of endothelial cell-cell junctions through effects on myosin light chain phosphorylation and cell contractility. We now show that blocking PAK function inhibits fluid leak in a mouse model of acute lung injury. In cultured endothelial cells, induction of myosin light chain phosphorylation by PAK is mediated by mitogen-activated protein kinase kinase and extracellular signal-regulated kinase (Erk). Erk in lipopolysaccharide (LPS)-treated mouse lung is activated in a PAK-dependent manner in several cell types, most prominently vascular endothelium. Activation of Erk requires the integrity of the complex between PAK, PIX, and GIT1. Several means of disrupting this complex inhibit stimulation of vascular permeability in vitro. A cell-permeant peptide that blocks binding of PAK to PIX inhibits LPS-induced fluid leak in the mouse lung injury model. We conclude that the PAK-PIX-GIT1 complex is critical for Erk-dependent myosin phosphorylation and vascular permeability.
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PMID:Induction of vascular permeability: beta PIX and GIT1 scaffold the activation of extracellular signal-regulated kinase by PAK. 1742 73


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