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 recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.
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PMID:Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases. 1764 1

The inwardly rectifying potassium channel Kir2.1 is inhibited by a variety of G-protein-coupled receptors (GPCRs). However, the mechanisms underlying the inhibition have not been fully elucidated. In this study the role of the small GTPase, Rho, in mediating this inhibition was determined. Stimulation of the m1 muscarinic receptor inhibited Kir2.1, when both receptor and channel were coexpressed in tsA201 cells. The inhibition of Kir2.1 by carbachol was reversible and atropine-sensitive. Cotransfection with a dominant-negative mutant of the small GTPase Rho abolished the inhibition of Kir2.1 with current amplitudes remaining at control levels in the presence of carbachol. Conversely, cotransfection with the constitutively activated mutant of Rho resulted in a reduction in basal Kir2.1 current amplitudes, suggesting that Rho inhibits Kir2.1. To further confirm the involvement of Rho in the signal transduction pathway, cotransfection with C3 transferase (EFC3), a selective inhibitor of Rho, abolished the reduction in Kir2.1 currents noted upon application of carbachol under control conditions. Preincubation with the phosphatidylinositol 3-kinase inhibitor wortmannin or the Rho kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2 HCl (Y-27632) had no effect on agonist-induced inhibition of Kir2.1, precluding these kinases as downstream effectors of Rho in mediation of the signal. In addition, 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), had no effect on the m1 receptor-induced inhibition of Kir2.1, suggesting that MAP kinases are not involved in the signaling pathway. In conclusion, these data indicate that the small GTPase, Rho, transduces the m1 muscarinic receptor-induced inhibition of Kir2.1 via an unidentified mechanism.
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PMID:Role of the small GTPase Rho in modulation of the inwardly rectifying potassium channel Kir2.1. 1450 Jul 55

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.
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PMID:Cytoplasmic p21Cip1 is involved in Ras-induced inhibition of the ROCK/LIMK/cofilin pathway. 1455 14

A key task for the multifunctional von Hippel-Lindau protein (pVHL) is regulation of the activity of hypoxia-inducible factor-1alpha (HIF-1alpha) by targeting it to the proteasome for degradation under normoxia. pVHL binding to HIF-1alpha is lost under low O2 tension, leading to transcription of several genes involved in the hypoxia response. However, regulation of pVHL by hypoxia remains to be investigated. We evaluated the effects of hypoxia on pVHL expression in carcinoma and endothelial cells. We showed that hypoxia stimulates pVHL levels (2.5-fold) in renal Caki-1 cells expressing wild-type VHL (VHL+/+). This upregulation was independent of VHL status, because hypoxia also increased pVHL expression in renal 786-O cells carrying mutated VHL (VHL-/-). Hypoxia did not affect pVHL expression in endothelial cells. Hypoxia-induced pVHL in Caki-1 cells was RhoA dependent, because inhibition by exotoxin C3 prevented pVHL stimulation. Furthermore, inhibition of Rho kinase by Y-27632 blocked pVHL induction by hypoxia. During normoxia, pVHL expression was also induced in cells transfected with dominant-active RhoA. Furthermore, disruption of actin organization by chemical agents or by hypoxia stimulated pVHL expression in kidney cells. On the other hand, inhibition of MAP kinases p38 and JNK, but not MAP kinase kinase (MEK1/2), reduced pVHL upregulation by 30 and 72%, respectively, during hypoxia, supporting a significant role for these signaling pathways. Expression and phosphorylation of c-Jun were stimulated in cells transfected with dominant-active RhoA. Together, these findings demonstrate that hypoxia induces pVHL expression in renal cancer cells, and this induction is mediated by RhoA-dependent pathways.
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PMID:Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma. 1458 36

There is a group of cells, called hyalocytes, in the cortical vitreous. Although hyalocytes were discovered more than a hundred years ago, the molecular and cellular biological characteristics of hyalocytes have yet to be elucidated. In this study, we investigated various aspects of hyalocytes and, also performed triamcinolone acetonide (TA)-assisted vitrectomy to remove the hyalocytes for diabetic macular edema. Immunohistochemical analysis of rat eyes showed that 90% of hyalocytes were negative for ED1 but positive for ED2, indicating that hyalocyte is a tissue macrophage. Chimeric mice were created by transplanting bone marrow from green fluorescent protein (GFP)-transgenic mice into irradiated wild-type mice, showing the origin of hyalocyte to be bone marrow cells. Bovine hyalocytes were cultured successfully. The proliferation of hyalocytes was significantly enhanced by hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF-2) and inhibited by transforming growth factor(TGF)-beta. Among these, PDGF-BB stimulated the proliferation most potently through the MEK 1 pathway. Hyalocyte migration assessed by double chamber assay was also stimulated by PDGF-BB and it was mediated by the PI3K and p38 MAPK pathways. Cellular contraction of hyalocyte was significantly enhanced by PDGF-BB and TGF-beta through Rho kinase, p44/42 MAPK, and protein kinase C pathways, as measured by collagen gel contraction assay. Next, the relationship between the vitreous cavity(VC) and the immune system was studied after intravitreous inoculation with ovalbumin (OVA). Injection of OVA into the VC of C 57 BL/6 mice resulted in suppressed systemic cell-mediated immunity to OVA as determined by the ear swelling assay. This aberrant immune responsiveness following VC injection of OVA was termed VC-associated immune deviation or VCAID. The phenomenon of VCAID was mediated by intravitreous antigen-presenting cells. The histological study of chimeric mice showed these cells to be intravitreous residential cells, namely hyalocytes. VCAID was abolished by intravitreous inflammation such as experimental autoimmune uveitis. Finally, TA-assisted vitrectomy for diabetic macular edema was performed to remove cortical vitreous, because it contained many hyalocytes which could secrete inflammatory cytokines including VEGF. Although the number of treated eyes was limited, the surgical results have been favorable so far. The investigation of hyalocytes would open a new avenue for better understanding and development of treatment for various vitreo-retinal diseases.
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PMID:[Cell biology of hyalocytes]. 1473 34

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

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.
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PMID:Smooth muscle actin determines mechanical force-induced p38 activation. 1559 Oct 55

Isoprostanes are prostaglandin (PG)-like compounds produced nonenzymatically by free radical-catalyzed peroxidation of arachidonic acid. Isoprostanes evoke potent vascular effects but their actions in the neonatal vasculature are poorly known. We aimed to study the effects of 8-iso-PGE(1), 8-iso-PGE(2), 8-iso-PGF(1alpha), 8-iso-PGF(1beta), 8-iso-PGF(2alpha), and 8-iso-PGF(2beta) in pulmonary arteries (PA), pulmonary veins (PV), and mesenteric arteries (MA) from newborn and 2-wk-old piglets. Isoprostanes produced concentration-dependent contractions of PA, PV, and MA (magnitudes up to 1.5- to 2-fold greater than the responses to 62.5 mM KCl) but they were markedly less potent vasoconstrictors than the thromboxane A(2) (TXA(2)) mimetic U46619. Neonatal PA were more sensitive to 8-iso-PGF(1alpha), 8-iso-PGF(1beta), and 8-iso-PGF(2beta) than 2-wk-old PA. Neonatal PV were more sensitive to 8-iso-PGE(2) and 8-iso-PGF(1alpha), and neonatal MA were more sensitive to 8-iso-PGE(2), 8-iso-PGF(1alpha), 8-iso-PGF(1beta), 8-iso-PGF(2alpha), and 8-iso-PGF(2beta) than the corresponding 2-wk-old vessels. The sensitivity to U46619 decreased with postnatal age in MA but did not change in PA and PV. The contractile responses to all the isoprostanes and to U46619 were reverted by the TXA(2) receptor (TP) antagonist SQ 29,548. Moreover, isoprostane-evoked contractions in 2-wk-old PA were reduced by inhibitors of tyrosine kinase (genistein) and Rho kinase (Y 27632 and hydroxyfasudil) but not by inhibitors of protein kinase C (chelerythrine), mitogen-activated protein kinase kinase (PD 98059) or p38-kinase (SB 203580). In conclusion, isoprostanes produced compound-, tissue-, and age-dependent constriction of neonatal porcine pulmonary and mesenteric vascular smooth muscle. Isoprostane-evoked PA vasoconstriction involved TP receptors and activation of tyrosine kinases and Rho kinases.
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PMID:Age-related differences in vasoconstrictor responses to isoprostanes in piglet pulmonary and mesenteric vascular smooth muscle. 1584 38

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.
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PMID:The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals. 1592 28

Lipoxins (LX) are arachidonic acid metabolites able to induce monocyte chemotaxis in vitro and in vivo. Nonetheless, the signaling pathways mediating this process are yet unclear. In this study, we have investigated the mechanisms associated with human monocyte activation in response to 15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATL-1), a stable 15-epi-LXA4 analog. Our results demonstrate that ATL-1-induced monocyte chemotaxis (10-300 nM) is inhibited by pertussis toxin, suggesting an effect via the G-protein-linked LXA4 receptor. Monocytes stimulated with the analog presented an increased ERK-2 phosphorylation, which was reduced by PD98059, a selective inhibitor of the MEK 1/2 pathway. After exposure of the cells to ATL-1, myosin L chain kinase (MLCK) phosphorylation was evident and this effect was inhibited by PD98059 or Y-27632, a specific inhibitor of Rho kinase. In addition, Y-27632 abolished ERK-2 activation, suggesting that the MAPK pathway is downstream of Rho/Rho kinase in MLCK activation induced by ATL-1. The specific MLCK inhibitor ML-7, as well as Y-27632, abrogated monocyte chemotaxis stimulated by the analog, confirming the central role of the Rho kinase/MLCK pathway on ATL-1 action. Together, these results indicate that ATL-1 acts as a potent monocyte chemoattractant via Rho kinase and MLCK. The present study clarifies some of the mechanisms involved on the activation of monocytes by LXs and opens new avenues for investigation of these checkpoint controllers of inflammation.
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PMID:Involvement of the Rho-kinase/myosin light chain kinase pathway on human monocyte chemotaxis induced by ATL-1, an aspirin-triggered lipoxin A4 synthetic analog. 1603 27


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