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)

In the course of screening for inhibitors of tumorigenic phenotype of K-ras-transformed NIH3T3 cells (DT cells), we found a novel compound, oxamflatin, an aromatic sulfonamide hydroxamate derivative, which induces flat phenotype in these cells and suppresses their anchorage-independent growth. In contrast to DT cells, in v-raf-transformed NIH3T3 cells, no change in their morphology and no specific inhibition of their anchorage-independent growth was observed. Interestingly, oxamflatin was effective to NIH3T3 cells transformed by constitutively activated mutant of MEK, indicating the possibility that oncogene-induced morphological change is not necessarily induced by common signaling pathway such as MAP kinase cascade. In oxamflatin-treated DT cells, the expression of transcription factor junD was highly augmented, resulting in trans-activation of fibronectin gene by junD via cyclic AMP responsive element in its promoter. This behavior of junD was confirmed to correlate well with partial blocking of malignant phenotype in DT cells. Thus, oxamflatin can be categorized as the first reagent which induces genes whose products can interfere with oncogene-dependent transformation.
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PMID:Oxamflatin: a novel compound which reverses malignant phenotype to normal one via induction of JunD. 870 May 40

Cell anchorage strongly affects the signal transduction cascade initiated by peptide mitogens. For both epidermal growth factor and platelet-derived growth factor, activation of the consensus mitogen-activated protein kinase cascade is impaired when cells are held in suspension as compared with cells anchored to a fibronectin substratum. Upstream events in the signaling cascade, including tyrosine phosphorylation of the mitogen receptor and GTP loading of Ras, are similar in anchored and suspended cells. However, propagation of the signal to Raf and subsequently to the downstream kinases MEK and mitogen-activated protein kinase is markedly attenuated in suspended cells. Thus, there seems to be a distinct anchorage-dependent step between Ras and Raf in the signaling cascade initiated by peptide mitogens. These observations may have important implications for understanding the anchorage dependence of cell growth.
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PMID:Cell anchorage permits efficient signal transduction between ras and its downstream kinases. 908 99

Adhesion molecules such as VLA-4 are important not only for monocyte adhesion to extracellular matrix proteins, but also for subsequent cell activation. Monocyte adherence to fibronectin or engagement of VLA-4 has been demonstrated to stimulate production of potent inflammatory mediators such as tumor necrosis factor-alpha, interleukin-1, and the procoagulant tissue factor protein. However, the intracellular signaling cascades leading to gene expression have not been elucidated. Using the human monocytic THP-1 cell line, VLA-4 cross-linking by monoclonal antibodies directed against its alpha4 and beta1 subunits produced a time-dependent increase in tyrosine phosphorylation of a broad range of cellular proteins. Using Western blot analysis directed against the phosphorylated form of the extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase proteins, as well as immunoprecipitation and in vitro kinase assays, we found that VLA-4 cross-linking increased ERK1/ERK2 tyrosine phosphorylation and activity. In conjunction, integrin cross-linking also increased NF-kappaB nuclear translocation and 4-h expression of tissue factor. Inhibition of tyrosine kinase activity with genistein (10 microg/ml) as well as selective MAP kinase inhibition with the MEK-1 inhibitor PD98059 abolished the VLA-4-dependent ERK tyrosine phosphorylation, inhibited NF kappaB nuclear binding, and abrogated tissue factor expression induced by both VLA-4 cross-linking and adhesion to fibronectin in THP-1 cells and human peripheral blood monocytes. These studies point to the involvement of the MAP kinase pathway in the activation of monocytic cells during transmigration to inflammatory sites.
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PMID:VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase. 909 80

Smooth muscle cell (SMC) proliferation is dependent on both anchorage to the extracellular matrix by integrins and the presence of growth factors. Integrins and growth factor receptors transduce signals that seem to converge on the extracellular signal-regulated (ERK) pathway, but the molecular basis for this interaction is not known. SMC proliferation has previously been shown to be supported by culture on fibronectin (FN), whereas cells cultured on laminin (LN) are growth inhibited. In the present study, we examined the mitogenic response to platelet-derived growth factor BB (PDGF-BB) in baboon SMCs cultured on FN vs. LN. Induction of DNA synthesis and the activity of ERK and the ERK activating kinase MKK-1 were reduced only slightly after stimulation with PDGF-BB in cells cultured on LN vs. those cultured on FN. We tested the possibility that endogenous FN secretion contributes to the ability of the cells to respond to PDGF stimulation during culture on LN. Inhibition of interactions between FN and integrin alpha 5 beta 1 by the competitive GRGDSP-peptide or anti-alpha 5 integrin antibody restricted cell spreading, reduced cell-surface staining for alpha 5 beta 1 and FN fibrils, and inhibited PDGF-BB-induced DNA synthesis. These results showed that SMC growth on LN required a provisional FN matrix. Although disruption of interactions between alpha 5 beta 1 and FN by the GRGDSP-peptide prevented PDGF-BB-induced DNA synthesis, neither ERK activity nor translocation of ERKs into the nucleus was inhibited. These results show that integrins regulate SMC growth through pathways that function in parallel with, but distinct from, growth factor-mediated ERK signaling.
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PMID:Disruption of integrin alpha 5 beta 1 signaling does not impair PDGF-BB-mediated stimulation of the extracellular signal-regulated kinase pathway in smooth muscle cells. 920 31

The MAP kinase pathway is a major regulator of both normal and oncogenic growth. We report that activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf show that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion-dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras-transformed cells in suspension but not when adherent. These results identify a novel synergism between cell adhesion- and growth factor-regulated pathways, and explain how oncogenic activation of MAP kinases induces both serum- and anchorage-independent growth.
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PMID:Growth factor activation of MAP kinase requires cell adhesion. 931 18

Directed cell migration is essential for a variety of important biological processes ranging from development and angiogenesis to metastasis. Ras plays a pivotal role in the signaling cascade that governs chemotaxis of fibroblasts toward platelet-derived growth factor-BB (PDGF-BB). Ras activates multiple downstream pathways, which include the extracellular signal-regulated kinase (ERK), Rac, and Ral signaling cascades. We therefore investigated the role of the Rac and ERK pathways in cell migration. We showed that migration of fibroblasts toward PDGF-BB is inhibited by expression of dominant negative Asn-17 Rac1. Blocking of the ERK pathway by either expression of dominant negative Ala-218/Ala-222-mitogen-activated protein kinase kinase (A218/A222-MEK1) or by a MEK-specific inhibitor did not inhibit migration toward PDGF-BB. In contrast, migration toward soluble fibronectin was suppressed by inhibition of the ERK pathway but not by Asn-17 Rac1 expression. These results indicate that directed cell migration mediated by different receptor classes in response to different ligands differentially utilizes the Rac and ERK pathways and suggest that Rac might play a critical role in pathological processes such as angiogenesis and metastasis.
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PMID:Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways. 938 4

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130(Cas), two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130(Cas) association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130(Cas) and its subsequent binding to several SH2 domains, which depended on both the p130(Cas) binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130(Cas) further increased cell migration on FN and coexpression of the p130(Cas) SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130(Cas), but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130(Cas) complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.
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PMID:Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. 942 68

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.
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PMID:Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase. 943 Jul 10

The alphavbeta3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-kappaB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and beta3 integrin ligation rapidly increased NF-kappaB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a beta1-integrin ligand) did not induce NF-kappaB activity. The alphavbeta3 integrin was most important for osteopontin-mediated NF-kappaB induction and survival, since adding a neutralizing anti-beta3 integrin antibody blocked NF-kappaB activity and induced endothelial cell death when cells were plated on osteopontin. NF-kappaB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-kappaB activity with nonphosphorylatable IkappaB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-kappaB was not required for fibronectin, laminin, and collagen type I-induced survival. Activation of NF-kappaB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-kappaB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-kappaB activation. These studies identify NF-kappaB as an important signaling molecule in alphavbeta3 integrin-mediated endothelial cell survival.
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PMID:NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival. 958 25

Treatment of confluent contact inhibited 10T1/2 cells with TPA or OAG induced a dramatic increase of the number of migrating cells, on cover slides inserted into culture dishes. When cover slides were coated with collagen IV or fibronectin, there was a similar increase of the number of migrating cells. RT PCR showed the presence of alpha PKC gene transcripts and the lack of beta and gamma PKC. Western blot analysis showed translocation of 80 kD alpha PKC to membranous fraction following brief treatment with TPA, and down-regulation of PKC after longer exposure to TPA. Collagen IV and fibronectin treatment of 10T1/2 cells induced MAP kinase, (MEK) kinase in the presence and in absence of FCS. Signal transduction pathway depending on protein kinase C and integrin receptors activation appears to facilitate migration of 10T1/2 cells and may be involved in the mechanism of the escape from contact inhibition of movement.
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PMID:Migration induction of contact inhibited C3H 10T1/2 cells by protein kinase C (PKC) dependent process. 968 86


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