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)

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.
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PMID:Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes. 953 50

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.
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PMID:Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1. 953 45

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
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PMID:Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. 966 Jul

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.
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PMID:Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation. 956 49

MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.
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PMID:The MEK1 proline-rich insert is required for efficient activation of the mitogen-activated protein kinases ERK1 and ERK2 in mammalian cells. 967 29

We examined epidermal growth factor (EGF)- and epinephrine-stimulated mitogen-activated protein kinase kinase (MEK) 1 and MEK2 activities, DNA polymerase alpha activity, and EGF-stimulated E2F DNA binding activity in primary cultured hepatocytes from 6- and 24-mo-old rats. MEK stimulation by either EGF or epinephrine was not altered with aging. However, stimulation of DNA polymerase alpha activity by these agents was 70% and 50% lower, respectively, in cells of aged compared with cells of young rats, consistent with a lesser increase in [3H]thymidine incorporation. EGF-stimulated E2F (a transcription factor that regulates expression of the DNA polymerase alpha gene) binding to DNA was reduced with age. PD-098059, a specific inhibitor of MEK, inhibited EGF-stimulated MEK1 and MEK2 activities in hepatocytes from 6- and 24-mo-old rats. Although PD-098059 inhibited EGF-stimulated DNA synthesis in hepatocytes from 6-mo-old rats, it had no effect in 24-mo-old rats. Thus the age-related impairment appears to occur before E2F activation, and signal transduction sequences other than the mitogen-activated protein kinase pathway may be involved in stimulated DNA synthesis in hepatocytes from old rats.
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PMID:Molecular mechanisms of impaired stimulation of DNA synthesis in cultured hepatocytes of aged rats. 968 45

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.
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PMID:Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen. 974 82

The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L).
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PMID:Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. 984 Sep 24

The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.
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PMID:Thrombopoietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets. 999 Mar 15

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.
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PMID:Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element. 1019 63


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