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)

Both MAP kinases and the protein kinase p74raf-1 are activated by many growth factors in a c-ras-dependent manner and by oncogenic p21ras. We were therefore interested in determining the relationship between MAP kinases and raf. The MAP kinase ERK2 is activated by expression of oncogenically activated raf, independently of cellular ras. Overexpressed p74raf-1 potentiates activation of ERK2 by EGF and TPA. MAP kinase kinase inactivated by phosphatase 2A treatment is phosphorylated and reactivated by incubation with p74raf-1 immunoprecipitated from phorbol ester-treated cells. We conclude that raf protein kinase is upstream of MAP kinases and is either a MAP kinase kinase kinase or a MAP kinase kinase kinase kinase.
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PMID:Activation of the MAP kinase pathway by the protein kinase raf. 133 Mar 21

Adult mammalian ventricular cardiomyocytes are terminally differentiated cells that enlarge adaptively by hypertrophy. In this situation, genes normally expressed in the fetal ventricular cardiomyocyte (e.g. atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM) alpha-actin) are re-expressed, and there is transient expression of immediate early genes (e.g. c-fos). Using appropriate reporter plasmids, we studied the effects of transfection of the constitutively active or dominant negative mitogen-activated protein kinase kinase MEK1 on ANF, beta-MHC, and SkM alpha-actin promoter activities in cultured ventricular cardiomyocytes. ANF expression was stimulated (maximally 75-fold) by the hypertrophic agonist phenylephrine in a dose-dependent manner (EC50, 10 microM), and this stimulation was inhibited by dominant negative MEK1. Cotransfection of dominant negative MEK1 with a dominant negative mitogen-activated protein kinase (extracellular signal-regulated protein kinase (ERK2)) increased this inhibition. Transfection with constitutively active MEK1 constructs doubled ANF promoter activity. The additional cotransfection of wild-type ERK2 stimulated ANF promoter activity by about 5-fold. Expression of beta-MHC and SkM alpha-actin was also stimulated. Promoter activity regulated by activator protein-1 or c-fos serum response element consensus sequences was also increased. We conclude that the MEK1/ERK2 cascade may play a role in regulating gene expression during hypertrophy.
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PMID:The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes. 749 96

The prototype mitogen-activated protein (MAP) kinase module is a three-kinase cascade consisting of the MAP kinase, extracellular signal-regulated protein kinase (ERK) 1 or ERK2, the MAP/ERK kinase (MEK) MEK1 or MEK2, and the MEK kinase, Raf-1 or B-Raf. This and other MAP kinase modules are thought to be critical signal transducers in major cellular events including proliferation, differentiation, and stress responses. To identify novel mammalian MAP kinase modules, polymerase chain reaction was used to isolate a new MEK family member, MEK5, from the rat. MEK5 is more closely related to MEK1 and MEK2 than to the other known mammalian MEKs, MKK3 and MKK4. MEK5 is thought to lie in an uncharacterized MAP kinase pathway, because MEK5 does not phosphorylate the ERK/MAP kinase family members ERK1, ERK2, ERK3, JNK/SAPK, or p38/HOG1, nor will Raf-1, c-Mos, or MEKK1 highly phosphorylate it. Alternative splicing results in a 50-kDa alpha and a 40-kDa beta isoform of MEK5. MEK5 beta is ubiquitously distributed and primarily cytosolic. MEK5 alpha is expressed most highly in liver and brain and is particulate. The 23 amino acids encoded by the 5' exon in the larger alpha isoform are similar to a sequence found in certain proteins believed to associate with the actin cytoskeleton; this alternatively spliced modular domain may lead to the differential subcellular localization of MEK5 alpha.
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PMID:Isolation of MEK5 and differential expression of alternatively spliced forms. 749 18

Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.
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PMID:Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2). 752 93

In KB epidermoid cells, we previously showed that interleukin-1 alpha (IL-1) and various mitogens activate the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate both myelin basic protein (MBP) and a peptide containing Thr669 of the epidermal growth factor receptor. In cell-free extracts made from gingival fibroblasts treated with platelet-derived growth factor or HepG2 hepatoma cells stimulated with phorbol myristate acetate, MBP and Thr669 kinase were both elevated 4-fold, and ERK1 and ERK2 were tyrosine-phosphorylated. In these cells IL-1 activated a kinase(s) that phosphorylated Thr669 peptide but not MBP and failed to cause tyrosine phosphorylation of ERK1/ERK2. Ceramide has been proposed as an intracellular mediator of IL-1 action, but C2-ceramide or sphingosine stimulated predominantly MBP-specific kinase activity in fibroblasts and had no effect in HepG2 cells. p54 MAP kinase (also called stress-activated protein kinase) is a c-Jun kinase first isolated from livers of cycloheximide-treated rats. After IL-1 stimulation, immunoprecipitates of lysates made from all three cell types with specific anti-p54 MAP kinase serum contained Thr669 and c-Jun phosphorylating activity, whereas precipitates from unstimulated cells contained no detectable p54 kinase activity. The major peak of IL-1-stimulated HepG2 Thr669 kinase activity co-chromatographed on Mono Q and phenyl-Superose with immunodetectable p54 MAP kinase. IL-1 did not cause p21ras activation in any cell type. Induction of Thr 669 kinase activity was not abrogated by elevation of cAMP levels, which has been shown to interfere with the activation of Raf-1. We could not detect MAP kinase kinase phosphorylating activity in unfractionated lysates made from IL-1-stimulated fibroblasts or HepG2 cells. KB cells contained a small amount of this activity, but it was not precipitated with an anti-Raf-1 antibody. We conclude that most of the IL-1-activated Thr669 kinase activity in fibroblasts and HepG2 cells, and a portion in KB cells, is due to p54 MAP kinase and that its activation is Ras-, Raf-, and MAP kinase kinase-independent.
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PMID:Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase. 752 98

Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.
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PMID:Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine. 753 1

The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation.
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PMID:Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase. 753 68

Although signaling by the epidermal growth factor (EGF) receptor is thought to be dependent on receptor tyrosine kinase activity, it is clear that mitogen-activated protein (MAP) kinase can be activated by receptors lacking kinase activity. Since analysis of the signaling pathways used by kinase-defective receptors could reveal otherwise masked capabilities, we examined in detail the tyrosine phosphorylations and enzymes of the MAP kinase pathway induced by kinase-defective EGF receptors. Following EGF stimulation of B82L cells expressing a kinase-defective EGF receptor mutant (K721M), we found that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all activated, and SHC became prominently tyrosine-phosphorylated. By contrast, kinase-defective receptors failed to induce detectable phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or p91STAT1, all of which were robustly phosphorylated by wild-type receptors. These data demonstrate that kinase-defective receptors induce several protein tyrosine phosphorylations, but that these represent only a subset of those seen with wild-type receptors. This suggests that kinase-defective receptors activate a heterologous tyrosine kinase with a specificity different from the EGF receptor. We found that kinase-defective receptors induced ErbB2/c-Neu enzymatic activation and ErbB2/c-Neu binding to SHC at a level even greater than that induced by wild-type receptors. Thus, heterodimerization with and activation of endogenous ErbB2/c-Neu is a possible mechanism by which kinase-defective receptors stimulate the MAP kinase pathway.
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PMID:An incomplete program of cellular tyrosine phosphorylations induced by kinase-defective epidermal growth factor receptors. 753 32

The expression of the urokinase-type plasminogen activator, which plays a crucial role in tissue remodeling by controlling the synthesis of the broadly acting plasmin serine protease, is regulated by several tyrosine kinases. Since the actions of these tyrosine kinases is dependent on the activation of ras proteins, we undertook a study to identify signaling events downstream of ras responsible for the stimulation of urokinase promoter activity. Transient expression of an activated c-Ha-ras in OVCAR-3 cells, which do not harbor the mutated oncogene, led to a dose-dependent trans-activation of the urokinase promoter. A sequence residing between -2109 and -1964 was critical for the stimulation of the urokinase promoter by c-Ha-ras. Mutation of an AP-1 and a PEA3 site at -1967 and -1973, respectively, or the co-expression of a transactivation domain-lacking c-jun substantially impaired the ability of c-Ha-ras to stimulate urokinase promoter activity. The induction of the urokinase promoter by ras was completely blocked by expression of a dominant negative c-raf expression vector and substantially reduced in cells made to co-express a catalytically inactive mitogen-activated protein kinase kinase. Further, the expression of an ERK1/ERK2-inactivating phosphatase (CL100) abrogated the stimulation of the urokinase promoter by c-Ha-ras. These data argue for a role of a mitogen-activated protein kinase-dependent signaling pathway in the regulation of urokinase promoter activity by ras.
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PMID:Involvement of a mitogen-activated protein kinase signaling pathway in the regulation of urokinase promoter activity by c-Ha-ras. 755 39

We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-dependent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrates cross-talk between G protein-coupled second messengers and protein tyrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate (PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increments in tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatment of osteoblasts, which raised intracellular cAMP levels and inhibited DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) as well as inhibited PKC-stimulated MAPK and Raf-1 activities. Despite this, PMA activated the intermediate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presence of a additional distal cAMP-dependent inhibitory mechanisms.
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PMID:Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts. 758 14


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