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 adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. It has previously been shown that several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. We set out to determine which MAPK signaling pathways are activated in our system and which MAPK pathways are required for TNF-alpha gene transcription or TNF-alpha mRNA translation. We confirm activation of the MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). We demonstrate that LPS also activates MEK2, MEK3, and MEK6. Furthermore, we demonstrate that dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocked translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Finally, overexpression of wild-type SAPKbeta was able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed.
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PMID:Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK. 934 88

The effects of activating the Gq protein-coupled cholecystokinin (CCK) receptor on different proteins/signaling molecules in the mitogen-activated protein kinase (MAPK) cascade in pancreatic acinar cells were analyzed and compared with the effects of activating the tyrosine kinase-coupled epidermal growth factor (EGF) receptor. Both EGF and CCK octapeptide rapidly increased the activity of the MAPKs [extracellular signal-regulated kinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-induced increase of MAPK activity was transient, with only a slight elevation after 30 min, whereas CCK-stimulated MAPK remained at a high level of activation to 60 min. The protein kinase C inhibitor GF-109203X abolished the activation by phorbol ester and inhibited the effect of CCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a much larger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereas EGF activated both MEKs by only 2-fold. Immunoblotting revealed three different forms of Raf in pancreatic acinar cells. Of the total basal Raf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% was c-Raf-1. All three forms of Raf were stimulated to a greater extent by CCK than by EGF, which was especially evident for Raf-A and c-Raf-1. The effect of CCK in activating Rafs was at least partially mimicked by stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantly increased GTP-bound Ras by 183 and 164% at 2.5 and 10 min, respectively; CCK and TPA had no measurable effect. Our study suggests that CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.
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PMID:Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells. 937 31

The extracellularly-responsive kinase (ERK) subfamily of mitogen-activated protein kinases (MAPKs) has been implicated in the regulation of cell growth and differentiation. Activation of ERKs involves a two-step protein kinase cascade lying upstream from ERK, in which the Raf family are the MAPK kinase kinases and the MEK1/MEK2 isoforms are the MAPK kinases. The linear sequence of Raf --> MEK --> ERK constitutes the ERK cascade. Although the ERK cascade is activated through growth factor-regulated receptor protein tyrosine kinases, they are also modulated through G protein-coupled receptors (GPCRs). All four G protein subfamilies (Gq/11 Gi/o, Gs and G12/13) influence the activation state of ERKs. In this review, we describe the ERK cascade and characteristics of its activation through GPCRs. We also discuss the identity of the intervening steps that may couple agonist binding at GPCRs to activation of the ERK cascade.
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PMID:Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. 937 13

The observation that mitogen-activated protein (MAP) kinases ERK1 and ERK2 are constitutively activated in a number of oncogene-transformed cell lines has led to the hypothesis that prolonged activation of these enzymes is required for the transformation process. To investigate this question, we have examined the regulation of the ERK pathway in Rat1 fibroblasts transformed with activated c-Raf-1 (Raf22W), v-Ha-Ras, and v-Src. Expression of these oncoproteins had no effect on the enzymatic activity of ERK1 and ERK2 in either serum-starved or exponentially growing cells. Moreover, the stimulatory effect of serum on ERK1/ERK2 activity was substantially reduced or abrogated in these cells; this impairment was associated with a strong attenuation of c-fos gene induction. In contrast, expression of Raf22w, v-Ha-Ras, or v-Src resulted in the constitutive activation of the upstream kinases MEK1 and MEK2. Treatment of the cells with vanadate completely restored the activation of ERK1/ERK2 in oncogene-transformed cells, suggesting the involvement of a vanadate-sensitive tyrosine phosphatase. Northern blot analysis of VH1-like dual-specificity MAP kinase phosphatases did not reveal any significant difference in the mRNA expression pattern of these genes between parental and transformed Rat1 cells. Phosphoamino acid analysis indicated that ERK1 is phosphorylated on threonine, but not on tyrosine, in oncogene-transformed cells and that vanadate treatment restores tyrosine phosphorylation. We conclude from these results that ERK1/ERK2 activity is repressed by a single-specificity tyrosine phosphatase in oncogene-transformed rat fibroblasts.
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PMID:Repression of mitogen-activated protein kinases ERK1/ERK2 activity by a protein tyrosine phosphatase in rat fibroblasts transformed by upstream oncoproteins. 939 54

The mitogen-activated protein kinase (MAPK) cascade acts as a focal point for signal transduction following activation of both G-protein-linked and tyrosine kinase growth factor receptors. A common intermediate between both of these diverse receptor subtypes includes the small guanosine triphosphate (GTP)-binding protein, p21ras. Point mutations of p21ras have been identified in various tumor types and lead to constitutive activation of this protein and subsequent activation of downstream pathways including the MAPK cascade. Using an in vivo model of hepatocellular carcinoma (HCC), we investigated the abundance and function of individual components of the MAPK cascade and the presence of specific p21ras mutations in this model. Expression of components of the MAPK cascade were determined in tumor and adjacent, non-neoplastic liver specimens by Western blot analysis and functional activity confirmed by substrate phosphorylation assays. Mutations in p21ras were analyzed using an enzyme-linked immunosorbent assay. In tumor, extracellular regulated kinases (ERKs) ERK1, ERK2, and mitogen-activated ERK-regulated kinase-1 (MEK1) were elevated by three- to fourfold as compared with adjacent nontumorigenic normal liver. In contrast, MEK2 was elevated by only 28%. Substrate phosphorylation and detection of phosphorylated ERK1/2 proteins showed increased functional activity of these proteins of the same magnitude as that observed for protein expression. Mutations in p21ras were not detected in this experimental model of HCC. We conclude that HCC is associated with marked changes in expression and function of components of the MAPK cascade independent of common p21ras mutations.
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PMID:Altered expression of mitogen-activated protein kinases in a rat model of experimental hepatocellular carcinoma. 939 88

The present work was initiated with the aim of identifying nuclear genes whose expression is sensitive to the mitochondrial DNA (mtDNA) status of transformed chicken DU24 cells. We cloned and sequenced cDNAs for the mitogen-activated protein kinase kinase type 2, MEK2, a protein involved in the mitogenic growth factor signal transduction pathway in vertebrates. Sequence comparisons between the chicken protein and its mammalian counterparts indicated that MEK2 proteins are highly conserved among vertebrates. Southern blot analysis of endonuclease-digested genomic DNA from primary chick embryo fibroblasts (CEF) suggested that MEK2 is a single-copy gene in this vertebrate species. The steady-state level of MEK2 transcripts is decreased in DUS3 mtDNA-less (rho0) cells developed by long-term exposure of DU24 rho+ cells to ethidium bromide (EtdBr). Run-on in vitro transcription assays and mRNA stability studies indicated that the decrease in MEK2 mRNA content is associated with post-transcriptional regulation. In parental DU24 cells, MEK2 mRNA content decreased after inhibition of mtDNA transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol (CAM). Cytoplasmic hybrids (cybrids) constructed by fusion of chicken rho0 cells with enucleated parental cells and CEF recovered a basal level of MEK2 expression. The MEK2 protein content is decreased in DUS3 rho0 cells and in parental DU24 rho+ cells treated with EtdBr and CAM for 6 days, while that of MEK1, a closely related kinase, remained unchanged. On the basis of these observations, we propose that mitochondria participate in the mitogenic signal transduction pathway in chicken cells through regulation of MEK2 expression.
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PMID:Cloning and characterization of cDNAs encoding chicken mitogen-activated protein kinase kinase type 2, MEK2: downregulation of MEK2 in response to inhibition of mitochondrial DNA expression. 939 67

We investigated subcellular distribution of ERK2 in osteoblast-like UMR-106 cell and explored to determine if its activities are regulated by insulin. 23%, 34% and 43% of total ERK2 were distributed in membrane, cytosol and nucleus, respectively. Insulin caused 40% increase of ERK2 content in membrane in 10 min whereas it induced approximately 50% decrease of ERK2 in cytosol in 10 min. In terms of kinase activity, insulin stimulated phosphorylation of the membrane-associated ERK2 by 2-fold and 1.8-fold in 1 min and 10 min and cytosolic ERK2 by 2.7-fold and 2.3-fold in 1 min and 10 min, respectively. In contrast, the phosphorylation of nuclear ERK2 was stimulated by insulin in time-dependent manner with maximal (3-fold) activity observed at 30 min. Insulin also increased the content of MEK2 in membrane by 2.2- to 2.6-fold in 10 min. MEK2 translocated into membrane in response to insulin may play a role in the activation of the membrane-associated ERK2 via phosphorylation.
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PMID:Insulin rapidly stimulates ERK2 in the membrane of osteoblast-like UMR-106 cell. 941 11

Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.
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PMID:Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs. 941 3

Two competitive enzyme immunoassays using digoxigenin-labeled peptides have been developed for the quantification of the protein kinase MEK2 in cell extracts. Rabbit polyclonal antibodies directed against either the amino-terminal or proline-rich amino acid sequences of MEK2 were used for the immunoconcentration of the protein. Anti-digoxigenin Fab fragments labeled with horseradish peroxidase allowed the detection of the immune complexes. Amino-terminal and proline-rich enzyme immunoassays exhibited a sensitivity level of 63 and 71 fmol/mL, respectively, and displayed a half-maximal saturation value of 1320 and 1780 fmol/mL. The intra- and inter-assay coefficients of variation for both assays assessed at three different concentrations of MEK2 were lower than 6% and 12%, respectively. The amount of MEK2 measured by the two methods demonstrated an excellent correlation with the expression level of the protein detected by immunoblot analyses when tested on different cell lysates.
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PMID:Digoxigenin-labeled peptides for the immunological quantification of intracellular signaling proteins: application to the MAP kinase kinase isoform MEK2. 942 43

Inactivation of growth factor-regulated mitogen-activated protein (MAP) kinases (ERK1 and ERK2) has been proposed to occur in part through dephosphorylation by the dual specificity MAP kinase phosphatase-1 (MKP-1), an immediate early gene that is induced by mitogenic signaling. In this study, we examined the effect of MKP-1 on signaling components upstream of ERK1 and ERK2. Coexpression of MKK1 or MKK2 with MKP-1 resulted in 7-10-fold activation of mitogen-activated protein kinase kinase (MKK), which required the presence of regulatory serine phosphorylation sites. Endogenous MKK1 and MKK2 were also activated upon MKP-1 expression. Raf-1, a direct regulator of MKK1 and MKK2, was activated under these conditions, and a synergistic activation of MKK was observed upon coexpression of Raf-1 and MKP-1. This effect did not appear to involve synthesis of autocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activity but was inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependent activation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulation of Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activating Raf-1/MKK signaling pathways alternative to those involving ERK.
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PMID:Feedback regulation of Raf-1 and mitogen-activated protein kinase (MAP) kinase kinases 1 and 2 by MAP kinase phosphatase-1 (MKP-1). 943 Jul 28


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