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)

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

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.
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PMID:Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase. 942 18

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf --> MEK pathway and has an impact on physiologically relevant genes in these cells.
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PMID:Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3-L1 cell lines. 942 50

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

Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-alpha, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.
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PMID:Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. 944 75

Stimulation of cell proliferation by mitogens involves tyrosine phosphorylation of proteins at the cell membrane by receptor tyrosine kinases. This promotes formation of multi-protein complexes that can activate the small G-protein, Ras. Activation of Ras, in turn, leads to sequential activation of the following three serine-threonine kinases: Raf, extracellular signal-regulated kinase kinase (MEK), and members of the family of mitogen-activated protein (MAP) kinases. Prior studies have shown that intraperitoneal injection of epidermal growth factor (EGF) leads to rapid activation of hepatic MAP kinases in adult rats but not in late gestation (E19) fetal rats (Boylan, J. M., and Gruppuso, P. A. (1996) Cell Growth & Differ. 7, 1261-1269). The present studies were undertaken to determine the mechanism for this "uncoupling" of the MAP kinase pathway. E19 fetal rats and adult male rats were injected with EGF (0.5 microg/g body weight, intraperitoneally) or with saline. After 15 min, livers were removed and prepared for kinase analyses. EGF injection led to a rapid and marked activation of hepatic Raf and MEK in both fetal and adult rats, whereas MAP kinase activation was minimal in fetal as opposed to adult rats. Examination of the ontogeny of this dissociation of MAP kinase activation from MEK activation showed gradual acquisition of intact signaling as an adult hepatocyte phenotype was attained during the first 4 postnatal weeks. Over this period, MAP kinase content as determined by Western immunoblotting was constant. Recombination experiments using partially purified fetal and adult rat liver MEK and MAP kinase showed intact MAP kinase activation in vitro, indicating that neither enzyme was irreversibly altered in the fetus. In studies using primary cultures of E19 fetal rat hepatocytes, uncoupling of MAP kinase activation from MEK activation could be induced by incubation of fetal hepatocytes for 24 h with a potent fetal hepatocyte mitogen, transforming growth factor-alpha. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.
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PMID:Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat. 945 12

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

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M. In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.
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PMID:Mitogen-activated protein kinases (Erk1,2) phosphorylate Lys-Ser-Pro (KSP) repeats in neurofilament proteins NF-H and NF-M. 959 82

The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-kappaB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 12P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes.
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PMID:Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: co-stimulation of AP-1 and CRE nuclear binding proteins. 962 Jan 67

SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.
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PMID:Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases. 962 Aug 46


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