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 addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.
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PMID:Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants. 798 67

The MAP kinase module (Raf/MAPKKK-MAPKK-MAPK) has been shown to be sequentially activated after mitogenic stimulation. Here we demonstrate, by site directed mutagenesis, that MAPK is able to retrophosphorylate its own activator, MAPKK, on two threonine residues Thr-292 and Thr-386 in vitro, and that these sites are also phosphorylated in vivo. A comparison of the kinetics of serum-mediated activation of a wild-type MAPKK and of a mutant unable to undergo phosphorylation by MAPK suggests that this retrophosphorylation may be involved in a negative feedback control of the cascade in vivo.
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PMID:Growth factor-stimulated MAP kinase induces rapid retrophosphorylation and inhibition of MAP kinase kinase (MEK1). 801 50

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.
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PMID:MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity. 801 5

The signal transduction kinase MEK (mitogen-activated protein (MAP) or extracellular signal-regulated (Erk) kinase)-1 is activated via phosphorylation by MEKK (MEK kinase) and raf kinases. We show here that these two kinases phosphorylate rat MEK-1 exclusively on two serine codons, Ser218 and Ser222. Phosphorylation of MEK-1 on serines 218 and 222 is both necessary and sufficient for MEK-1 to be activated and able to phosphorylate MAP kinase. A mutant form of MEK-1 that replaces these two codons with alanine cannot be activated, and one that substitutes glutamic acid residues in place of these 2 serines is active independent of activation by phosphorylation. These sites of activation occur in a region of MEK-1 that is similar to sites of activating phosphorylation in several other serine/threonine kinases, suggesting that this region may represent a conserved "activating domain" of many kinases. MEKK and raf display differences in site preference between these two codons, with MEKK showing preference for the amino acid at codon 218 and raf phosphorylating each residue approximately equally. This site preference might result in differences in the temporal or subsequent substrate patterns of MEK activation that result from these two activation pathways.
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PMID:Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. 803 65

In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.
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PMID:Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1). 803 96

In the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.
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PMID:The torso pathway in Drosophila: a model system to study receptor tyrosine kinase signal transduction. 804 87

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.
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PMID:Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization. 807 75

Intracellular signalling following mitogenic stimulation of quiescent cells involves the initiation of a phosphorylation cascade that leads to the rapid and reversible activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. MAP kinase activation is mediated by dual phosphorylation within the motif Thr-Glu-Tyr by MAP kinase kinase (MEK). Following activation, the MAP kinases translocate into the nucleus where they phosphorylate several transduction targets, including transcription factors. We have previously identified PAC1 as an immediate-early mitogen-inducible tyrosine phosphatase in nuclei of T cells. Here we present several lines of evidence indicating that PAC1 is a physiologically relevant MAP kinase phosphatase. Recombinant PAC1 in vitro is a dual-specific Thr/Tyr phosphatase with stringent substrate specificity for MAP kinase. Constitutive expression of PAC1 in vivo leads to inhibition of MAP kinase activity normally stimulated by epidermal growth factor, phorbol myristyl acetate, or T-cell receptor crosslinking. The inactivation of MAP kinase by PAC1 results in inhibition of MAP kinase-regulated reporter gene expression.
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PMID:Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1. 810 50

The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.
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PMID:Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. 812 67

MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets. MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts. Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells. In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation. Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms.
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PMID:MAP kinases: universal multi-purpose signaling tools. 812 84


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