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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 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

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.
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PMID:Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. 754 86

Protein phosphorylation has evolved as the most versatile posttranslational modification widely used by cells. Signal transduction pathways mediated by activation of MAP kinases and protein kinase C trigger the exit of cells from the quiscence (Go-->G1 transition). Indeed, binding of growth factors at the cell surface triggers their receptors, usually possessing a tyrosine kinase on the cytoplasmic side, to phosphorylate other molecules passing on the information sequentially to GRB2 protein, to p21ras, to c-Raf-1, to MAP kinase kinase, to MAP kinase, to p90rsk, to transcription factors. Activated PKC, MAP kinase, and pp90src can translocate to the nucleus where they phosphorylate a number of protein transcription regulators in a cell cycle-dependent manner or in response to cell stimulation for exit from quiescence. The cell cycle is mainly regulated by p34cdc2 or otherwise called cdc2 in association with cyclins B at G2/M and by Cdk2 in association with cyclins A, D1, and E at G1/S checkpoints; phosphorylation of histone H1 and lamins by cdc2 triggers chromosome assembly and nuclear envelope breakdown, respectively, as a prelude to mitosis. Cdc2 activities functioning as a G2/M regulator are controlled by its phosphorylation and dephosphorylation at Ser/Thr residues. MAP kinases might be the missing link in the chain connecting the Go to G1 transition with the cell cycle regulation, whereas phosphorylation of replication protein factors, retinoblastoma, and p53 might link the G1 to S transition with the control of DNA synthesis. A number of transcription factors are known to stimulate DNA replication, including p53, c-Myc, AP-1, Oct-1, T-antigen; the DNA binding activities of all these proteins and their interaction with other transcription factors are controlled by phosphorylation. The nuclear import of several proteins including NF kappa B, Dorsal, glucocorticoid receptor, ISGF3, rNFIL-6, T antigen, and the kinases PKC, MAP, and p90rsk, are dependent on their phosphorylation at specific sites. Histone phosphorylation stimulated at discrete stages of the cell cycle or in response to cAMP or other stimuli might induce profound changes in chromatin organization.
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PMID:Phosphorylation of transcription factors and control of the cell cycle. 754 80

As they respond to numerous extracellular and intracellular stimuli, plants develop various morphological features and the capacity for a large variety of physiological processes during their growth. If we are to understand the molecular basis of such developments, we must elucidate the way in which signals generated by such stimuli can be transduced into plant cells and transmitted by cellular components to induce the appropriate terminal events. In yeast and animal systems, signal pathways that are known collectively as MAPK (mitogen-activated protein kinase) cascades have been shown to play a central role in the transmission of various signals. The components of these pathways include the MAPK family, the activator kinases of the MAPK family (the MAPKK family) and the activator kinases of the MAPKK family (the MAPKKK family). The members of each respective family are structurally conserved and signals are transmitted by similar phosphotransfer reactions at corresponding steps that are mediated by a specific member of each family in turn. Both cDNAs and genes that encode putative homologues of these components have recently been isolated from plant sources. Some of them have been shown to be related not only structurally but also functionally to members of the MAPK cascades of other organisms. These findings suggest that plants have signal pathways that are analogues to the MAPK cascades in yeast and animal cells but it remains to be proven that plant homologues do in fact constitute kinase cascades.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells. 755 83

Mechanical stress induces cardiac hypertrophy and expression of specific genes in the cardiac myocytes. External stimuli are generally transduced into the nucleus through the activation of a protein kinase cascade. We have previously shown that stretching cardiomyocytes stimulates the activity of protein kinase C (PKC), mitogen-activated protein (MAP) kinase and S6 protein kinase. In the present study, we examined two other kinases, Raf-1 kinase and MAP kinase kinase, which are supposed to lie between PKC and MAP kinase in the protein kinase cascade. Stretching cardiocytes by using the in vitro system induced hyperphosphorylation of Raf-1 kinase and activation of MAP kinase kinase. The protein kinases activated by mechanical stress are similar to those activated by growth factors. We examined the possible involvement of angiotensin II (Ang II) in the protein synthesis and gene expression induced by mechanical stress. CV11974, an Ang II-receptor antagonist, partially suppressed the increases in amino acid incorporation, c-fos gene expression and MAP kinase activity induced by stretching. These results suggest that a variety of protein kinases are activated by mechanical stress and that locally produced Ang II may in part play important roles in converting mechanical stimuli into biochemical signals.
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PMID:Protein kinase cascade activated by mechanical stress in cardiocytes: possible involvement of angiotensin II. 755 78

The 14.3.3 zeta protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A2, which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably 1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 zeta polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 zeta binds primarily to Raf noncatalytic sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3 zeta binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 zeta (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 zeta polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 zeta in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 zeta binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 zeta, are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c-Raf. Overexpression of 14.3.3 zeta enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated.
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PMID:Identification of the 14.3.3 zeta domains important for self-association and Raf binding. 755 37

Analysis of genes involved in yeast cell wall beta-glucan assembly has led to the isolation of EXG1, PBS2 and PTC1. EXG1 and PBS2 were isolated as genes that, when expressed from multicopy plasmids, led to a dominant killer toxin-resistant phenotype. The PTC1 gene was cloned by functional complementation of the calcofluor white-hypersensitive mutant cwh47-1. PTC1/CWH47 is the structural gene for a type 2C serine/threonine phosphatase, EXG1 codes for an exo-beta-glucanase, and PBS2 encodes a MAP kinase kinase in the Pbs2p-Hog1p signal transduction pathway. Overexpression of EXG1 on a 2 mu plasmid led to reduction in a cell wall beta 1,6-glucan and caused killer resistance in wild type cells; while the exg1 delta mutant displayed modest increases in killer sensitivity and beta 1,6-glucan levels. Disruption of PTC1/CWH47 and overexpression of PBS2 gave rise to similar beta-glucan related phenotypes, with higher levels of EXG1 transcription, increased exo-beta-glucanase activity, reduced beta 1,6-glucan levels, and resistance to killer toxin. Genetic analysis revealed that loss of function of the PBS2 gene was epistatic to PTC1/CWH47 disruption, indicating a functional role for the Ptc1p/Cwh47p phosphatase in the Pbs2p-Hog1p signal transduction pathway. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in cell wall glucan assembly, and that this is effected in part by modulating Exg1p activity.
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PMID:Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription. 756 87

The MPK1 (SLT2) gene of Saccharomyces cerevisiae encodes a mitogen-activated protein kinase that is regulated by a kinase cascade whose known elements are Pkc1 (a homolog of protein kinase C), Bck1 (Slk1) (a homolog of MEK kinase), and the functionally redundant Mpk1 activators Mkk1 and Mkk2 (homologs of MEK). An activated mutation of MKK1, MKK1P386, inhibits growth when overexpressed. This growth-inhibitory effect was suppressed by the mpk1 delta mutation, suggesting that hyperactivation of the Mpk1 pathway is toxic to cells. To search for genes that interact with the Mpk1 pathway, we isolated both chromosomal mutations and dosage suppressor genes that ameliorate the growth-inhibitory effect of overexpressed Mkk1P386. One of the genes identified by the analysis of chromosomal mutations is RLM1 (resistance to lethality of MKK1P386 overexpression), which encodes a protein homologous to a conserved domain of the MADS (Mcm1, Agamous, Deficiens, and serum response factor) box family of transcription factors. Although rlm1 delta cells grow normally at any temperature, they display a caffeine-sensitive phenotype similar to that observed in mutants defective in BCK1, MKK1/MKK2, or MPK1. A gene fusion that provides Rlm1 with a transcriptional activation domain of Gal4 suppresses bck1 delta and mpk1 delta. A screening for dosage suppressors yielded the MSG5 genes, which encode a dual-specificity protein phosphatase. Our results suggest that Rlm1 functions as a transcription factor downstream of Mpk1 that is subject to activation by the Mpk1 mitogen-activated protein kinase pathway.
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PMID:Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. 756 26

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.
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PMID:Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. 756 95

Because cAMP exerts opposite effects on cell proliferation in different cell types, we undertook to study its effect on the mitogen-activated protein kinase (MAPK) pathway in three cell lines (Rat-1, Swiss-3T3, and COS-7) chosen for their different mitogenic responses to cAMP. We measured the effect of cAMP on MAPK, MEK, and Raf-1 activities after stimulation by agonists acting through a tyrosine kinase receptor (epidermal growth factor) or a G protein-coupled receptor (lysophosphatidic acid). In Rat-1 cells we found that cAMP strongly inhibited all three activities (MAPK, MEK, and Raf-1), in good agreement with its effect on cell proliferation in these cells. In Swiss-3T3 and COS-7 cells, on the contrary, cAMP did not inhibit epidermal growth factor- and lysophosphatidic acid-induced stimulation of MAPK and MEK activities, and even stimulated MAPK activity slightly on its own. Again these results are in good agreement with the proliferative effect of cAMP in Swiss-3T3 cells. Raf-1 activity on the hand, was inhibited by cAMP in Swiss-3T3 and COS-7 as it was in Rat-1 cells. This result indicates that signaling pathways in Swiss-3T3 and COS-7 cells can activate MEK and MAPK in a Raf-1-independent and cAMP-insensitive manner. Our results add to growing evidence for the existence of Ras- and/or Raf-1-independent pathways leading to MEK and MAPK activation.
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PMID:Differential effects on cAMP on the MAP kinase cascade: evidence for a cAMP-insensitive step that can bypass Raf-1. 757 5


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