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)

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 serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.
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PMID:Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. 762 7

The c-Rmil/B-raf proto-oncogene belongs to the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We previously showed that the avian c-Rmil gene encodes two proteins of 94 and 95 kDa resulting from the alternative splicing of a 120 bp exon encoding 40 aminoacids (exon 10). We isolated from a mouse brain library B-raf cDNAs containing this exon 10 and a previously unidentified 36 bp insert which constitutes an additional alternatively spliced exon designated exon 8b. These two exons are located between the CR2 region and the catalytic domain of the protein. By using specific sera generated against different regions of the B-Raf protein, we identified 10 B-Raf isoforms and we defined their structure and their expression pattern in adult mouse tissues. The B-Raf proteins are mainly expressed in neural tissues and, interestingly, isoforms containing aminoacids encoded by exon 10 are specifically expressed in these tissues. We also show that several B-Raf isoforms interact with the Mek-1 protein (MAP kinase kinase) and phosphorylate this protein on serine residues 218 and 222.
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PMID:[B-raf gene encodes for multiple isoforms with Mek-1 kinase activity]. 764 69

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.
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PMID:Desensitization of Ras activation by a feedback disassociation of the SOS-Grb2 complex. 767 8

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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PMID:Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1. 768 43

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.
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PMID:The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line. 771 78

An evolutionarily conserved signal transduction pathway that utilizes a receptor tyrosine kinase and a Ras protein mediates the induction of vulval cell fates in the nematode Caenorhabditis elegans. We sought new genes that function in this pathway by screening for suppressors of the Multivulva phenotype caused by a mutation that activates the let-60 ras gene. Seven such suppressor mutations defined a new gene involved in vulval induction. We named this gene mek-2, because its predicted protein product is most similar to MEK, a protein-serine/threonine and tyrosine kinase. mek-2 mutations can be arranged in an allelic series. A probable null mutation eliminated vulval induction, and the strongest mutations alter codons conserved in most or all protein kinases. Our genetic analysis showed that mek-2 functions downstream of let-60 ras and is required for ras-mediated signal transduction in vivo. The MEK-2 protein may interact with the products of the lin-45 raf and mpk-1 MAP kinase genes, which also mediate vulval induction.
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PMID:The Caenorhabditis elegans gene mek-2 is required for vulval induction and encodes a protein similar to the protein kinase MEK. 772 91

Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.
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PMID:Characterization of MEK1 phosphorylation by the v-Mos protein. 773 26

Activation of the mitogen activated protein kinase (MAPK) plays essential roles in many signal transduction pathways. MAPK has been demonstrated to phosphorylate and regulate numerous cellular proteins, including growth factor receptor, transcription factors, cytoskeletal proteins, phospholipase and other protein kinases. Activation of MAPK requires phosphorylation of both threonine and tyrosine residues, which are catalysed by a single protein kinase known as MAPK kinase or MEK. MEK itself is activated by phosphorylation on two conserved serine residues. Three distinct mammalian Ser/Thr kinases, including Raf, Mos and MEKK (for MEK kinase), have been demonstrated to phosphorylate and activate MEK. The MAP kinase cascade is highly conserved in all eukaryotes and involved in numerous cellular responses. Activation of MAPK is a transient event that is tightly regulated by both kinases and phosphatases. A growth factor induced dual specific phosphatase is likely to play an important role in MAPK regulation.
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PMID:The mitogen activated protein kinase signal transduction pathway: from the cell surface to the nucleus. 785 62

The Mos protein kinase is a serine-/threonine-specific protein kinase with a crucial role in meiotic cell divisions in vertebrates. Several oncogenic derivatives of the c-Mos protein have been discovered in murine retroviruses. These proteins have acquired mutations and exhibit different degrees of protein kinase activity in vitro. In an attempt to understand the factors governing Mos protein kinase activity we have compared the kinase activities of the wild-type c-Mos protein and two v-Mos proteins (strain HT1 and MSV124) after expression in insect cells. Only the 124 v-Mos protein showed kinase activity in vitro as measured by autophosphorylation, vimentin phosphorylation or by phosphorylation and activation of MAP kinase kinase. By domain swapping and site-directed mutagenesis we identified a single point mutation in the 124 v-Mos protein (Arg145-->Gly) which is responsible for its constitutive activity. This residue is located in the alpha-helix C of the kinase domain close to the ATP binding fold and is conserved in all known c-Mos proteins. Introduction of the corresponding mutation into HT1 v-Mos and into murine c-Mos activated both proteins for autophosphorylation, vimentin phosphorylation and for signalling via MAP kinase kinase in vitro. We hypothesize that the Arg145-->Gly mutation found in 124 v-Mos mimicks a conformational change which might be an obligatory step in the activation of c-Mos in vivo.
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PMID:Kinase activities of c-Mos and v-Mos proteins: a single amino acid exchange is responsible for constitutive activation of the 124 v-Mos kinase. 786 39


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