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 this study, we report the cloning and characterization of a novel human Ste20-related kinase that we designated MST4. The 416 amino acid full-length MST4 contains an amino-terminal kinase domain, which is highly homologous to MST3 and SOK, and a unique carboxy-terminal domain. Northern blot analysis indicated that MST4 is highly expressed in placenta, thymus, and peripheral blood leukocytes. Wild-type but not kinase-dead MST4 can phosphorylate myelin basic protein in an in vitro kinase assay. MST4 specifically activates ERK but not JNK or p38 MAPK in transient transfected cells or in stable cell lines. Overexpression of dominant negative MEK1 or treatment with PD98059 abolishes MST4-induced ERK activity, whereas dominant-negative Ras or c-Raf-1 mutants failed to do so, indicating MST4 activates MEK1/ERK via a Ras/Raf-1 independent pathway. HeLa and Phoenix cell lines overexpressing wild-type, but not kinase-dead, MST4 exhibit increased growth rate and form aggressive soft-agar colonies. These phenotypes can be inhibited by PD98059. These results provide the first evidence that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation.
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PMID:MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulating ERK pathway. 1164 81

Rice (Oryza sativa) anther development is easily damaged by moderately low temperatures above 12 degrees C. Subtractive screening of cDNA that accumulated in 12 degrees C-treated anthers identified a cDNA clone, OsMEK1, encoding a protein with features characteristic of a mitogen-activated protein (MAP) kinase kinase. The putative OsMEK1 protein shows 92% identity to the maize (Zea mays) MEK homolog, ZmMEK1. OsMEK1 transcript levels were induced in rice anthers by 12 degrees C treatment for 48 h. Similar OsMEK1 induction was observed in shoots and roots of seedlings that were treated at 12 degrees C for up to 24 h. It is interesting that no induction of OsMEK1 transcripts was observed in 4 degrees C-treated seedlings. In contrast, rice lip19, encoding a bZIP protein possibly involved in low temperature signal transduction, was not induced by 12 degrees C treatment but was induced by 4 degrees C treatment. Among the three MAP kinase homologs cloned, only OsMAP1 displayed similar 12 degrees C-specific induction pattern as OsMEK1. A yeast two-hybrid system revealed that OsMEK1 interacts with OsMAP1, but not with OsMAP2 and OsMAP3, suggesting that OsMEK1 and OsMAP1 probably function in the same signaling pathway. An in-gel assay of protein kinase activity revealed that a protein kinase (approximately 43 kD), which preferentially uses myelin basic protein as a substrate, was activated by 12 degrees C treatment but not by 4 degrees C treatment. Taken together, these results lead us to conclude that at least two signaling pathways for low temperature stress exist in rice, and that a MAP kinase pathway with OsMEK1 and OsMAP1 components is possibly involved in the signaling for the higher range low-temperature stress.
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PMID:Two novel mitogen-activated protein signaling components, OsMEK1 and OsMAP1, are involved in a moderate low-temperature signaling pathway in rice. 1217 2

To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (M(r) 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP-MBP-S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP-MBP-S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin-axonal interactions.
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PMID:Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelin. 1223 60

The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.
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PMID:Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis. 1249 39

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).
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PMID:A quantitative method for measuring protein phosphorylation. 1257 52

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.
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PMID:LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation. 1588 12

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2'3'nucleotide-cyclic 3'phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.
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PMID:A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching. 1972 58

SUMMARY Lipopolysaccharides (LPS) are indispensable cell surface components of Gram-negative bacteria and have diverse roles in plant-microbe interactions. Treatment of Nicotiana tabacum with the LPS of an endophytic strain of Burkholderia cepacia results in an enhanced defensive capacity in the tissue. In this study the rapid and transient phosphorylation of an extracellular signal-regulated (ERK)-like mitogen-activated protein (MAP) kinase in response to LPS from B. cepacia (LPS(B.cep.)) elicitation is reported. Based on in-gel kinase assays it was found that this 43-kDa LPS(B.cep.)-responsive kinase is optimally activated following 7 min elicitation with 100 microg/mL LPS. Its identity as an ERK MAPK was supported by tyrosine-phosphorylated association with induction, immunodetection with pTEpY-specific MAPK antibodies and inhibition of phosphorylation by U0126, an upstream MAPKK inhibitor. The kinase utilized myelin basic protein, but not casein or histone, as substrate. Ca(2+) did not modulate the phosphorylation, nor did wounding. To date, other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates produced during the oxidative burst. It was found that hydrogen peroxide is either not generated in leaf tissue in response to LPS elicitation or, if generated, does not trigger the phosphorylation of the kinase. Physicochemical characterization of the ERK-like MAPK indicated a molecular mass of 43 kDa and a pI of 6.3; two-dimensional gel analysis indicated two charge isomers. This is the first demonstration of such an LPS-responsive MAP kinase phosphorylation in plants.
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PMID:Identification of a lipopolysaccharide responsive erk-like MAP kinase in tobacco leaf tissue. 2056

Many extracellular and intrinsic factors regulate oligodendrocyte development, but their signaling pathways remain poorly understood. Although the p38 mitogen-activated protein kinase (MAPK)-dependent pathway is implicated in oligodendrocyte progenitor cell (OPC) lineage progression, its molecular targets involved in myelinogenesis are mostly unidentified. We have analyzed mechanisms by which p38MAPK regulates oligodendrocyte development and demonstrate that p38MAPK inhibition prevents OPC lineage progression and inhibits MBP (myelin basic protein) promoter activity and Sox10 function. In white-matter tissue, differential levels of MAPK phosphorylation are observed in oligodendrocyte lineage cells. Phosphorylated p38MAPK was found in CC1- and CNP-expressing differentiated oligodendrocytes of the adult brain and was temporally associated with a decline in the levels of phosphorylated extracellular signal-regulated kinase (ERK) in cells of this lineage. PDGF stimulates the phosphorylation of ERK, p38MAPK, and c-Jun N-terminal kinase (JNK), and p38MAPK inhibition was associated with increased ERK, JNK, and c-Jun phosphorylation. In the presence of PDGF, simultaneous inhibition of p38MAPK and either MAPK kinase (MEK) or JNK significantly alleviates the repression of myelin gene expression and lineage progression induced by p38MAPK inhibition alone. Dominant-negative c-Jun reverses the inhibition of myelin promoter activity by active MEK1 or dominant-negative p38MAPKalpha mutants, and phosphorylated c-Jun was detected at the MBP promoter after p38MAPK inhibition, indicating c-Jun as a negative mediator of p38MAPK action. Our findings indicate that p38MAPK activity in the brain supports myelin gene expression through distinct mechanisms via positive and negative regulatory targets. We show that oligodendrocyte differentiation involves p38-mediated Sox10 regulation and cross talk with parallel ERK and JNK pathways to repress c-Jun activity.
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PMID:Mechanisms of regulation of oligodendrocyte development by p38 mitogen-activated protein kinase. 2072 Jan 8

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg(2+) and Mn(2+) cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3's dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.
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PMID:Plasmodium falciparum possesses a unique dual-specificity serine/threonine and tyrosine kinase, Pfnek3. 2211 21


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