Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51812 (mitogen-activated protein)
 10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Spatiotemporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate. An understanding of the mechanisms by which these cellular phenomena are regulated during palate development involves the identification of the various signal transduction pathways. In the present study, the presence and activation of mitogen-activated protein (MAP) kinases were investigated during the development of quail secondary palate. The palatal shelves were dissected on days 5-9 of incubation, homogenized, and centrifuged, after which the samples were separated by anion exchange fast protein liquid chromatography. The fractions were analyzed for myelin basic protein (MBP) phosphorylation. In addition, primary cultures of quail palate mesenchymal cells (QPMCs) were treated with epidermal growth factor (EGF) and prepared for MBP phosphorylation assays. A temporally regulated pattern of phosphotransferase activity, characterized by a three-fold increase in phosphotransferase activity toward MBP between days 5 and 8 of incubation, was observed during quail palate development. Western blotting, using MAP kinase antibodies, demonstrated the presence of a 42-kDa isoform between days 5 and 9 of incubation, during which the level of protein remained constant. Antityrosine immunoblotting with 4G10 also detected a 42-kDa protein. Phosphotransferase assays, using either a MAP kinase-specific substrate peptide (S5) or a protein kinase C inhibitor (R3), further confirmed the presence of a MAP kinase in the developing palate of quail. Because diverse biological processes occur concurrently during in vivo palate morphogenesis, the involvement of MAP kinase was explored further in primary cell culture. The data showed that EGF stimulated proliferation and activated 42-kDa MAP kinase in QPMCs. It is suggested that MAP kinase cascade may be involved in growth factor-regulated cell proliferation during morphogenesis of quail secondary palate.
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PMID:In vivo and in vitro assessment of mitogen activated protein kinase involvement during quail secondary palate formation. 977 74

AG957, a tyrphostin tyrosine kinase inhibitor, has been shown previously to inhibit p210(bcr-abl) phosphorylation with concurrent inhibition of p210(bcr-abl)-expressing K562 cell growth (Kaur G and Sausville EA, Anticancer Drugs 7: 815-824, 1996). To assess the specificity of the action of AG957, we have examined its effect in another tyrosine kinase-mediated system, anti CD-3-stimulated Jurkat T Acute Lymphoblastic Leukemia cells. We also compared the effects of AG957 with those of geldanamycin, which can disrupt tyrosine kinase signaling through binding to heat shock protein (hsp90), and two geldanamycin analogs, 17-amino-17-demethoxygeldanamycin (17AG) and 17-allylamino-17-demethoxygeldanamycin (17AAG). At concentrations found to produce 90% inhibition of Jurkat T-cell growth, AG957 within 4 hr of addition inhibited mitogen-activated protein (MAP) kinase activation and activity, as shown by a decreased anti CD-3-stimulated erk-2 mobility shift in lysates of treated cells and a decrease in the stimulated myelin basic protein peptide kinase activity in erk-2 immunoprecipitates, respectively. AG957 did not inhibit this activity when added directly to immunoprecipitates. Effects in cells were found to be accompanied by a decrease in the anti CD-3-stimulated phosphorylation of p120cbl. Under conditions of a similar degree of growth inhibition, geldanamycin initially did not inhibit MAP kinase activation. Geldanamycin analogs did not decrease anti CD-3-induced cbl phosphorylation, but did reduce basal p120cbl tyrosine phosphorylation. The action of AG957 occurred with an apparent shift of several tyrosine-phosphorylated proteins to apparent higher molecular weights, which also did not occur with the geldanamycins. These results suggest that growth inhibition by AG957 can alter tyrosine kinase signaling systems unrelated to p210(bcr-abl) with a prominent early effect on MAP kinase activation in T-lymphoblasts. AG957 and geldanamycin affect tyrosine kinase signaling by distinct mechanisms.
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PMID:Different early effets of tyrphostin AG957 and geldanamycins on mitogen-activated protein kinase and p120cbl phosphorylation in anti CD-3-stimulated T-lymphoblasts. 989 May 55

We tested the coupling of endothelin receptors to mitogen-activated protein kinases (MAPK) and nuclear factor c-Jun in intact canine pulmonary artery smooth muscle. Muscle rings denuded of endothelium were stimulated with 10(-7)M ET-1 and frozen during contraction. An <<in-gel>> kinase assay with myelin basic protein as substrate revealed protein kinase activities at 98, 75, 55, 50, 44 and 40 kDa. Erk1 and Erk2 MAPK were activated by ET-1 to 5.4+/-0.97 and 4.03+/-1. 54 times basal activity at 10 min. Using phospho-specific antibodies, we found increased threonine/tyrosine phosphorylation of p38 and JNK1 MAPK to 2.04+/-0.47 and 2.56+/-0.72 times basal. ET-1 increased the phosphorylation level of nuclear factor c-Jun with a time-course closely matching the activation of JNK1 and p38 MAPK. Therefore, endothelin receptors initiate intracellular signals leading to activation of Erk, p38 and JNK1 MAPK pathways and ultimately to nuclear targets. The activation of JNK1 MAPK seems closely related to the phosphorylation of nuclear transcription factor c-Jun.
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PMID:Endothelin-1 activates MAP kinases and c-Jun in pulmonary artery smooth muscle. 991 57

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.
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PMID:Rapid Avr9- and Cf-9 -dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses. 992 44

The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli. The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms. The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts. Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity. Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector. Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity. Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location. This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2. The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting.
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PMID:An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature. 1051 73

Wheat cultured cells were used to study the role of Ca2+ in regulating protein kinases during the induction of defense-related genes by fungal elicitor treatments. Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1, a gene encoding mitogen-activated protein (MAP) kinase. The induction of WCK-1 mRNA by A23187 did not occur when extracellular Ca2+ was chelated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The WCK-1 mRNA was also induced by Typhula ishikariensis-derived elicitors, suggesting a possible involvement of WCK-1 in the plant defense response against pathogens. BAPTA and a calcium channel blocker, La3+, inhibited the elicitor-induced expression of the WCK-1 mRNA. A recombinant fusion protein of WCK-1 (GST-WCK-1) autophosphorylated at the Tyr residue and exhibited an autophosphorylation-dependent protein kinase activity towards myelin basic protein. Alteration of Tyr-196 in the conserved 'TEY' motif in GST-WCK-1 to Phe by site-directed mutagenesis abolished the autophosphorylation. The GST-WCK-1 protein was activated by elicitor-treated wheat cell extracts but not by the control extract. These results suggest that fungal elicitors activate WCK-1, a specific MAP kinase in wheat. Furthermore, the results suggest a possible involvement of Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts.
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PMID:Elicitor- and A23187-induced expression of WCK-1, a gene encoding mitogen-activated protein kinase in wheat. 1052 17

Four species of protein kinase were identified in senescent maize leaves using a gel assay for kinase activity with myelin basic protein (MBP) as the substrate. Most of these kinases were also found in healthy green leaves that had been exposed to low-temperature stress (5 degrees C) and then returned to 25 degrees C. A 41-kDa protein was activated in senescent leaves, whereas a 45-kDa protein was activated 3 h after up-shift from 5 degrees C to 25 degrees C as well as in senescent leaves. A 39-kDa protein was activated by cold stress. The other two proteins, of 35 kDa and 52 kDa, constitutively phosphorylated MBP during senescence and temperature up-shift. Judging from their molecular masses, cation requirements and substrate specificities, it seemed likely that the 39-kDa, 41-kDa and 45-kDa proteins represented mitogen-activated protein kinases (MAPKs). Subsequently two MAPK cDNAs were isolated from a cDNA library constructed using mRNAs from senescent leaves. Northern analysis showed that the transcript corresponding to one of the cDNAs, designated ZmMPK5, accumulated in healthy leaves 3 h after the up-shift to 25 degrees C as well as in senescent leaves, suggesting that the 45-kDa protein kinase is encoded by ZmMPK5. Western analysis using an antiserum against the C-terminal region of ZmMPK5 showed that the level of the ZmMPK5 protein increased in senescent leaves. These results indicate that a 45-kDa MAPK is involved in the process of senescence and in recovery from low-temperature stress in maize plants.
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PMID:Involvement of a MAP kinase, ZmMPK5, in senescence and recovery from low-temperature stress in maize. 1058 42

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (</=0.5 microM) is significantly lower than that of the measured K(m(MBP)) (4.2 +/- 0.8 microM). Furthermore, MBP binds to the ERK2 x ATP complex at least 1500-fold more tightly than does ERKtide (K(d(ERKtide)) >/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (</=1.2 s(-1)) versus that of the synthetic peptide (>/=56 s(-1)), from the ERK2 active site.
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PMID:Catalytic reaction pathway for the mitogen-activated protein kinase ERK2. 1082 2

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.
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PMID:WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II. 1082 64

The mitogen-activated protein (MAP) kinases are characterized by their requirement for dual phosphorylation at a conserved threonine and tyrosine residue for catalytic activation. The structural consequences of dual-phosphorylation in the MAP kinase ERK2 (extracellular signal-regulated kinase 2) include active site closure, alignment of key catalytic residues that interact with ATP, and remodeling of the activation loop. In this study, we report the specific effects of dual phosphorylation on the individual catalytic reaction steps in ERK2. Dual phosphorylation leads to an increase in overall catalytic efficiency and turnover rate of approximately 600,000- and 50,000-fold, respectively. Solvent viscosometric studies reveal moderate decreases in the equilibrium dissociation constants (K(d)) for both ATP and myelin basic protein. However, the majority of the overall rate enhancement is due to an increase in the rate of the phosphoryl group transfer step by approximately 60,000-fold. By comparison, the rate of the same step in the ATPase reaction is enhanced only 2000-fold. This suggests that optimizing the position of the invariant residues Lys(52) and Glu(69), which stabilize the phosphates of ATP, accounts for only part of the enhanced rate of phosphoryl group transfer in the kinase reaction. Thus, significant stabilization of the protein phosphoacceptor group must also occur. Our results demonstrate similarities between the activation mechanisms of ERK2 and the cell cycle control enzyme, Cdk2 (cyclin-dependent kinase 2). Rather than dual phosphorylation, however, activation of the latter is controlled by cyclin binding followed by phosphorylation at Thr(160).
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PMID:Mechanism of activation of ERK2 by dual phosphorylation. 1101 42


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