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Sample records for arabidopsis map kinase

  1. Arabidopsis map kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

    Brodersen, P; Johansen, Bo; Petersen, M;

    2000-01-01

    Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) levels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern...... of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression....

  2. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

    Petersen, M.; Brodersen, P.; Naested, H.;

    2000-01-01

    Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) revels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern...... of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression....

  3. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    Science.gov (United States)

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  4. The wheat MAP kinase phosphatase 1 alleviates salt stress and increases antioxidant activities in Arabidopsis.

    Science.gov (United States)

    Zaidi, Ikram; Ebel, Chantal; Belgaroui, Nibras; Ghorbel, Mouna; Amara, Imène; Hanin, Moez

    2016-04-01

    Mitogen-activated protein kinase phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, development and stress responses. We have previously shown that the heterologous expression of a durum wheat MKP, TMKP1, results in increased tolerance to salt stress in yeast but its particular contribution in salt stress tolerance in plants was not investigated. Here, TMKP1 was overexpressed in Arabidopsis thaliana and physiological changes were assessed in transgenic plants exposed to stress conditions. Under salt stress and especially LiCl, the TMKP1 overexpressors displayed higher germination rates in comparison to wild type plants. The enhancement of salt stress tolerance was accompanied by increased antioxidant enzyme activities, namely superoxide dismutase, catalase and peroxydases. Such increases in antioxidant activities were concomitant with lower malondialdehyde, superoxide anion O2(-) and hydrogen peroxide levels in the TMKP1 transgenic seedlings. Moreover, we provide evidence that, in contrast to the Arabidopsis ortholog AtMKP1, TMKP1 acts as a positive regulator of salt stress tolerance via its ectopic expression in the Arabidopsis mkp1 mutant. PMID:26927025

  5. Arabidopsis MAP Kinase 4 regulates gene expression via transcription factor release in the nucleus

    DEFF Research Database (Denmark)

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus;

    2008-01-01

    kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from...... MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further...... supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation....

  6. Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana

    KAUST Repository

    Danquah, Agyemang

    2015-04-01

    Summary Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling. Significance Statement We report in this article the identification of a complete MAPK module, composed of MAP3K17/18, MKK3 and MPK1/2/7/14, which is activated by ABA through the ABA core signalling complex. We showed that the activation of this module requires the MAP3K protein synthesis which occurs after hours of stress treatment, suggesting that the pathway is involved in a delayed wave of cellular responses to ABA and drought. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  7. Genetic analysis of the Arabidopsis protein kinases MAP3Kε1 and MAP3Kε2 indicates roles in cell expansion and embryo development.

    Science.gov (United States)

    Chaiwongsar, Suraphon; Strohm, Allison K; Su, Shih-Heng; Krysan, Patrick J

    2012-01-01

    MAP3Kε1 and MAP3Kε2 are a pair of Arabidopsis thaliana genes that encode protein kinases related to cdc7p from Saccharomyces cerevisiae. We have previously shown that the map3kε1;map3kε2 double-mutant combination causes pollen lethality. In this study, we have used an ethanol-inducible promoter construct to rescue this lethal phenotype and create map3kε1(-/-);map3kε2(-/-) double-mutant plants in order to examine the function of these genes in the sporophyte. These rescued double-mutant plants carry a yellow fluorescent protein (YFP)-MAP3Kε1 transgene under the control of the alcohol-inducible AlcA promoter from Aspergillus nidulans. The double-mutant plants were significantly smaller and had shorter roots than wild-type when grown in the absence of ethanol treatment. Microscopic analysis indicated that cell elongation was reduced in the roots of the double-mutant plants and cell expansion was reduced in rosette leaves. Treatment with ethanol to induce expression of YFP-MAP3Kε1 largely rescued the leaf phenotypes. The double-mutant combination also caused embryos to arrest in the early stages of development. Through the use of YFP reporter constructs we determined that MAP3Kε1 and MAP3Kε2 are expressed during embryo development, and also in root tissue. Our results indicate that MAP3Kε1 and MAP3Kε2 have roles outside of pollen development and that these genes affect several aspects of sporophyte development.

  8. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.

    Science.gov (United States)

    Brodersen, Peter; Petersen, Morten; Bjørn Nielsen, Henrik; Zhu, Shijiang; Newman, Mari-Anne; Shokat, Kevan M; Rietz, Steffen; Parker, Jane; Mundy, John

    2006-08-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA-ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4-EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

  9. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4

    DEFF Research Database (Denmark)

    Brodersen, Klaus Peter; Petersen, Morten; Nielsen, Henrik Bjørn;

    2006-01-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective...

  10. Map kinases in fungal pathogens.

    Science.gov (United States)

    Xu, J R

    2000-12-01

    MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens. PMID:11273677

  11. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  12. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  13. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  14. Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

    Directory of Open Access Journals (Sweden)

    Pieterse Corné MJ

    2007-02-01

    Full Text Available Abstract Background Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. Results Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities. Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. Conclusion We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown kinases that play a role in P. syringae infection. This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.

  15. The MAP kinase substrate MKS1 is a regulator of plant defense responses

    DEFF Research Database (Denmark)

    Andreasson, E.; Jenkins, T.; Brodersen, P.;

    2005-01-01

    Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used...

  16. MAP Kinase Cascades in Plant Innate Immunity

    Directory of Open Access Journals (Sweden)

    Magnus Wohlfahrt Rasmussen

    2012-07-01

    Full Text Available Plant mitogen-activated protein kinase (MAPK cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs by host transmembrane pattern recognition receptors (PRRs which trigger MAPK-dependent innate immune responses. In the model Arabidopsis, molecular genetic evidence implicates a number of MAPK cascade components in PAMP signaling, and in responses to immunity-related phytohormones such as ethylene, jasmonate and salicylate. In a few cases, cascade components have been directly linked to the transcription of target genes or to the regulation of phytohormone synthesis. Thus MAPKs are obvious targets for bacterial effector proteins and are likely guardees of resistance (R proteins, which mediate defense signaling in response to the action of effectors, or effector-triggered immunity (ETI. This mini-review discusses recent progress in this field with a focus on the Arabidopsis MAPKs MPK3, 4, 6 and 11 in their apparent pathways.

  17. The protein kinase TOUSLED facilitates RNAi in Arabidopsis.

    Science.gov (United States)

    Uddin, Mohammad Nazim; Dunoyer, Patrice; Schott, Gregory; Akhter, Salina; Shi, Chunlin; Lucas, William J; Voinnet, Olivier; Kim, Jae-Yean

    2014-07-01

    RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference. PMID:24920830

  18. MAP KinaseCascades Responding to Environmental Stress in Plants

    Institute of Scientific and Technical Information of China (English)

    YUShun—Wu; TANGKe—Xuan

    2004-01-01

    Plant mitogen-activated protein kinases(MAPKs) are involved in growth,evelopment and responses to endogenous and environmental cues.which link stimuli that areactivated by external sensors to cellular responses.In Arabidopsis,as amodel,all of MAP kinase genes have been listed and classified.Based on the Arabidopsis MAPK families.a number of MAPk inase genes in other plant species have been recently isolated and classified.Most of the cloned MAPk inase genes can be activated by avariety of stresss timuli including pathogen infection.wounding.temperature,drought.salinity.osmolarity.UV irradiation.ozone and reactive oxygen species.Some tools and strategies are used to investigate their functions and signal pathways under different environmental stresses.indicating complexity and crosstalk of plant MAPk inase signaling pathways.It is still necessary to explore more novel tools and strategies to clarify MAPK signaling pathways,and how to apply the MAPK cascade to improve the resistance of crop to abiotic and biotic stress

  19. MAP kinases and histone modification

    Institute of Scientific and Technical Information of China (English)

    Tamaki Suganuma; Jerry L. Workman

    2012-01-01

    Signal transduction pathways alter the gene expression program in response to extracellular or intracellular cues.Mitogen-activated protein kinases (MAPKs) govern numerous cellular processes including cell growth,stress response,apoptosis,and differentiation.In the past decade,MAPKs have been shown to regulate the transcription machinery and associate with chromatin-modifying complexes.Moreover,recent studies demonstrate that several MAPKs bind directly to chromatin at target genes.This review highlights the recent discoveries of MAPK signaling in regard to histone modifications and chromatin regulation.Evidence suggesting that further unknown mechanisms integrate signal transduction with chromatin biology is discussed.

  20. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    OpenAIRE

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  1. The MEKK1-MKK1/MKK2-MPK4 Kinase Cascade Negatively Regulates Immunity Mediated by a Mitogen-Activated Protein Kinase Kinase Kinase in Arabidopsis[C][W

    Science.gov (United States)

    Kong, Qing; Qu, Na; Gao, Minghui; Zhang, Zhibin; Ding, Xiaojun; Yang, Fan; Li, Yingzhong; Dong, Oliver X.; Chen, She; Li, Xin; Zhang, Yuelin

    2012-01-01

    In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding–leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses. PMID:22643122

  2. Constitutive activation of AtMEK5, a MAPK kinase, induces salicylic acid-independent cell death in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    LIU Hongxia; WANG Ying; ZHOU Tianhong; SUN Yujing; LIU Guoqin; REN Dongtao

    2004-01-01

    AtMEK5DD is an active mutant of AtMEK5, a MAP kinase kinase in Arabidopsis. Induction of AtMEK5DD expression in transgenic plants leads to activation of 44 and 48 kD MAPKs and causes a rapid cell death. To compare the cell death induced by the expression of AtMEK5DD with the HR-cell death induced by avirulence pathogen infection, we analyzed the activation of downstream MAP Kinase and induction of PR genes expression in permanent transgenic Arabidopsis plants. In-gel kinase activity assay revealed that the infection of Pseudomonas syringae DC3000 harboring Avr Rpt2 gene also lead to activation of 44 and 48 kD MAPKs. PAL, PR1 and PR5 were strongly induced in plants undergoing HR-cell death caused by the infection of P. Syringae DC3000, while only the expression of PR5 was strongly induced in transgenic plants expressing AtMEK5DD protein. NahG protein in AtMEK5DD×NahG plants cannot suppress the cell death induced by AtMEK5DD. And AtMEK5DD protein expressed AtMEK5DD×NahG plants showed no significant change in salicylic acid (SA)level.All these suggest that the cell death induced by the activation of AtMEK5 is salicylic acid-independent.

  3. Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase.

    Science.gov (United States)

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

    Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane. PMID:21388955

  4. Measuring MAP kinase activity in immune complex assays.

    Science.gov (United States)

    Cherkasova, Vera A

    2006-11-01

    I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins. PMID:16890454

  5. Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

    NARCIS (Netherlands)

    Ritsema, T.; Joore, J.; Workum, W. van; Pieterse, C.M.J.

    2007-01-01

    Background: Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian s

  6. A comparative map viewer integrating genetic maps for Brassica and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Erwin Timothy A

    2007-07-01

    Full Text Available Abstract Background Molecular genetic maps provide a means to link heritable traits with underlying genome sequence variation. Several genetic maps have been constructed for Brassica species, yet to date, there has been no simple means to compare this information or to associate mapped traits with the genome sequence of the related model plant, Arabidopsis. Description We have developed a comparative genetic map database for the viewing, comparison and analysis of Brassica and Arabidopsis genetic, physical and trait map information. This web-based tool allows users to view and compare genetic and physical maps, search for traits and markers, and compare genetic linkage groups within and between the amphidiploid and diploid Brassica genomes. The inclusion of Arabidopsis data enables comparison between Brassica maps that share no common markers. Analysis of conserved syntenic blocks between Arabidopsis and collated Brassica genetic maps validates the application of this system. This tool is freely available over the internet on http://bioinformatics.pbcbasc.latrobe.edu.au/cmap. Conclusion This database enables users to interrogate the relationship between Brassica genetic maps and the sequenced genome of A. thaliana, permitting the comparison of genetic linkage groups and mapped traits and the rapid identification of candidate genes.

  7. Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

    DEFF Research Database (Denmark)

    Peraldi, P; Frödin, M; Barnier, J V;

    1995-01-01

    In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the r...

  8. A Brassinosteroid-Signaling Kinase Interacts withMultiple Receptor-Like Kinases in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Dear Editor, Higher plants have evolved hundreds of genes encodingreceptor-like kinases (RLKs), which function as cell surfacereceptors perceiving developmental and environmental sig-nals (Shiu et al., 2004). Many RLKs have been shown to playspecific roles in hormone responses, developmental regula-tion, defense against pathogen infection, and adaptationto abiotic stresses (Chae et al., 2009; Antolin-Llovera et al.,2012). The mechanisms that ensure specific signal transduc-tion from each RLK to target cellular responses remain poorlyunderstood. Recent studies revealed that many RLKs trans-duce signals by phosphorylating receptor-like cytoplasmickinases (RLCKs), which lack the transmembrane domainsbut are anchored at the plasma membrane through lipidmodification (Tang et al., 2008; Zhang et al., 2010; Shi et al.,2013). There are over 400 RLKs and only about 150 RLCKs inArabidopsis (Shiu et al., 2004). One outstanding question iswhether each RLCK mediates signaling downstream of a spe-cific RLK, participates in multiple RLK pathways, or mediatescrosstalk between RLK pathways.

  9. The receptor kinase IMPAIRED OOMYCETE SUSCEPTIBILITY1 attenuates abscisic acid responses in Arabidopsis.

    Science.gov (United States)

    Hok, Sophie; Allasia, Valérie; Andrio, Emilie; Naessens, Elodie; Ribes, Elsa; Panabières, Franck; Attard, Agnès; Ris, Nicolas; Clément, Mathilde; Barlet, Xavier; Marco, Yves; Grill, Erwin; Eichmann, Ruth; Weis, Corina; Hückelhoven, Ralph; Ammon, Alexandra; Ludwig-Müller, Jutta; Voll, Lars M; Keller, Harald

    2014-11-01

    In plants, membrane-bound receptor kinases are essential for developmental processes, immune responses to pathogens and the establishment of symbiosis. We previously identified the Arabidopsis (Arabidopsis thaliana) receptor kinase IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as required for successful infection with the downy mildew pathogen Hyaloperonospora arabidopsidis. We report here that IOS1 is also required for full susceptibility of Arabidopsis to unrelated (hemi)biotrophic filamentous oomycete and fungal pathogens. Impaired susceptibility in the absence of IOS1 appeared to be independent of plant defense mechanism. Instead, we found that ios1-1 plants were hypersensitive to the plant hormone abscisic acid (ABA), displaying enhanced ABA-mediated inhibition of seed germination, root elongation, and stomatal opening. These findings suggest that IOS1 negatively regulates ABA signaling in Arabidopsis. The expression of ABA-sensitive COLD REGULATED and RESISTANCE TO DESICCATION genes was diminished in Arabidopsis during infection. This effect on ABA signaling was alleviated in the ios1-1 mutant background. Accordingly, ABA-insensitive and ABA-hypersensitive mutants were more susceptible and resistant to oomycete infection, respectively, showing that the intensity of ABA signaling affects the outcome of downy mildew disease. Taken together, our findings suggest that filamentous (hemi)biotrophs attenuate ABA signaling in Arabidopsis during the infection process and that IOS1 participates in this pathogen-mediated reprogramming of the host. PMID:25274985

  10. Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

    KAUST Repository

    Kim, Dongjin

    2015-10-09

    Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

  11. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Science.gov (United States)

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  12. An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

    OpenAIRE

    Fujii, Hiroaki; Zhu, Jian-Kang

    2009-01-01

    The protein kinase SOS2 (Salt Overly Sensitive 2) is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed ...

  13. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  14. Fine-mapping of an Arabidopsis cell death mutation locus

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    An Arabidopsis cell death mutation locus was mapped to chromosome 2 between IGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.

  15. Fine-mapping of an Arabidopsis cell death mutation locus

    Institute of Scientific and Technical Information of China (English)

    牟中林; 戴亚; 李家洋

    2000-01-01

    An Arabidopsis cell death mutation locus was mapped to chromosome 2 between lGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.

  16. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  17. Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Sandra Franz; Britta Ehlert; Anja Liese; Joachim Kurth; Anne-Claire Cazalé; Tina Romeis

    2011-01-01

    Calcium-dependent protein kinases(CDPKs)comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule.So far,a biological function in abiotic stress signaling has only been reported for few CDPK isoforms,whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown.Here,we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress.Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation.In transgenic Arabidopsis complementation lines in the cpk21 mutant background,in which either CPK21 wildtype,or a full-length enzyme variant carrying an amino-acid substitution were stably expressed,stress responsitivity was restored by CPK21 but not with the kinase inactive variant.The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain,N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity,suggesting a crucial role for the N-terminal EF-hand pair.Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

  18. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq;

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  19. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq;

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.......7.1.21)‐like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T‐DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a...

  20. SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana

    OpenAIRE

    Umezawa, Taishi; Yoshida, Riichiro; Maruyama, Kyonoshin; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2004-01-01

    Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Addition...

  1. Importance of MAP kinases during protoperithecial morphogenesis in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Alexander Lichius

    Full Text Available In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.

  2. Positional signaling mediated by a receptor-like kinase in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Shen, Ronglai; Schiefelbein, John

    2005-02-18

    The position-dependent specification of root epidermal cells in Arabidopsis provides an elegant paradigm for cell patterning during development. Here, we describe a new gene, SCRAMBLED (SCM), required for cells to appropriately interpret their location within the developing root epidermis. SCM encodes a receptor-like kinase protein with a predicted extracellular domain of six leucine-rich repeats and an intracellular serine-threonine kinase domain. SCM regulates the expression of the GLABRA2, CAPRICE, WEREWOLF, and ENHANCER OF GLABRA3 transcription factor genes that define the cell fates. Further, the SCM gene is expressed throughout the developing root. Therefore, SCM likely enables developing epidermal cells to detect positional cues and establish an appropriate cell-type pattern.

  3. Somatic Embryogenesis Receptor Kinases Control Root Development Mainly via Brassinosteroid Independent Actions in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Junbo Du; Hongju Yin; Shasha Zhang; ZhuoyunWei; Baolin Zhao; Jinghua Zhang; Xiaoping Gou; Honghui Lin; Jia Li

    2012-01-01

    Brassinosteroids (BRs),a group of plant steroidal hormones,play critical roles in many aspects of plant growth and development.Previous studies showed that BRI1-mediated BR signaling regulates cell division and differentiation during Arabidopsis root development via interplaying with auxin and other phytohormones.Arabidopsis somatic embryogenesis receptor-like kinases (SERKs),as co-receptors of BRI1,were found to play a fundamental role in an early activation step of BR signaling pathway.Here we report a novel function of SERKs in regulating Arabidopsis root development.Genetic analyses indicated that SERKs control root growth mainly via a BR-independent pathway.Although BR signaling pathway is completely disrupted in the serk1 bak1 bkk1 triple mutant,the root growth of the triple mutant is much severely damaged than the BR deficiency or signaling null mutants.More detailed analyses indicated that the triple mutant exhibited drastically reduced expression of a number of genes critical to polar auxin transport,cell cycle,endodermis development and root meristem differentiation,which were not observed in null BR biosynthesis mutant cpd and null BR signaling mutant bri1-701.

  4. The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Iris Camehl

    2011-05-01

    Full Text Available Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1 gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H₂O₂ and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1. A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H₂O₂ formation is even reduced by the fungus. Importantly, phospholipase D (PLDα1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

  5. An Arabidopsis example of association mapping in structured samples.

    Directory of Open Access Journals (Sweden)

    Keyan Zhao

    2007-01-01

    Full Text Available A potentially serious disadvantage of association mapping is the fact that marker-trait associations may arise from confounding population structure as well as from linkage to causative polymorphisms. Using genome-wide marker data, we have previously demonstrated that the problem can be severe in a global sample of 95 Arabidopsis thaliana accessions, and that established methods for controlling for population structure are generally insufficient. Here, we use the same sample together with a number of flowering-related phenotypes and data-perturbation simulations to evaluate a wider range of methods for controlling for population structure. We find that, in terms of reducing the false-positive rate while maintaining statistical power, a recently introduced mixed-model approach that takes genome-wide differences in relatedness into account via estimated pairwise kinship coefficients generally performs best. By combining the association results with results from linkage mapping in F2 crosses, we identify one previously known true positive and several promising new associations, but also demonstrate the existence of both false positives and false negatives. Our results illustrate the potential of genome-wide association scans as a tool for dissecting the genetics of natural variation, while at the same time highlighting the pitfalls. The importance of study design is clear; our study is severely under-powered both in terms of sample size and marker density. Our results also provide a striking demonstration of confounding by population structure. While statistical methods can be used to ameliorate this problem, they cannot always be effective and are certainly not a substitute for independent evidence, such as that obtained via crosses or transgenic experiments. Ultimately, association mapping is a powerful tool for identifying a list of candidates that is short enough to permit further genetic study.

  6. Genetic mapping of adaptation reveals fitness tradeoffs in Arabidopsis thaliana.

    Science.gov (United States)

    Ågrena, Jon; Oakley, Christopher G; McKay, John K; Lovell, John T; Schemske, Douglas W

    2013-12-24

    Organisms inhabiting different environments are often locally adapted, and yet despite a considerable body of theory, the genetic basis of local adaptation is poorly understood. Unanswered questions include the number and effect sizes of adaptive loci, whether locally favored loci reduce fitness elsewhere (i.e., fitness tradeoffs), and whether a lack of genetic variation limits adaptation. To address these questions, we mapped quantitative trait loci (QTL) for total fitness in 398 recombinant inbred lines derived from a cross between locally adapted populations of the highly selfing plant Arabidopsis thaliana from Sweden and Italy and grown for 3 consecutive years at the parental sites (>40,000 plants monitored). We show that local adaptation is controlled by relatively few genomic regions of small to modest effect. A third of the 15 fitness QTL we detected showed evidence of tradeoffs, which contrasts with the minimal evidence for fitness tradeoffs found in previous studies. This difference may reflect the power of our multiyear study to distinguish conditionally neutral QTL from those that reflect fitness tradeoffs. In Sweden, but not in Italy, the local genotype underlying fitness QTL was often maladaptive, suggesting that adaptation there is constrained by a lack of adaptive genetic variation, attributable perhaps to genetic bottlenecks during postglacial colonization of Scandinavia or to recent changes in selection regime caused by climate change. Our results suggest that adaptation to markedly different environments can be achieved through changes in relatively few genomic regions, that fitness tradeoffs are common, and that lack of genetic variation can limit adaptation.

  7. Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases

    Directory of Open Access Journals (Sweden)

    Yu-Hung eYeh

    2015-05-01

    Full Text Available Upon recognition of microbe-associated molecular patterns (MAMPs such as the bacterial flagellin (or the derived peptide flg22 by pattern-recognition receptors (PRRs such as the FLAGELLIN SENSING2 (FLS2, plants activate the pattern-triggered immunity (PTI response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2 is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs possess two copies of the C-X8-C-X2-C (DUF26 motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6 and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1 was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6 and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity.

  8. Activation and signaling of the p38 MAP kinase pathway

    Institute of Scientific and Technical Information of China (English)

    Tyler ZARUBIN; Jiahuai HAN

    2005-01-01

    The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.

  9. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  10. The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis.

    Science.gov (United States)

    Kwak, Su-Hwan; Schiefelbein, John

    2007-02-01

    Cell-type patterning in the Arabidopsis root epidermis is achieved by a network of transcription factors and influenced by a position-dependent mechanism. The SCRAMBLED receptor-like kinase is required for the normal pattern to arise, but its precise role is not understood. Here we describe genetic and molecular studies to define the spatial and temporal role of SCM in epidermal patterning and its relationship to the transcriptional network. Our results suggest that SCM helps unspecified epidermal cells interpret their position in relation to the underlying cortical cells and establish distinct cell identities. Furthermore, SCM loss-of-function and overexpression analyses suggest that SCM influences cell fate through its negative transcriptional regulation of the WEREWOLF MYB gene in epidermal cells at the H position. We also find that SCM function is specifically required for patterning the post-embryonic root epidermis and not for the analogous epidermal cell-type patterning during embryogenesis or hypocotyl development. In addition, we show that two closely related SCM-like genes in Arabidopsis (SRF1 and SRF3) are not required alone or together with SCM for proper epidermal patterning. These findings help define the developmental and mechanistic role of SCM and suggest a new model for its action in root epidermal cell patterning.

  11. An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hiroaki Fujii; Jian-Kang Zhu

    2009-01-01

    The protein kinase SOS2 (Salt Overly Sensitive 2) is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the S228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.

  12. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    Science.gov (United States)

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  13. LIK1, a CERK1-interacting kinase, regulates plant immune responses in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Mi Ha Le

    Full Text Available Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1 as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190 and a leucine-rich repeat receptor like kinase (At3g14840, which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1. The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.

  14. 3D gel map of Arabidopsis complex I

    OpenAIRE

    Katrin ePeters; Katharina eBelt; Hans-Peter eBraun

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves and roots. Subunits of complex I were resolved by 3D blue native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, 7 of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of su...

  15. The MAP kinase substrate MKS1 is a regulator of plant defense responses.

    Science.gov (United States)

    Andreasson, Erik; Jenkins, Thomas; Brodersen, Peter; Thorgrimsen, Stephan; Petersen, Nikolaj H T; Zhu, Shijiang; Qiu, Jin-Long; Micheelsen, Pernille; Rocher, Anne; Petersen, Morten; Newman, Mari-Anne; Bjørn Nielsen, Henrik; Hirt, Heribert; Somssich, Imre; Mattsson, Ole; Mundy, John

    2005-07-20

    Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.

  16. Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin.

    Science.gov (United States)

    Wan, Jinrong; Zhang, Shuqun; Stacey, Gary

    2004-03-01

    SUMMARY Chitin, a polysaccharide composed of beta-1-->4-linked N-acetyl-d-glucosamine, has been shown or implicated as a signal in plant defence and development. However, the key components of chitin perception and downstream signalling in non-leguminous plants are largely unknown. In recent years, mitogen-activated protein kinases (MAPKs) and their cascades were shown to transduce various extracellular stimuli into internal cellular responses. To investigate the possible involvement of MAPKs in chitin signalling in plants, the model plant Arabidopsis thaliana was treated with crab-shell chitin and also with the purified chitin oligomers (degree of polymerization, d.p. = 2-8). Both mRNA levels and kinase activity of two MAPK genes, AtMPK6 and AtMPK3, were monitored after treatment. The mRNA of AtMPK3 was strongly up-regulated by both chitin and its larger oligomers (d.p. = 6-8), but the mRNA of AtMPK6 did not appear to be regulated by these treatments. However, the kinase activity of both MAPKs was induced by chitin and the larger oligomers (d.p. = 6-8), with AtMPK6 much more strongly induced. In addition, WRKY22, WRKY29, WRKY33 and WRKY53, which encode four WRKY transcription factors that recognize TTGAC(C/T) W-box elements in promoters of numerous plant defence-related genes, were up-regulated by these treatments. WRKY33 and WRKY53 expression was induced by the transgenic expression of the tobacco MAPKK NtMEK2 active mutant NtMEK2(DD), suggesting a potential role for these WRKY transcription factors in relaying the signal generated from the MAPK cascade to downstream genes. These data suggest that AtMPK6/AtMPK3 and WRKY transcription factors (such as WRKY33 and WRKY53) may be important components of a pathway involved in chitin signalling in Arabidopsis plants.

  17. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

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    Nasar Virk

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

  18. Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein

    Institute of Scientific and Technical Information of China (English)

    Nicholas TRAKUL; Marsha R. ROSNER

    2005-01-01

    Proteins like Raf kinase inhibitory protein (RKIP) that serve as modulators of signaling pathways, either by promoting or inhibiting the formation of productive signaling complexes through protein-protein interactions, have been demonstrated to play an increasingly important role in a number of cell types and organisms. These proteins have been implicated in development as well as the progression of cancer. RKIP is a particularly interesting regulator, as it is a highly conserved, ubiquitously expressed protein that has been shown to play a role in growth and differentiation in a number of organisms and can regulate multiple signaling pathways. RKIP is also the first MAP kinase signaling modulator to be identified as playing a role in cancer metastasis, and identification of the mechanism by which it regulates Raf-1 activation provides new targets for therapeutic intervention.

  19. Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

    Science.gov (United States)

    Huang, Yafan; Li, Hui; Hutchison, Claire E.; Laskey, James; Kieber, Joseph J.

    2003-01-01

    CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

  20. Regulation of the S-locus receptor kinase and self-incompatibility in Arabidopsis thaliana.

    Science.gov (United States)

    Strickler, Susan R; Tantikanjana, Titima; Nasrallah, June B

    2013-02-01

    Intraspecific mate selectivity often is enforced by self-incompatibility (SI), a barrier to self-pollination that inhibits productive pollen-pistil interactions. In the Brassicaceae, SI specificity is determined by two highly-polymorphic proteins: the stigmatic S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich protein (SCR). Arabidopsis thaliana is self fertile, but several of its accessions can be made to express SI, albeit to various degrees, by transformation with functional SRK-SCR gene pairs isolated from its close self-incompatible relative, Arabidopsis lyrata. Here, we use a newly identified induced mutation that suppresses the SI phenotype in stigmas of SRK-SCR transformants of the Col-0 accession to investigate the regulation of SI and the SRK transgene. This mutation disrupts NRPD1a, a gene that encodes a plant-specific nuclear RNA polymerase required for genomic methylation and production of some types of silencing RNAs. We show that NRPD1a, along with the RNA-dependent RNA polymerase RDR2, is required for SI in some A. thaliana accessions. We also show that Col-0 nrpd1a mutants exhibit decreased accumulation of SRK transcripts in stigmas, which is not, however, responsible for loss of SI in these plants. Together, our analysis of the nrpd1a mutation and of SRK promoter activity in various accessions reveals that the SRK transgene is subject to several levels of regulation, which vary substantially by tissue type and by accession. This study thus helps explain the well-documented differences in expression of SI exhibited by SRK-SCR transformants of different A. thaliana accessions.

  1. The NDR kinase scaffold HYM1/MO25 is essential for MAK2 map kinase signaling in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Anne Dettmann

    2012-09-01

    Full Text Available Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete Neurospora crassa, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell-cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell-cell contact. hym-1 mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in hym-1, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.

  2. A maize mitogen-activated protein kinase kinase, ZmMKK1, positively regulated the salt and drought tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Cai, Guohua; Wang, Guodong; Wang, Li; Liu, Yang; Pan, Jiaowen; Li, Dequan

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules in animals, plants and yeast. MAPK cascades are complicated networks and play vital roles in signal transduction pathways involved in biotic and abiotic stresses. In this study, a maize MAPKK gene, ZmMKK1, was characterized. Quantitative real time PCR (qRT-PCR) analysis demonstrated that ZmMKK1 transcripts were induced by diverse stresses and ABA signal molecule in maize root. Further study showed that the ZmMKK1-overexpressing Arabidopsis enhanced the tolerance to salt and drought stresses. However, seed germination, post-germination growth and stomatal aperture analysis demonstrated that ZmMKK1 overexpression was sensitive to ABA in transgenic Arabidopsis. Molecular genetic analysis revealed that the overexpression of ZmMKK1 in Arabidopsis enhanced the expression of ROS scavenging enzyme- and ABA-related genes, such as POD, CAT, RAB18 and RD29A under salt and drought conditions. In addition, heterologous overexpression of ZmMKK1 in yeast (Saccharomyces cerevisiae) improved the tolerance to salt and drought stresses. These results suggested that ZmMKK1 might act as an ABA- and ROS-dependent protein kinase in positive modulation of salt and drought tolerance. Most importantly, ZmMKK1 interacted with ZmMEKK1 as evidenced by yeast two-hybrid assay, redeeming a deficiency of MAPK interaction partners in maize. PMID:24974327

  3. In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

    Institute of Scientific and Technical Information of China (English)

    Stuart Sullivan; Catriona E. Thomson; Douglas J.Lamont; Matthew A. Jones; John M.Christie

    2008-01-01

    Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2,located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser58, Ser85, Ser350, and Ser410), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

  4. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

    Science.gov (United States)

    Jia, Weiyan; Li, Baohua; Li, Shujia; Liang, Yan; Wu, Xiaowei; Ma, Mei; Wang, Jiyao; Gao, Jin; Cai, Yueyue; Zhang, Yuanya; Wang, Yingchun; Li, Jiayang; Wang, Yonghong

    2016-09-01

    Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development. PMID:27618482

  5. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  6. Cellular pattern formation by SCRAMBLED, a leucine-rich repeat receptor-like kinase in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Schiefelbein, John

    2008-02-01

    The appropriate specification of distinct cell types is important for generating the proper tissues and bodies of multicellular organisms. In the root epidermis of Arabidopsis, cell fate determination is accomplished by a transcriptional regulatory circuit that is influenced by positional signaling. A leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), has been shown to be responsible for the position-dependent aspect of this epidermal pattern. In a recent report, we find that SCM affects the transcriptional regulatory network by down-regulating the WEREWOLF (WER) MYB gene expression in a set of epidermal cells located in a specific position. We also find that SCM and the SCM-related SRF1 and SRF3 are not required for embryonic epidermal patterning and that SRF1 and SRF3 do not act redundantly with SCM. This suggests that distinct positional signaling mechanisms exist for embryonic and post-embryonic epidermal patterning. In this addendum, we discuss the implications of our recent findings and extend our working model for epidermal cell pattering.

  7. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    Science.gov (United States)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  8. INHIBITING MAP KINASE ACTIVITY PREVENTS CALCIUM TRANSIENTS AND MITOSIS ENTRY IN EARLY SEA URCHIN EMBRYOS

    OpenAIRE

    Philipova, Rada; Larman, Mark G.; Leckie, Calum P.; Harrison, Patrick K.; Groigno, Laurence; Whitaker, Michael

    2005-01-01

    A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos both kinases show a similar activation profile, peaking at the time of mitosis entry.

  9. Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

    Directory of Open Access Journals (Sweden)

    Guan Kunliang

    2003-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

  10. The cAMP Signaling and MAP Kinase Pathways in Plant Pathogenic Fungi

    NARCIS (Netherlands)

    Mehrabi, R.; Zhao, X.; Kim, Y.; Xu, J.R.

    2009-01-01

    The key components of the well conserved cyclic AMP signaling and MAP kinase pathways have been functionally characterized in the corn smut Ustilago maydis, rice blast fungus Magnaporthe grisea, and a few other fungal pathogens. In general, the cAMP signaling and the MAP kinase cascade homologous to

  11. Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

    Science.gov (United States)

    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill

    2016-03-25

    TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction.

  12. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    KAUST Repository

    Yunta, Cristina

    2011-11-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  13. Genome wide association mapping for the tolerance to the polyamine oxidase inhibitor guazatine in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kostadin Evgeniev eAtanasov

    2016-04-01

    Full Text Available Guazatine is a potent inhibitor of polyamine oxidase (PAO activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines. Here we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1 within this locus was studied as candidate gene, together with its paralog (CLH2. The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2 and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine.

  14. Genome Wide Association Mapping for the Tolerance to the Polyamine Oxidase Inhibitor Guazatine in Arabidopsis thaliana.

    Science.gov (United States)

    Atanasov, Kostadin E; Barboza-Barquero, Luis; Tiburcio, Antonio F; Alcázar, Rubén

    2016-01-01

    Guazatine is a potent inhibitor of polyamine oxidase (PAO) activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines). Here, we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA) mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1) within this locus was studied as candidate gene, together with its paralog (CLH2). The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2, and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine. PMID:27092150

  15. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

    Science.gov (United States)

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Kumar, Narender; Churchman, Michelle; Larkin, John C; Kwon, Ashley; Lu, Hua

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. PMID:26561564

  16. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    Science.gov (United States)

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  17. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eLeprince

    2015-01-01

    Full Text Available Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signalling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K, VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1, a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.

  18. Integration of linkage maps for the Amphidiploid Brassica napus and comparative mapping with Arabidopsis and Brassica rapa

    Directory of Open Access Journals (Sweden)

    Delourme Régine

    2011-02-01

    Full Text Available Abstract Background The large number of genetic linkage maps representing Brassica chromosomes constitute a potential platform for studying crop traits and genome evolution within Brassicaceae. However, the alignment of existing maps remains a major challenge. The integration of these genetic maps will enhance genetic resolution, and provide a means to navigate between sequence-tagged loci, and with contiguous genome sequences as these become available. Results We report the first genome-wide integration of Brassica maps based on an automated pipeline which involved collation of genome-wide genotype data for sequence-tagged markers scored on three extensively used amphidiploid Brassica napus (2n = 38 populations. Representative markers were selected from consolidated maps for each population, and skeleton bin maps were generated. The skeleton maps for the three populations were then combined to generate an integrated map for each LG, comparing two different approaches, one encapsulated in JoinMap and the other in MergeMap. The BnaWAIT_01_2010a integrated genetic map was generated using JoinMap, and includes 5,162 genetic markers mapped onto 2,196 loci, with a total genetic length of 1,792 cM. The map density of one locus every 0.82 cM, corresponding to 515 Kbp, increases by at least three-fold the locus and marker density within the original maps. Within the B. napus integrated map we identified 103 conserved collinearity blocks relative to Arabidopsis, including five previously unreported blocks. The BnaWAIT_01_2010a map was used to investigate the integrity and conservation of order proposed for genome sequence scaffolds generated from the constituent A genome of Brassica rapa. Conclusions Our results provide a comprehensive genetic integration of the B. napus genome from a range of sources, which we anticipate will provide valuable information for rapeseed and Canola research.

  19. An atypical kinase under balancing selection confers broad-spectrum disease resistance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Carine Huard-Chauveau

    Full Text Available The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1, conferring broad-spectrum resistance to Xanthomonas campestris (Xc, a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops.

  20. Cyclin-dependent kinase-activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana.

    Science.gov (United States)

    Takatsuka, Hirotomo; Umeda-Hara, Chikage; Umeda, Masaaki

    2015-06-01

    For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK-activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate-type CAKs, three CDKDs (CDKD;1-CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post-embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1-1 cdkd;3-1 pollen grains were defective in pollen mitosis I and II, producing one-cell or two-cell pollen grains that lacked fertilization ability. We also found that the double knock-out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post-embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1-1 cdkd;3-1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post-embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.

  1. Diverse Transcriptional Programs Associated with Environmental Stress and Hormones in the Arabidopsis Receptor-Like Kinase Gene Family

    Institute of Scientific and Technical Information of China (English)

    Lee Chae; Sylvia Sudat; Sandrine Dudoit; Tong Zhu; Sheng Luan

    2009-01-01

    The genome of Arabidopsis thaliana encodes more than 600 receptor-like kinase (RLK) genes, by far the dominant class of receptors found in land plants. Although similar to the mammalian receptor tyrosine kinases, plant RLKs are serine/threonine kinases that represent a novel signaling innovation unique to plants and, consequently, an excellent opportunity to understand how extracellular signaling evolved and functions in plants as opposed to animals. RLKs are predicted to be major components of the signaling pathways that allow plants to respond to environmental and developmental conditions. However, breakthroughs in identifying these processes have been limited to only a handful of individual RLKs. Here, we used a Syngenta custom Arabidopsis GeneChip array to compile a detailed profile of the transcriptional activity of 604 receptor-like kinase genes after exposure to a cross-section of known signaling factors in plants,including abiotic stresses, biotic stresses, and hormones. In the 68 experiments comprising the study, we found that 582 of the 604 RLK genes displayed a two-fold or greater change in expression to at least one of 12 types of treatments, thereby providing a large body of experimental evidence for targeted functional screens of individual RLK genes. We investigated whether particular subfamilies of RLK genes are responsive to specific types of signals and found that each subfamily displayed broad ranges of expression, as opposed to being targeted towards particular signal classes. Finally, by analyzing the divergence of sequence and gene expression among the RLK subfamilies, we present evidence as to the functional basis for the expansion of the RLKs and how this expansion may have affected conservation and divergences in their function. Taken as a whole, our study represents a preliminary, working model of processes and interactions in which the members of the RLK gene family may be involved, where such information has remained elusive for so many

  2. Crystal Structure of the MAP3K TAO2 Kinase Domain Bound by an Inhibitor Staurosporine

    Institute of Scientific and Technical Information of China (English)

    Tian-Jun ZHOU; Li-Guang SUN; Yan GAO; Elizabeth J. GOLDSMITH

    2006-01-01

    Mitogen-activated protein kinase (MAPK) signal transduction pathways are ubiquitous in eukaryotic cells, which transfer signals from the cell surface to the nucleus, controlling multiple cellular programs. MAPKs are activated by MAPK kinases [MAP2Ks or MAP/extracellular signal-regulated kinase (ERK) kinases (MEK)], which in turn are activated by MAPK kinase kinases (MAP3Ks). TAO2 is a MAP3K level kinase that activates the MAP2Ks MEK3 and MEK6 to activate p38 MAPKs. Because p38 MAPKs are key regulators of expression of inflammatory cytokines, they appear to be involved in human diseases such as asthma and autoimmunity. As an upstream activator of p38s, TAO2 represents a potential drug target. Here we report the crystal structure of active TAO2 kinase domain in complex with staurosporine, a broadrange protein kinase inhibitor that inhibits TAO2 with an IC50 of 3 μM. The structure reveals that staurosporine occupies the position where the adenosine of ATP binds in TAO2, and the binding of the inhibitor mimics many features of ATP binding. Both polar and nonpolar interactions contribute to the enzyme-inhibitor recognition. Staurosporine induces conformational changes in TAO2 residues that surround the inhibitor molecule, but causes very limited global changes in the kinase. The structure provides atomic details for TAO2-staurosporine interactions, and explains the relatively low potency of staurosporine against TAO2. The structure presented here should aid in the design of inhibitors specific to TAO2 and related kinases.

  3. Finding a missing link in MAP kinase cascade

    OpenAIRE

    Chung, Kwi-Mi; Sano, Hiroshi

    2008-01-01

    Mitogen-activated protein kinase (MAPK) cascade is one of the major signaling systems in eukaryotes. External signals are tranduced through three protein kinases, which successively relay phosphorylation to finally activate target genes/proteins. However, few information on targets of MAPK have so far been available. In this study, we identified a novel transcription factor, NtWIF, which is directly phosphorylated by a wound-induced protein kinase (WIPK), a typical MAPK from tobacco plants. P...

  4. Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform1[w

    Science.gov (United States)

    Turner, William L.; Waller, Jeffrey C.; Vanderbeld, Barb; Snedden, Wayne A.

    2004-01-01

    NAD kinase (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca2+-dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (KmNAD = 0.20 mM, KmMg2+−ATP = 0.17 mM). The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nm) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations. PMID:15247403

  5. The S-Domain Receptor Kinase Arabidopsis Receptor Kinase2 and the U Box/Armadillo Repeat-Containing E3 Ubiquitin Ligase9 Module Mediates Lateral Root Development under Phosphate Starvation in Arabidopsis.

    Science.gov (United States)

    Deb, Srijani; Sankaranarayanan, Subramanian; Wewala, Gayathri; Widdup, Ellen; Samuel, Marcus A

    2014-06-25

    When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots (LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients, reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate for synthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. In our screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, the Arabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this module in regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plants lacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grown under phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin in their primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin was sufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for this process. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interaction between ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plants resulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role for autophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions

  6. Arabidopsis lectin receptor kinases LecRK-IX.1 and LecRK-IX.2 are functional analogs in regulating phytophthora resistance and plant cell death

    NARCIS (Netherlands)

    Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

    2015-01-01

    L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showi

  7. Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis.

    Directory of Open Access Journals (Sweden)

    Prasad Vaddepalli

    Full Text Available Tissue morphogenesis in plants requires the coordination of cellular behavior across clonally distinct histogenic layers. The underlying signaling mechanisms are presently being unraveled and are known to include the cell surface leucine-rich repeat receptor-like kinase STRUBBELIG in Arabidopsis. To understand better its mode of action an extensive structure-function analysis of STRUBBELIG was performed. The phenotypes of 20 EMS and T-DNA-induced strubbelig alleles were assessed and homology modeling was applied to rationalize their possible effects on STRUBBELIG protein structure. The analysis was complemented by phenotypic, cell biological, and pharmacological investigations of a strubbelig null allele carrying genomic rescue constructs encoding fusions between various mutated STRUBBELIG proteins and GFP. The results indicate that STRUBBELIG accepts quite some sequence variation, reveal the biological importance for the STRUBBELIG N-capping domain, and reinforce the notion that kinase activity is not essential for its function in vivo. Furthermore, individual protein domains of STRUBBELIG cannot be related to specific STRUBBELIG-dependent biological processes suggesting that process specificity is mediated by factors acting together with or downstream of STRUBBELIG. In addition, the evidence indicates that biogenesis of a functional STRUBBELIG receptor is subject to endoplasmic reticulum-mediated quality control, and that an MG132-sensitive process regulates its stability. Finally, STRUBBELIG and the receptor-like kinase gene ERECTA interact synergistically in the control of internode length. The data provide genetic and molecular insight into how STRUBBELIG regulates intercellular communication in tissue morphogenesis.

  8. Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo

    KAUST Repository

    Fujii, Hiroaki

    2011-01-10

    Osmotic stress associated with drought or salinity is a major factor that limits plant productivity. Protein kinases in the SNF1-related protein kinase 2 (SnRK2) family are activated by osmotic stress, suggesting that the kinases are involved in osmotic stress signaling. However, due to functional redundancy, their contribution to osmotic stress responses remained unclear. In this report, we constructed an Arabidopsis line carrying mutations in all 10 members of the SnRK2 family. The decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10 grew poorly under hyperosmotic stress conditions but was similar to the wild type in culture media in the absence of osmotic stress. The mutant was also defective in gene regulation and the accumulation of abscisic acid (ABA), proline, and inositol 1,4,5-trisphosphate under osmotic stress. In addition, analysis of mutants defective in the ABA-activated SnRK2s (snrk2.2/3/6) and mutants defective in the rest of the SnRK2s (snrk2.1/4/5/7/8/9/10) revealed that SnRK2s are a merging point of ABA-dependent and -independent pathways for osmotic stress responses. These results demonstrate critical functions of the SnRK2s in mediating osmotic stress signaling and tolerance.

  9. Roles of MAP kinase signaling pathway in oocyte meiosis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.

  10. Identification of prior candidate genes for Sclerotinia local resistance in Brassica napus using Arabidopsis cDNA microarray and Brassica-Arabidopsis comparative mapping

    Institute of Scientific and Technical Information of China (English)

    LIU; Renhu; ZHAO; Jianwei; XIAO; Yong; MENG; Jinling

    2005-01-01

    Arabidopsis cDNA arrays were used to screen the local-defense-associated genes in oilseed rape (Brassica napus L.) at the challenge of Sclerotinia sclerotiorum. 61 genes with two-fold expression changes were screened out from the local tissue around the necrosis. Among them, 36 unique genes were up-regulated and 25 unique genes were down-regulated. RT-PCR and Northern blot results were consistent with the array results, suggesting Arabidopsis arrays were useful for transcriptional profiling of B. napus genes. Some of these genes were located in the interval of some QTLs for Sclerotinia resistance in B. napus by Brassica- Arabidopsis comparative mapping. These genes may have priority to be pursued for more intensive research.

  11. Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Arondel, V.; Lemieux, B.; Hwang, I. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1992-11-20

    A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1 tabs.

  12. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  13. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis.

    Science.gov (United States)

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-10-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction.

  14. Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development.

    Science.gov (United States)

    Lin, Lin; Zhong, Si-Hui; Cui, Xiao-Feng; Li, Jianming; He, Zu-Hua

    2012-12-01

    The balance between cell proliferation and cell differentiation is essential for leaf patterning. However, identification of the factors coordinating leaf patterning and cell growth behavior is challenging. Here, we characterized a temperature-sensitive Arabidopsis mutant with leaf blade and venation defects. We mapped the mutation to the sub-2 allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) receptor-like kinase gene whose functions in leaf development have not been demonstrated. The sub-2 mutant displayed impaired blade development, asymmetric leaf shape and altered venation patterning under high ambient temperature (30°C), but these defects were less pronounced at normal growth temperature (22°C). Loss of SCM/SUB function results in reduced cell proliferation and abnormal cell expansion, as well as altered auxin patterning. SCM/SUB is initially expressed throughout leaf primordia and becomes restricted to the vascular cells, coinciding with its roles in early leaf patterning and venation formation. Furthermore, constitutive expression of the SCM/SUB gene also restricts organ growth by inhibiting the transition from cell proliferation to expansion. We propose the existence of a SCM/SUB-mediated developmental stage-specific signal for leaf patterning, and highlight the importance of the balance between cell proliferation and differentiation for leaf morphogenesis.

  15. The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Nabila eDjafi

    2013-08-01

    Full Text Available Phosphoinositide-dependent phospholipases C (PI-PLCs are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII to produce inositol triphosphate and diacylglycerol (DAG that is phosphorylated into phosphatidic acid (PA by DAG-kinases (DGKs. The roles of PI4KIIIs, PI-PLCs and DGKs in basal signalling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 µM wortmannin or R59022, inhibitors of PI-PLCs, PI4KIIIs and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements, that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs. We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

  16. A protein–protein interaction network linking the energy-sensor kinase SnRK1 to multiple signaling pathways in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Madlen Nietzsche

    2016-04-01

    Full Text Available In plants, the sucrose non-fermenting (SNF1-related protein kinase 1 (SnRK1 represents a central integrator of low energy signaling and acclimation towards many environmental stress responses. Although SnRK1 acts as a convergent point for many different environmental and metabolic signals to control growth and development, it is currently unknown how these many different signals could be translated into a cell-type or stimulus specific response since many components of SnRK1-regulated signaling pathways remain unidentified. Recently, we have demonstrated that proteins containing a domain of unknown function (DUF 581 interact with the catalytic α subunits of SnRK1 (AKIN10/11 from Arabidopsis thaliana and could potentially act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation. To further extend the SnRK1 signaling network in plants, we systematically screened for novel DUF581 interaction partners using the yeast two-hybrid system. A deep and exhaustive screening identified 17 interacting partners for 10 of the DUF581 proteins tested. Many of these novel interaction partners are implicated in cellular processes previously associated with SnRK1 signaling. Furthermore, we mined publicly available interaction data to identify additional DUF581 interacting proteins. A protein–protein interaction network resulting from our studies suggests connections between SnRK1 signaling and other central signaling pathways involved in growth regulation and environmental responses. These include TOR and MAP-kinase signaling as well as hormonal pathways. The resulting protein–protein interaction network promises to be effective in generating hypotheses to study the precise mechanisms SnRK1 signaling on a functional level.

  17. The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

    International Nuclear Information System (INIS)

    The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

  18. Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Calcium and protein kinase serve as the common mediators to regulate plant responses to multiple stresses including salt and ABA stimulus. Here we reported a novel protein kinase (CIPK14) that regulated the responses to ABA treatment and salt stress in Arabidopsis. CIPK14 transcripts, capable been checked in roots, stems, leaves and flowers, were highly expressed in flowers and roots. CIPK14 was induced by ABA and salt treatments. The disruption of CIPK14 altered the transcriptional pattern of a gene marker line related to ABA and salt responses, and the results suggested that CIPK14 probably was responsible to the control of the salt and ABA responses. Comparing with wild types, the lines inserted with the T-DNA in which CIPK14 gene expression was knocked out were also more sensitive to ABA and salt stimulus, showing low germination rate and the less root elongation. While, when these conditioned seeds were treated with norflurazon, their germination percentages could recover to a certain extent. We also found that exogenous calcium could have an effect on the transcription of CIPK14 under ABA and salt treatments, and it seemed that calcium ion might work upstream CIPK14 to regulate the plant response to ABA and salt response.

  19. Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

    Science.gov (United States)

    Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

    2015-12-01

    Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development.

  20. QTL mapping in new Arabidopsis thaliana advanced intercross-recombinant inbred lines.

    Directory of Open Access Journals (Sweden)

    Sureshkumar Balasubramanian

    Full Text Available BACKGROUND: Even when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs. METHODOLOGY/PRINCIPAL FINDINGS: We present two new AI-RIL populations of Arabidopsis thaliana genotyped with an average intermarker distance of 600 kb. The advanced intercrossing design led to expansion of the genetic map in the two populations, which contain recombination events corresponding to 50 kb/cM in an F(2 population. We used the AI-RILs to map QTL for light response and flowering time, and to identify segregation distortion in one of the AI-RIL populations due to a negative epistatic interaction between two genomic regions. CONCLUSIONS/SIGNIFICANCE: The two new AI-RIL populations, EstC and KendC, derived from crosses of Columbia (Col to Estland (Est-1 and Kendallville (Kend-L provide an excellent resource for high precision QTL mapping. Moreover, because they have been genotyped with over 100 common markers, they are also excellent material for comparative QTL mapping.

  1. Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes.

    Science.gov (United States)

    Shukla, Shivendra D; Lee, Youn Ju; Park, Pil-hoon; Aroor, Annayya R

    2007-01-01

    Although both oxidative and non-oxidative metabolites of ethanol are involved in generating ethanol matabolic stress (Emess), the oxidative metabolite acetaldehyde plays a critical role in the cellular actions of ethanol. We have investigated the effects of acetaldehyde on p42/44 MAP kinase, p46/p54 c-jun N-terminal kinase (JNK1/JNK2) and p38 MAP kinase in hepatocytes. Acetaldehyde caused temporal activation of p42/44 MAPK followed by JNK, but the activation of the p42/44 MAPK was not a prerequisite for the JNK activation. Activation ofJNK1 by acetaldehyde was greater than JNK2. Ethanol and acetaldehyde activatedJNK have opposing roles; ethanol-induced JNK activation increased apoptosis whereas that by acetaldehyde decreased apoptosis. Acetaldehyde also caused histone H3 acetylation at Lys9 and phosphorylation of histone H3 at Serl0 and 28, the latter being dependent on p38 MAP kinase. Phosphorylation at Ser28 was higher than at Serl0. Thus acetaldehyde distinctively alters MAP kinase signalling and histone modifications, processes involved in transcriptional activation. PMID:17590997

  2. Brf1 posttranscriptionally regulates pluripotency and differentiation responses downstream of Erk MAP kinase

    OpenAIRE

    Tan, Frederick E.; Elowitz, Michael B.

    2014-01-01

    AU-rich element mRNA-binding proteins (AUBPs) are key regulators of development, but how they are controlled and what functional roles they play depends on cellular context. Here, we show that Brf1 (zfp36l1), an AUBP from the Zfp36 protein family, operates downstream of FGF/Erk MAP kinase signaling to regulate pluripotency and cell fate decision making in mouse embryonic stem cells (mESCs). FGF/Erk MAP kinase signaling up-regulates Brf1, which disrupts the expression of core pluripotency-asso...

  3. Specificity of MAP Kinase Signaling in Yeast Differentiation Involves Transient versus Sustained MAPK Activation

    OpenAIRE

    Sabbagh, Walid; Flatauer, Laura J.; Bardwell, A. Jane; Bardwell, Lee

    2001-01-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options—mating and invasive growth—are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosp...

  4. Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yuan Tong

    2010-01-01

    Full Text Available Abstract Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs, representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

  5. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    OpenAIRE

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  6. MAP Kinase 4 Substrates and Plant Innate Immunity

    DEFF Research Database (Denmark)

    Rasmussen, Magnus Wohlfahrt

    . For example, Arabidopsis MPK4 regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with (i) PAT1, component of the mRNA decapping machinery, (ii) AOC3, a component in the biosynthesis pathway of JA and (iii) e......IF4E, a component in the translational initiation protein complex. For PAT1 and eIF4E we show that MPK4 phosphorylates specific Ser and Thr residues in vitro, and that MPK4 also phosphorylates AOC3 at an unmapped residue. Specific in vivo phosphorylation for PAT1 is shown in response to pathogen...... recognition, which also induce its localization to cytoplasmic processing bodies. All three proteins; PAT1, AOC3 and eIF4E also interacts with MPK4 in vivo although the functional outcome of these interactions are still elusive. The thesis comprise a general introduction to plant innate immunity followed...

  7. MAP kinase phosphatase 2 regulates macrophage-adipocyte interaction.

    Directory of Open Access Journals (Sweden)

    Huipeng Jiao

    Full Text Available Inflammation is critical for the development of obesity-associated metabolic disorders. This study aims to investigate the role of mitogen-activated protein kinase phosphatase 2 (MKP-2 in inflammation during macrophage-adipocyte interaction.White adipose tissues (WAT from mice either on a high-fat diet (HFD or normal chow (NC were isolated to examine the expression of MKP-2. Murine macrophage cell line RAW264.7 stably expressing MKP-2 was used to study the regulation of MKP-2 in macrophages in response to saturated free fatty acid (FFA and its role in macrophage M1/M2 activation. Macrophage-adipocyte co-culture system was employed to investigate the role of MKP-2 in regulating inflammation during adipocyte-macrophage interaction. c-Jun N-terminal kinase (JNK- and p38-specific inhibitors were used to examine the mechanisms by which MKP-2 regulates macrophage activation and macrophage-adipocytes interaction.HFD changed the expression of MKP-2 in WAT, and MKP-2 was highly expressed in the stromal vascular cells (SVCs. MKP-2 inhibited the production of proinflammatory cytokines in response to FFA stimulation in macrophages. MKP-2 inhibited macrophage M1 activation through JNK and p38. In addition, overexpression of MKP-2 in macrophages suppressed inflammation during macrophage-adipocyte interaction.MKP-2 is a negative regulator of macrophage M1 activation through JNK and p38 and inhibits inflammation during macrophage-adipocyte interaction.

  8. TaSK5, an abiotic stress-inducible GSK3/shaggy-like kinase from wheat, confers salt and drought tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Christov, Nikolai Kirilov; Christova, Petya Koeva; Kato, Hideki; Liu, Yuelin; Sasaki, Kentaro; Imai, Ryozo

    2014-11-01

    A novel cold-inducible GSK3/shaggy-like kinase, TaSK5, was isolated from winter wheat using a macroarray-based differential screening approach. TaSK5 showed high similarity to Arabidopsis subgroup I GSK3/shaggy-like kinases ASK-alpha, AtSK-gamma and ASK-epsilon. RNA gel blot analyses revealed TaSK5 induction by cold and NaCl treatments and to a lesser extent by drought treatment. TaSK5 functionally complemented the cold- and salt-sensitive phenotypes of a yeast GSK3/shaggy-like kinase mutant, △mck1. Transgenic Arabidopsis plants overexpressing TaSK5 cDNA showed enhanced tolerance to salt and drought stresses. By contrast, the tolerance of the transgenic plants to freezing stress was not altered. Microarray analysis revealed that a number of abiotic stress-inducible genes were constitutively induced in the transgenic Arabidopsis plants, suggesting that TaSK5 may function in a novel signal transduction pathway that appears to be unrelated to DREB1/CBF regulon and may involve crosstalk between abiotic and hormonal signals.

  9. Natural variation at Strubbelig Receptor Kinase 3 drives immune-triggered incompatibilities between Arabidopsis thaliana accessions

    NARCIS (Netherlands)

    Alcazar, R.; Garcia, A.V.; Kronholm, I.; Meaux, De J.; Koornneef, M.; Parker, J.E.; Reymond, M.

    2010-01-01

    Accumulation of genetic incompatibilities within species can lead to reproductive isolation and, potentially, speciation. In this study, we show that allelic variation at SRF3 (Strubbelig Receptor Family 3), encoding a receptor-like kinase, conditions the occurrence of incompatibility between Arabid

  10. MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.

    Science.gov (United States)

    Madhani, H D; Styles, C A; Fink, G R

    1997-11-28

    Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK. PMID:9393860

  11. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  12. Feedback phosphorylation of an RGS protein by MAP kinase in yeast.

    Science.gov (United States)

    Garrison, T R; Zhang, Y; Pausch, M; Apanovitch, D; Aebersold, R; Dohlman, H G

    1999-12-17

    Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling. PMID:10593933

  13. Using µPIXE for quantitative mapping of metal concentration in Arabidopsis thaliana seeds

    Directory of Open Access Journals (Sweden)

    Magali eSchnell Ramos

    2013-06-01

    Full Text Available Seeds are a crucial stage in plant life. They contain the nutrients necessary to initiate the development of a new organism. Seeds also represent an important source of nutrient for human beings. Iron (Fe and zinc (Zn deficiencies affect over a billion people worldwide. It is therefore important to understand how these essential metals are stored in seeds. In this work, Particle-Induced X–ray Emission with the use of a focused ion beam (µPIXE has been used to map and quantify essential metals in Arabidopsis seeds. In agreement with Synchrotron radiation X-Ray Fluorescence (SXRF imaging and Perls/DAB staining, µPIXE maps confirmed the specific pattern of Fe and Mn localization in the endodermal and subepidermal cell layers in dry seeds, respectively. Moreover, µPIXE allows absolute quantification revealing that the Fe concentration in the endodermal cell layer reaches ~800 µg•g-1 dry weight. Nevertheless, this cell layer accounts only for about half of Fe stores in dry seeds. Comparison between Arabidopsis wild type and mutant seeds impaired in Fe vacuolar storage (vit1-1 or release (nramp3nramp4 confirmed the strongly altered Fe localization pattern in vit1-1, whereas no alteration could be detected in nramp3nramp4 dry seeds. Imaging of imbibed seeds indicates a dynamic localization of metals as Fe and Zn concentrations increase in the subepidermal cell layer of cotyledons after imbibition. The complementarities between µPIXE and other approaches as well as the importance of being able to quantify the patterns for the interpretation of mutant phenotypes are discussed.

  14. Genome-Wide Association Mapping in Arabidopsis Identifies Previously Known Flowering Time and Pathogen Resistance Genes.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There is currently tremendous interest in the possibility of using genome-wide association mapping to identify genes responsible for natural variation, particularly for human disease susceptibility. The model plant Arabidopsis thaliana is in many ways an ideal candidate for such studies, because it is a highly selfing hermaphrodite. As a result, the species largely exists as a collection of naturally occurring inbred lines, or accessions, which can be genotyped once and phenotyped repeatedly. Furthermore, linkage disequilibrium in such a species will be much more extensive than in a comparable outcrossing species. We tested the feasibility of genome-wide association mapping in A. thaliana by searching for associations with flowering time and pathogen resistance in a sample of 95 accessions for which genome-wide polymorphism data were available. In spite of an extremely high rate of false positives due to population structure, we were able to identify known major genes for all phenotypes tested, thus demonstrating the potential of genome-wide association mapping in A. thaliana and other species with similar patterns of variation. The rate of false positives differed strongly between traits, with more clinal traits showing the highest rate. However, the false positive rates were always substantial regardless of the trait, highlighting the necessity of an appropriate genomic control in association studies.

  15. Genome-wide association mapping in Arabidopsis identifies previously known flowering time and pathogen resistance genes.

    Directory of Open Access Journals (Sweden)

    María José Aranzana

    2005-11-01

    Full Text Available There is currently tremendous interest in the possibility of using genome-wide association mapping to identify genes responsible for natural variation, particularly for human disease susceptibility. The model plant Arabidopsis thaliana is in many ways an ideal candidate for such studies, because it is a highly selfing hermaphrodite. As a result, the species largely exists as a collection of naturally occurring inbred lines, or accessions, which can be genotyped once and phenotyped repeatedly. Furthermore, linkage disequilibrium in such a species will be much more extensive than in a comparable outcrossing species. We tested the feasibility of genome-wide association mapping in A. thaliana by searching for associations with flowering time and pathogen resistance in a sample of 95 accessions for which genome-wide polymorphism data were available. In spite of an extremely high rate of false positives due to population structure, we were able to identify known major genes for all phenotypes tested, thus demonstrating the potential of genome-wide association mapping in A. thaliana and other species with similar patterns of variation. The rate of false positives differed strongly between traits, with more clinal traits showing the highest rate. However, the false positive rates were always substantial regardless of the trait, highlighting the necessity of an appropriate genomic control in association studies.

  16. Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    SUNYUE; YingLiGuo; 等

    2000-01-01

    Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.

  17. AthaMap-assisted transcription factor target gene identification in Arabidopsis thaliana.

    Science.gov (United States)

    Bülow, Lorenz; Brill, Yuri; Hehl, Reinhard

    2010-01-01

    The AthaMap database generates a map of potential transcription factor binding sites (TFBS) and small RNA target sites in the Arabidopsis thaliana genome. The database contains sites for 115 different transcription factors (TFs). TFBS were identified with positional weight matrices (PWMs) or with single binding sites. With the new web tool 'Gene Identification', it is possible to identify potential target genes for selected TFs. For these analyses, the user can define a region of interest of up to 6000 bp in all annotated genes. For TFBS determined with PWMs, the search can be restricted to high-quality TFBS. The results are displayed in tables that identify the gene, position of the TFBS and, if applicable, individual score of the TFBS. In addition, data files can be downloaded that harbour positional information of TFBS of all TFs in a region between -2000 and +2000 bp relative to the transcription or translation start site. Also, data content of AthaMap was increased and the database was updated to the TAIR8 genome release. Database URL: http://www.athamap.de/gene_ident.php. PMID:21177332

  18. Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

    Directory of Open Access Journals (Sweden)

    Debora Bogani

    2009-09-01

    Full Text Available Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY gonad, sex-determining region of the Y (SRY protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4, a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas. These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and

  19. Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    QIN YuZhi; LI Xu; GUO Ming; DENG KeQin; LIN dianZhong; TANG DongYing; GUO XinHong; LIU XuanMing

    2008-01-01

    Calcium and protein kinsse serve as the common mediators to regulate plant responses to multiple stresses including salt and ABA stimulus. Here we reported a novel protein kinase (CIPK14) that regulated the responses to ABA treatment and salt stress in Arabidopsis. CIPK14 transcripts, capable been checked in roots, stems, leaves and flowers, were highly expressed in flowers and roots. CIPK14 was induced by ABA and salt treatments. The disruption of CIPK14 altered the transcriptional pattern of a gene marker line related to ABA and salt responses, and the results suggested that CIPK14 probably was responsible to the control of the salt and ABA responses. Comparing with wild types, the lines inserted with the T-DNA in which CIPK14 gene expression was knocked out were also more sensitive to ABA and salt stimulus, showing low germination rate and the less root elongation. While, when these conditioned seeds were treated with norflurazon, their germination percentages could recover to a certain extent. We also found that exogenous calcium could have an effect on the transcription of CIPK14 under ABA end salt treatments, and it seemed that calcium ion might work upstream ClPK14 to regulate the plant response to ABA and salt response.

  20. Arabidopsis inositol 1,3,4-trisphosphate 5/6 kinase 2 is required for seed coat development

    Institute of Scientific and Technical Information of China (English)

    Yong Tang; Shutang Tan; Hongwei Xue

    2013-01-01

    Inositol 1,3,4-trisphosphate 5/6 kinase (ITPK) phosphorylates inositol 1,3,4-trisphosphate to form inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate which can be finally transferred to inositoi hexaphosphate (IP6) and play important roles during plant growth and development.There are 4 putative ITPK members in Arabidopsis.Expression pattern analysis showed that ITPK2 is constitutively expressed in various tissues.A TDNA knockout mutant of ITPK2 was identified and scanning electron microscopy (SEM) analysis showed that the epidermis structure of seed coat was irregularly formed in seeds of itpk2-1 mutant,resulting in the increased permeability of seed coat to tetrazolium salts.Further analysis by gas chromatography coupled with mass spectrometry of lipid polyester monomers in cell wall confirmed a dramatic decrease in composition of suberin and cutin,which relate to the permeability of seed coat and the formation of which is accompanied with seed coat development.These results indicate that ITPK2 plays an essential role in seed coat development and lipid polyester barrier formation.

  1. Plant protein kinase genes induced by drought, high salt and cold stresses

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

  2. A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway.

    Science.gov (United States)

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J

    2006-10-01

    Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.

  3. How can yeast cells decide between three activated MAP kinase pathways? A model approach.

    Science.gov (United States)

    Rensing, Ludger; Ruoff, Peter

    2009-04-21

    In yeast (Saccharomyces cerevisiae), the regulation of three MAP kinase pathways responding to pheromones (Fus3 pathway), carbon/nitrogen starvation (Kss1 pathway), and high osmolarity/osmotic stress (Hog1 pathway) is the subject of intensive research. We were interested in the question how yeast cells would respond when more than one of the MAP kinase pathways are activated simultaneously. Here, we give a brief overview over the regulatory mechanisms of the yeast MAP kinase pathways and investigate a kinetic model based on presently known molecular interactions and feedbacks within and between the three mitogen-activated protein kinases (MAPK) pathways. When two pathways are activated simultaneously with the osmotic stress response as one of them, the model predicts that the osmotic stress response (Hog1 pathway) is turned on first. The same is true when all three pathways are activated at the same time. When testing simultaneous stimulations by low nitrogen and pheromones through the Kss1 and Fus3 pathways, respectively, the low nitrogen response dominates over the pheromone response. Due to its autocatalytic activation mechanism, the pheromone response (Fus3 pathway) shows typical sigmoid response kinetics and excitability. In the presence of a small but sufficient amount of activated Fus3, a stimulation by pheromones will lead to a rapid self-amplification of the pheromone response. This 'excitability' appears to be a feature of the pheromone pathway that has specific biological significance. PMID:19322936

  4. The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection

    KAUST Repository

    Ederli, Luisa

    2011-10-01

    In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O3). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O3 and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O3 sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H2O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen. © 2011 Elsevier GmbH.

  5. RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

    Science.gov (United States)

    Zeng, Lingchun; Ehrenreiter, Karin; Menon, Jyotsana; Menard, Ray; Kern, Florian; Nakazawa, Yoko; Bevilacqua, Elena; Imamoto, Akira; Baccarini, Manuela; Rosner, Marsha Rich

    2013-05-01

    MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

  6. NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ.

    Science.gov (United States)

    Aoto, Phillip C; Martin, Bryan T; Wright, Peter E

    2016-01-01

    The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways. PMID:27353957

  7. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Wilfried Jonkers

    2014-11-01

    Full Text Available Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this

  8. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

    Science.gov (United States)

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H; Hickson, Gilles R; Archambault, Vincent

    2014-10-27

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

  9. Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation.

    Science.gov (United States)

    Sabbagh, W; Flatauer, L J; Bardwell, A J; Bardwell, L

    2001-09-01

    Signals transmitted by common components often elicit distinct (yet appropriate) outcomes. In yeast, two developmental options-mating and invasive growth-are both regulated by the same MAP kinase cascade. Specificity has been thought to result from specialized roles for the two MAP kinases, Kss1 and Fus3, and because Fus3 prevents Kss1 from gaining access to the mating pathway. Kss1 has been thought to participate in mating only when Fus3 is absent. Instead, we show that Kss1 is rapidly phosphorylated and potently activated by mating pheromone in wild-type cells, and that this is required for normal pheromone-induced gene expression. Signal identity is apparently maintained because active Fus3 limits the extent of Kss1 activation, thereby preventing inappropriate signal crossover. PMID:11583629

  10. The Potential for Signal Integration and Processing in Interacting Map Kinase Cascades

    OpenAIRE

    John H Schwacke; Voit, Eberhard O.

    2007-01-01

    The cellular response to environmental stimuli requires biochemical information processing through which sensory inputs and cellular status are integrated and translated into appropriate responses by way of interacting networks of enzymes. One such network, the Mitogen Activated Protein (MAP) kinase cascade is a highly conserved signal transduction module that propagates signals from cell surface receptors to various cytosolic and nuclear targets by way of a phosphorylation cascade. We have i...

  11. A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

    Science.gov (United States)

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

    2006-01-01

    Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

  12. Rapid Phosphatidic Acid Accumulation in Response to Low Temperature Stress in Arabidopsis is Generated through Diacylglycerol Kinase

    Directory of Open Access Journals (Sweden)

    Steven A. Arisz

    2013-01-01

    Full Text Available Phosphatidic acid (PtdOH is emerging as an important signalling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labelled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e. i via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH, ii via phospholipase D hydrolysis of structural phospholipids or iii via phosphorylation of diacylglycerol (DAG by DAG kinase (DGK. Using a differential 32P-labelling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1 and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

  13. Exploitation of latent protein allostery enables the evolution of novel and divergent MAP kinase regulation

    Science.gov (United States)

    Coyle, Scott M.; Flores, Jonathan; Lim, Wendell A.

    2013-01-01

    SUMMARY Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets co-evolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and the evolution of distinct signaling behaviors. PMID:23953117

  14. Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

    Science.gov (United States)

    Coyle, Scott M; Flores, Jonathan; Lim, Wendell A

    2013-08-15

    Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and to the evolution of distinct signaling behaviors. PMID:23953117

  15. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

    OpenAIRE

    Mei, Yu; Jia, Wen-Jing; Chu, Yu-Jia; Xue, Hong-Wei

    2011-01-01

    Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P2) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k...

  16. Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells.

    Science.gov (United States)

    Vaultier, Marie-Noëlle; Cantrel, Catherine; Vergnolle, Chantal; Justin, Anne-Marie; Demandre, Chantal; Benhassaine-Kesri, Ghouziel; Ciçek, Dominique; Zachowski, Alain; Ruelland, Eric

    2006-07-24

    Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced-membrane rigidification is upstream of DAGK pathway activation. PMID:16839551

  17. Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity

    Institute of Scientific and Technical Information of China (English)

    Hanbing Zhong; Danyang Wang; Nan Wang; Yesenia Rios; Haigen Huang; Song Li; Xinrong Wu; Shuo Lin

    2011-01-01

    Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)zn1 transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.

  18. A Dominant Allele of Arabidopsis Pectin-Binding Wall-Associated Kinase Induces a Stress Response Suppressed by MPK6 but Not MPK3 Mutations

    Institute of Scientific and Technical Information of China (English)

    Bruce D.Kohorn; Susan L.Kohorn; Tanya Todorova; Gillian Baptiste; Kevin Stansky; Meghan McCullough

    2012-01-01

    The plant cell wall is composed of a matrix of cellulose fibers,flexible pectin polymers,and an array of assorted carbohydrates and proteins.The receptor-like Wall-Associated Kinases(WAKs)of Arabidopsis bind pectin in the wall,and are necessary both for cell expansion during development and for a response to pathogens and wounding.Mitogen Activated Protein Kinases(MPKs)form a major signaling link between cell surface receptors and both transcriptional and enzyme regulation in eukaryotes,and Arabidopsis MPK6 and MPK3 indeed have important roles in development and the response to stress and pathogens.A dominant allele of WAK2 requires kinase activity and activates a stress response that includes an increased ROS accumulation and the up-regulation of numerous genes involved in pathogen resistance,wounding,and cell wall biogenesis.This dominant allele requires a functional pectin binding and kinase domain,indicating that it is engaged in a WAK signaling pathway.A null mutant of the major plasma membrane ROS-producing enzyme complex,rbohd/f does not suppress the WAK2cTAP-induced phenotype.A mpk6,but not a mpk3,null allele is able to suppress the effects of this dominant WAK2 mutation,thus distinguishing MPK3 and MPK6,whose activity previously was thought to be redundant.Pectin activation of gene expression is abated in a wak2-null,but is tempered by the WAK-dominant allele that induces elevated basal stress-related transcript levels.The results suggest a mechanism in which changes to the cell wall can lead to a large change in cellular responses and help to explain how pathogens and wounding can have general effects on growth.

  19. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.

    Science.gov (United States)

    Slaughter, Brian D; Schwartz, Joel W; Li, Rong

    2007-12-18

    Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation. PMID:18077328

  20. Involvement of the mitogen-activated protein (MAP kinase signalling pathway in host cell invasion by Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Robert-Gangneux F.

    2000-06-01

    Full Text Available Little is known about signalling in Toxoplasma gondii, but it is likely that protein kinases might play a key role in the parasite proliferation, differentiation and probably invasion. We previously characterized Mitogen-Activated Protein (MAP kinases in T. gondii lysates. In this study, cultured cells were tested for their susceptibility to Toxoplasma gondii infection after tachyzoite pretreatment with drugs interfering with AMP kinase activation pathways. Protein kinases inhibitors, i.e. genistein, R031-8220 and PD098059, reduced tachyzoite infectivity by 38 ± 4.5 %, 85.5 ± 9 % and 56 ± 10 %, respectively. Conversely, protein kinases activators, i.e. bombesin and PMA, markedly increased infectivity (by 202 ± 37 % and 258 ± 14 %, respectively. These results suggest that signalling pathways involving PKC and AAAP kinases play a role in host cell invasion by Toxoplasma.

  1. MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase MPK3 in both rice and Arabidopsis.

    Science.gov (United States)

    Raghuram, Badmi; Sheikh, Arsheed H; Rustagi, Yashika; Sinha, Alok K

    2015-02-01

    MicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein-protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.

  2. Gene expression related to synaptogenesis, neuritogenesis, and MAP kinase in behavioral sensitization to psychostimulants.

    Science.gov (United States)

    Ujike, Hiroshi; Takaki, Manabu; Kodama, Masafumi; Kuroda, Shigetoshi

    2002-06-01

    The most important characteristic of behavioral sensitization to psychostimulants, such as amphetamine and cocaine, is the very long-lasting hypersensitivity to the drug after cessation of exposure. Rearrangement and structural modification of neural networks in CNS must be involved in behavioral sensitization. Previous microscopic studies have shown that the length of dendrites and density of dendritic spines increased in the nucleus accumbens and frontal cortex after repeated exposure to amphetamine and cocaine, but the molecular mechanisms responsible are not well understood. We investigated a set of genes related to synaptogenesis, neuritogenesis, and mitogen-activated protein (MAP) kinase after exposure to methamphetamine. Synaptophysin mRNA, but not VAMP2 (synaptobrevin 2) mRNA, which are considered as synaptogenesis markers, increased in the accumbens, striatum, hippocampus, and several cortices, including the medial frontal cortex, after a single dose of 4 mg/kg methamphetamine. Stathmin mRNA, but not neuritin or narp mRNA, which are markers for neuritic sprouting, increased in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNA of arc, an activity-regulated protein associated with cytoskeleton, but not of alpha-tubulin, as markers for neuritic elongation, showed robust increases in the striatum, hippocampus, and cortices after a single dose of methamphetamine. The mRNAs of MAP kinase phosphatase-1 (MKP-1), MKP-3, and rheb, a ras homologue abundant in brain, were investigated to assess the MAP kinase cascades. MKP-1 and MKP-3 mRNAs, but not rheb mRNA, increased in the striatum, thalamus, and cortices, and in the striatum, hippocampus, and cortices, respectively, after a single methamphetamine. Synaptophysin and stathmin mRNAs did not increase again after chronic methamphetamine administration, whereas the increases in arc, MKP-1, and MKP-3 mRNAs persisted in the brain regions after chronic methamphetamine administration

  3. Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways.

    Science.gov (United States)

    Qiao, Xiaoyong; Yong Qiao, Xiao; Nie, Ying; Ma, Yaxian; Xian Ma, Ya; Chen, Yan; Cheng, Ran; Yin, Weiyao; Yao Yinrg, Wei; Hu, Ying; Xu, Wenming; Ming Xu, Wen; Xu, Liangzhi; Zhi Xu, Liang

    2016-01-01

    Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued. PMID:26738434

  4. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    Science.gov (United States)

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  5. P34^ Kinase and MAP Kinase Activities and Parthenogenetic Activation in Porcine Oocytes after Injection of Miniature Pig Sperm Extracts

    OpenAIRE

    Matsuura, Daizou; Maeda, Teruo

    2008-01-01

    The aim of the present study was to examine the rate of activation and time-dependent changes in p34cdc2 kinase and MAP kinase activities in porcine oocytes after injection of sperm extracts (SE) or treatment with Ca2+ ionophore to clarify whether SE injection is useful for porcine oocyte activation. SE was prepared from miniature pig sperm by non-ionic surfactant. Oocytes that were treated with Ca2+ ionophore and injected with SE were activated at rates of 41% and 46%, respectively. The acti...

  6. DETORQUEO, QUIRKY, and ZERZAUST represent novel components involved in organ development mediated by the receptor-like kinase STRUBBELIG in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Lynette Fulton

    2009-01-01

    Full Text Available Intercellular signaling plays an important role in controlling cellular behavior in apical meristems and developing organs in plants. One prominent example in Arabidopsis is the regulation of floral organ shape, ovule integument morphogenesis, the cell division plane, and root hair patterning by the leucine-rich repeat receptor-like kinase STRUBBELIG (SUB. Interestingly, kinase activity of SUB is not essential for its in vivo function, indicating that SUB may be an atypical or inactive receptor-like kinase. Since little is known about signaling by atypical receptor-like kinases, we used forward genetics to identify genes that potentially function in SUB-dependent processes and found recessive mutations in three genes that result in a sub-like phenotype. Plants with a defect in DETORQEO (DOQ, QUIRKY (QKY, and ZERZAUST (ZET show corresponding defects in outer integument development, floral organ shape, and stem twisting. The mutants also show sub-like cellular defects in the floral meristem and in root hair patterning. Thus, SUB, DOQ, QKY, and ZET define the STRUBBELIG-LIKE MUTANT (SLM class of genes. Molecular cloning of QKY identified a putative transmembrane protein carrying four C(2 domains, suggesting that QKY may function in membrane trafficking in a Ca(2+-dependent fashion. Morphological analysis of single and all pair-wise double-mutant combinations indicated that SLM genes have overlapping, but also distinct, functions in plant organogenesis. This notion was supported by a systematic comparison of whole-genome transcript profiles during floral development, which molecularly defined common and distinct sets of affected processes in slm mutants. Further analysis indicated that many SLM-responsive genes have functions in cell wall biology, hormone signaling, and various stress responses. Taken together, our data suggest that DOQ, QKY, and ZET contribute to SUB-dependent organogenesis and shed light on the mechanisms, which are dependent on

  7. Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis.

    Science.gov (United States)

    Hashimoto-Sugimoto, Mimi; Negi, Juntaro; Monda, Keina; Higaki, Takumi; Isogai, Yasuhiro; Nakano, Toshiaki; Hasezawa, Seiichiro; Iba, Koh

    2016-05-01

    HT1 (HIGH LEAF TEMPERATURE 1) is the first component associated with changes in stomatal aperture in response to CO2 to be isolated by forward genetic screening. The HT1 gene encodes a protein kinase expressed mainly in guard cells. The loss-of-function ht1-1 and ht1-2 mutants in Arabidopsis thaliana have CO2-hypersensitive stomatal closure with concomitant reductions in their kinase activities in vitro In addition to these mutants, in this study we isolate or obtaine five new ht1 alleles (ht1-3, ht1-4, ht1-5, ht1-6, and ht1-7). Among the mutants, only ht1-3 has a dominant mutant phenotype and has widely opened stomata due to CO2 insensitivity. The ht1-3 mutant has a missense mutation affecting a non-conserved residue (R102K), whereas the other six recessive mutants have mutations in highly conserved residues in the catalytic domains required for kinase activity. We found that the dominant mutation does not affect the expression of HT1 or the ability to phosphorylate casein, a universal kinase substrate, but it does affect autophosphorylation activity in vitro A 3D structural model of HT1 also shows that the R102 residue protrudes from the surface of the kinase, implying a role for the formation of oligomers and/or interaction with its targets. We demonstrate that both the loss-of-function and gain-of-function ht1 mutants have completely disrupted CO2 responses, although they have normal responses to ABA. Furthermore, light-induced stomatal opening is smaller in ht1-3 and much smaller in ht1-2 Taken together, these results indicate that HT1 is a critical regulator for CO2 signaling and is partially involved in the light-induced stomatal opening pathway. PMID:27034327

  8. Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae

    Science.gov (United States)

    Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

    2016-01-01

    The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes. PMID:27782169

  9. Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

    Science.gov (United States)

    Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

    2014-07-01

    The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway.

  10. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  11. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Jiang, Shanshan; Zhang, Dan; Wang, Li; Pan, Jiaowen; Liu, Yang; Kong, Xiangpei; Zhou, Yan; Li, Dequan

    2013-10-01

    Calcium-dependent protein kinases (CDPKs) play essential roles in calcium-mediated signal transductions in plant response to abiotic stress. Several members have been identified to be regulators for plants response to abscisic acid (ABA) signaling. Here, we isolated a subgroup I CDPK gene, ZmCPK4, from maize. Quantitative real time PCR (qRT-PCR) analysis revealed that the ZmCPK4 transcripts were induced by various stresses and signal molecules. Transient and stable expression of the ZmCPK4-GFP fusion proteins revealed ZmCPK4 localized to the membrane. Moreover, overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth and stomatal movement. The transgenic plants also enhanced drought stress tolerance. Taken together, the results suggest that ZmCPK4 might be involved in ABA-mediated regulation of stomatal closure in response to drought stress. PMID:23911729

  12. Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase I protein is present in sporophytic and gametophytic cells and undergoes endocytosis

    NARCIS (Netherlands)

    Kwaaitaal, M.A.C.J.; Vries, de S.C.; Russinova, E.T.

    2005-01-01

    Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected

  13. The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sergi Portolés

    2010-11-01

    Full Text Available Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1. CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2 are essential for clock temperature compensation in Arabidopsis.

  14. Molecular cloning and mRNA expression analysis of a novel rice (Oryzasativa L.) MAPK kinase kinase, OsEDR1, an ortholog of Arabidopsis AtEDR1, reveal its role in defense/stress signalling pathways and development.

    Science.gov (United States)

    Kim, Jung-A; Agrawal, Ganesh K; Rakwal, Randeep; Han, Keon Seon; Kim, Kyung-Nam; Yun, Choong-Hyo; Heu, Sunggi; Park, Sook-Young; Lee, Yong-Hwan; Jwa, Nam-Soo

    2003-01-24

    Mitogen-activated protein kinase (MAPK) cascade(s) is important for plant defense/stress responses. Though MAPKs have been identified and characterized in rice (Oryza sativa L.), a monocot cereal crop research model, the first upstream component of the kinase cascade, namely MAPK kinase kinase (MAPKKK) has not yet been identified. Here we report the cloning of a novel rice gene encoding a MAPKKK, OsEDR1, designated based on its homology with the Arabidopsis MAPKKK, AtEDR1. OsEDR1, a single copy gene in the genome of rice, encodes a predicted protein with molecular mass of 113046.13 and a pI of 9.03. Using our established two-week-old rice seedling in vitro model system, we show that OsEDR1 has a constitutive expression in seedling leaves and is further up-regulated within 15 min upon wounding by cut, treatment with the global signals jasmonic acid (JA), salicylic acid (SA), ethylene (ethephon, ET), abscisic acid, and hydrogen peroxide. In addition, protein phosphatase inhibitors, fungal elicitor chitosan, drought, high salt and sugar, and heavy metals also dramatically induce its expression. Moreover, OsEDR1 expression was altered by co-application of JA, SA, and ET, and required de novo synthesized protein factor(s) in its transient regulation. Furthermore, using an in vivo system we also show that OsEDR1 responds to changes in temperature and environmental pollutants-ozone and sulfur dioxide. Finally, OsEDR1 expression varied significantly in vegetative and reproductive tissues. These results suggest a role for OsEDR1 in defense/stress signalling pathways and development. PMID:12559953

  15. Functional Redundancy of ERK1 and ERK2 MAP Kinases during Development

    Directory of Open Access Journals (Sweden)

    Christophe Frémin

    2015-08-01

    Full Text Available ERK1 and ERK2 are the effector kinases of the ERK1/2 MAP-kinase signaling pathway, which plays a central role in transducing signals controlling cell proliferation, differentiation, and survival. Deregulated activity of the ERK1/2 pathway is linked to a group of developmental syndromes and contributes to the pathogenesis of various human diseases. One fundamental question that remains unaddressed is whether ERK1 and ERK2 have evolved unique physiological functions or whether they are used redundantly to reach a threshold of global ERK activity. Here, we show that the extent of development of the mouse placenta and embryo bearing different combinations of Erk1 and Erk2 alleles is strictly correlated with total ERK1/2 activity. We further demonstrate that transgenic expression of ERK1 fully rescues the embryonic and placental developmental defects associated with the loss of ERK2. We conclude that ERK1 and ERK2 exert redundant functions in mouse development.

  16. Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

    Directory of Open Access Journals (Sweden)

    Dennis J Templeton

    Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

  17. Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3.

    Directory of Open Access Journals (Sweden)

    Toni M Brand

    Full Text Available Nuclear localized HER family receptor tyrosine kinases (RTKs have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3 have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁ and 27 (B₂ amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

  18. Overexpression of a maize SNF-related protein kinase gene, ZmSnRK2.11, reduces salt and drought tolerance in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fan; CHEN Xun-ji; WANG Jian-hua; ZHENG Jun

    2015-01-01

    Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) is a unique family of protein kinases associated with abiotic stress signal transduction in plants. In this study, a maize SnRK2 gene ZmSnRK2.11 was cloned and characterized. The results showed that ZmSnRK2.11 is up-regulated by high-salinity and dehydration treatment, and it is expressed mainly in maize mature leaf. A transient expression assay using onion epidermal cel s revealed that ZmSnRK2.11-GFP fusion proteins are localized to both the nucleus and cytoplasm. Overexpressing-ZmSnRK2.11 in Arabidopsis resulted in salt and drought sensitivity phenotypes that exhibited an increased rate of water loss, reduced relative water content, delayed stoma closure, accumulated less free proline content and increased malondialdehyde (MDA) content relative to the phenotypes observed in wild-type (WT) control. Furthermore, overexpression of ZmSnRK2.11 up-regulated the expression of the genes ABI1 and ABI2 and decreased the expression of DREB2A and P5CS1. Taken together, our results suggest that ZmSnRK2.11 is a possible negative regulator involved in the salt and drought stress signal transduction pathways in plants.

  19. Scattering of MCF7 cells by heregulin ß-1 depends on the MEK and p38 MAP kinase pathway.

    Directory of Open Access Journals (Sweden)

    Rintaro Okoshi

    Full Text Available Heregulin (HRG β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell-cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell-cell adhesion.

  20. Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements.

    OpenAIRE

    Lagercrantz, U.

    1998-01-01

    Chromosome organization and evolution in the Brassicaceae family was studied using comparative linkage mapping. A total of 160 mapped Arabidopsis thaliana DNA fragments identified 284 homologous loci covering 751 cM in Brassica nigra. The data support that modern diploid Brassica species are descended from a hexaploid ancestor, and that the A. thaliana genome is similar in structure and complexity to those of each of the hypothetical diploid progenitors of the proposed hexaploid. Thus, the Br...

  1. Genome wide association mapping of time-dependent growth responses to moderate drought stress in Arabidopsis

    OpenAIRE

    Bac-Molenaar, Johanna A.; Granier, Christine; Keurentjes, Joost J.B.; Vreugdenhil, Dick

    2016-01-01

    Large areas of arable land are often confronted with irregular rainfall resulting in limited water availability for part(s) of the growing seasons, which demands research for drought tolerance of plants. Natural variation was observed for biomass accumulation upon controlled moderate drought stress in 324 natural accessions of Arabidopsis. Improved performance under drought stress was correlated with early flowering and lack of vernalization requirement, indicating overlap in the regulatory n...

  2. Routine mapping of Fusarium wilt resistance in BC1 populations of Arabidopsis thaliana

    OpenAIRE

    Andrew C Diener

    2013-01-01

    Abstract Background Susceptibility to Fusarium wilt disease varies among wild accessions of Arabidopsis thaliana. Six RESISTANCE TO FUSARIUM OXYSPORUM (RFO) quantitative trait loci (QTLs) controlling the resistance of accession Columbia-0 (Col-0) and susceptibility of Taynuilt-0 to Fusarium oxysporum forma specialis matthioli (FOM) are detected in a recombinant population derived from a single backcross of the F1 hybrid (BC1). In particular, ...

  3. Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

    LENUS (Irish Health Repository)

    McEneaney, Victoria

    2010-01-01

    Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1\\/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1\\/2-dependent. Aldosterone induced the rapid activation of ERK1\\/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1\\/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1\\/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1\\/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1\\/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1\\/2 was inhibited in cells suppressed in the expression of PKD1.

  4. Integration of linkage maps for the Amphidiploid Brassica napus and comparative mapping with Arabidopsis and Brassica rapa

    OpenAIRE

    Delourme Régine; Falentin Cyril; Parkin Isobel AP; Lydiate Derek J; Wang Jun; Carion Pierre WC; King Graham J

    2011-01-01

    Abstract Background The large number of genetic linkage maps representing Brassica chromosomes constitute a potential platform for studying crop traits and genome evolution within Brassicaceae. However, the alignment of existing maps remains a major challenge. The integration of these genetic maps will enhance genetic resolution, and provide a means to navigate between sequence-tagged loci, and with contiguous genome sequences as these become available. Results We report the first genome-wide...

  5. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    Science.gov (United States)

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

  6. Identification and map location of TTR1, a single locus in Arabidopsis thaliana that confers tolerance to tobacco ringspot nepovirus.

    Science.gov (United States)

    Lee, J M; Hartman, G L; Domier, L L; Bent, A F

    1996-11-01

    The interaction between Arabidopsis and the nepovirus tobacco ringspot virus (TRSV) was characterized. Of 97 Arabidopsis lines tested, all were susceptible when inoculated with TRSV grape strain. Even though there was systemic spread of the virs, there was a large degree of variation in symptoms as the most sensitive lines died 10 days after inoculation, while the most tolerant lines either were symptomless or developed only mild symptoms. Four lines were selected for further study based on their differential reactions to TRSV. Infected plants of line Col-0 and Col-0 gl1 flowered and produced seeds like noninfected plants, while those of lines Estland and H55 died before producing seeds. Symptoms appeared on sensitive plants approximately 5 to 6 days after inoculation. Serological studies indicated that in mechanically inoculated seedlings, the virus, as measured by coat protein accumulation, developed at essentially the same rates and to the same levels in each of the four lines, demonstrating that differences in symptom development were not due to a suppression of virus accumulation. Two additional TRSV strains gave similar results when inoculated on the four lines. Genetic studies with these four Arabidopsis lines revealed segregation of a single incompletely dominant locus controlling tolerance to TRSV grape strain. We have designated this locus TTR1. By using SSLP and CAPS markers, TTR1 was mapped to chromosome V near the nga129 marker. Seed transmission frequency of TRSV for Col-0 and Col-0 gl1 was over 95% and their progeny from crosses all had seed transmission frequencies of over 83%, which made it possible to evaluate the segregation of TTR1 in F2 progeny from infected F1 plants without inoculating F2 plants. Seed transmission of TRSV will be further exploited to streamline selection of individuals for fine mapping the TTR1 gene. The identification of tolerant and sensitive interactions between TRSV and A. thaliana lines provides a model system for

  7. Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity.

    Science.gov (United States)

    Lamson, Rachel E; Takahashi, Satoe; Winters, Matthew J; Pryciak, Peter M

    2006-03-21

    Distinct MAP kinase pathways in yeast share several signaling components , including the PAK Ste20 and the MAPKKK Ste11, yet signaling is specific. Mating pheromones trigger an initial step in which Ste20 activates Ste11 , and this requires plasma membrane recruitment of the MAP kinase cascade scaffold protein, Ste5 . Here, we demonstrate an additional role for Ste5 membrane localization. Once Ste11 is activated, signaling through the mating pathway remains minimal but is substantially amplified when Ste5 is recruited to the membrane either by the Gbetagamma dimer or by direct membrane targeting, even to internal membranes. Ste11 signaling is also amplified by Ste5 oligomerization and by a hyperactivating mutation in the Ste7 binding region of Ste5. We suggest a model in which membrane recruitment of Ste5 concentrates its binding partners and thereby amplifies signaling through the kinase cascade. We find similar behavior in the osmotically responsive HOG pathway. Remarkably, while both pheromone and hyperosmotic stimuli amplify signaling from constitutively active Ste11, the resulting signaling output remains pathway specific. These findings suggest a common mode of regulation in which pathway stimuli both initiate and amplify MAP kinase cascade signaling. The regulation of rate-limiting steps that lie after a branchpoint from shared components helps ensure signaling specificity.

  8. Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock.

    Science.gov (United States)

    Jiménez, Carlos; Berl, Tomas; Rivard, Christopher J; Edelstein, Charles L; Capasso, Juan M

    2004-02-01

    The microalga Dunaliella viridis has the ability to adapt to a variety of environmental stresses including osmotic and thermal shocks, UV irradiation and nitrogen starvation. Lacking a rigid cell wall, Dunaliella provides an excellent model to study stress signaling in eukaryotic unicellular organisms. When exposed to hyperosmotic stress, UV irradiation or high temperature, a 57-kDa protein is recognized by antibodies specific to mammalian p38, to its yeast homologue Hog1, and to the phospho-p38 MAP kinase motif. This 57-kDa protein appears to be both up-regulated and phosphorylated. Three other proteins (50, 45, 43 kDa) were transiently phosphorylated under stress conditions as detected with an antibody specific to the mammalian phospho c-Jun N-terminal kinase (JNK) motif. Treatment with specific inhibitors of p38 MAP kinase (SB203580) and JNK (SP600125) activities markedly impaired the adaptation of Dunaliella to osmotic stress. From an evolutionary standpoint, these data strongly suggest that MAP kinase signaling pathways, other than ERK, were already operating in the common ancestor of plant and animal kingdoms, probably as early as 1400 million years ago. PMID:14741745

  9. The Arabidopsis Homolog of the Mammalian OS-9 Protein Plays a Key Role in the Endoplasmic Reticulum-Associated Degradation of Misfolded Receptor-Like Kinases

    Institute of Scientific and Technical Information of China (English)

    Wei Su; Yidan Liu; Yang Xia; Zhi Hong; Jianming Li

    2012-01-01

    The endoplasmic reticulum-associated degradation(ERAD)is a highly conserved mechanism to remove misfolded membrane/secretory proteins from the endoplasmic reticulum(ER).While many of the individual components of the ERAD machinery are well characterized in yeast and mammals,our knowledge of a plant ERAD process is rather limited.Here,we report a functional study of an Arabidopsis homolog(AtOS9)of an ER luminal lectin Yos9(OS-9 in mammals)that recognizes a unique asparagine-linked glycan on misfolded proteins.We discovered that AtOS9 is an ER-localized glycoprotein that is co-expressed with many known/predicted ER chaperones.AT-DNA insertional atos9-t mutation blocks the degradation of a structurally imperfect yet biochemically competent brassinosteroid(BR)receptor bri1-9,causing its increased accumulation in the ER and its consequent leakage to the cell surface responsible for restoring the BR sensitivity and suppressing the dwarfism of the bri1-9 mutant.In addition,we identified a missense mutation in AtOS9 in a recently discovered ERAD mutant ems-mutagenized bri1 suppressor 6(ebs6-1).Moreover,we showed that atos9-t also inhibits the ERAD of bri1-5,another ER-retained BR receptor,and a misfolded EFR,a BRI1-like receptor for the bacterial translation elongation factor EF-Tu.Furthermore,we found that AtOS9 interacted biochemically and genetically with EBS5,an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L known to collaborate with Yos9/OS-9 to select ERAD clients.Taken together,our results demonstrated a functional role of AtOS9 in a plant ERAD process that degrades misfolded receptor-like kinases.

  10. The plastid-localized pfkB-type carbohydrate kinases FRUCTOKINASE-LIKE 1 and 2 are essential for growth and development of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Gilkerson Jonathan

    2012-07-01

    Full Text Available Abstract Background Transcription of plastid-encoded genes requires two different DNA-dependent RNA polymerases, a nuclear-encoded polymerase (NEP and plastid-encoded polymerase (PEP. Recent studies identified two related pfkB-type carbohydrate kinases, named FRUCTOKINASE-LIKE PROTEIN (FLN1 and FLN2, as components of the thylakoid bound PEP complex in both Arabidopsis thaliana and Sinapis alba (mustard. Additional work demonstrated that RNAi-mediated reduction in FLN expression specifically diminished transcription of PEP-dependent genes. Results Here, we report the characterization of Arabidopsis FLN knockout alleles to examine the contribution of each gene in plant growth, chloroplast development, and in mediating PEP-dependent transcription. We show that fln plants have severe phenotypes with fln1 resulting in an albino phenotype that is seedling lethal without a source of exogenous carbon. In contrast, fln2 plants display chlorosis prior to leaf expansion, but exhibit slow greening, remain autotrophic, can grow to maturity, and set viable seed. fln1 fln2 double mutant analysis reveals haplo-insufficiency, and fln1 fln2 plants have a similar, but more severe phenotype than either single mutant. Normal plastid development in both light and dark requires the FLNs, but surprisingly skotomorphogenesis is unaffected in fln seedlings. Seedlings genetically fln1-1 with dexamethasone-inducible FLN1-HA expression at germination are phenotypically indistinguishable from wild-type. Induction of FLN-HA after 24 hours of germination cannot rescue the mutant phenotype, indicating that the effects of loss of FLN are not always reversible. Examination of chloroplast gene expression in fln1-1 and fln2-1 by qRT-PCR reveals that transcripts of PEP-dependent genes were specifically reduced compared to NEP-dependent genes in both single mutants. Conclusions Our results demonstrate that each FLN protein contributes to wild type growth, and acting additively are

  11. Deactivation of the Arabidopsis BRI1 receptor kinase by autophosphorylation within the glycine-rich loop involved in ATP binding

    Science.gov (United States)

    The activity of the dual-specificity brassinosteroid receptor kinase, BRI1, reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we identify regions of the juxtamembrane domain that are essential f...

  12. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway

    Directory of Open Access Journals (Sweden)

    Zeljkica eJandric

    2013-11-01

    Full Text Available Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in Candida glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologues CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species, and required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  13. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Serena A D'Souza

    2016-04-01

    Full Text Available The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

  14. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

    Science.gov (United States)

    D’Souza, Serena A.; Rajendran, Luckshika; Bagg, Rachel; van Pel, Derek M.; Moshiri, Houtan; Roy, Peter J.

    2016-01-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  15. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    Science.gov (United States)

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

  16. Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

    DEFF Research Database (Denmark)

    Brockdorff, J; Nielsen, M; Kaltoft, K;

    2000-01-01

    found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis...... fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation...

  17. A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism.

    Science.gov (United States)

    Zeng, Fanyun; Gong, Xiaoyan; Hamid, Mahammad Imran; Fu, Yanping; Jiatao, Xie; Cheng, Jiasen; Li, Guoqing; Jiang, Daohong

    2012-05-01

    Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans. PMID:22426009

  18. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  19. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

    Science.gov (United States)

    Andrusiak, Matthew G; Jin, Yishi

    2016-04-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans.

  20. The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea.

    Science.gov (United States)

    Schamber, Astrid; Leroch, Michaela; Diwo, Janine; Mendgen, Kurt; Hahn, Matthias

    2010-01-01

    In all fungi studied so far, mitogen-activated protein (MAP) kinase cascades serve as central signalling complexes that are involved in various aspects of growth, stress response and infection. In this work, putative components of the yeast Fus3/Kss1-type MAP kinase cascade and the putative downstream transcription factor Ste12 were analysed in the grey mould fungus Botrytis cinerea. Deletion mutants of the MAP triple kinase Ste11, the MAP kinase kinase Ste7 and the MAP kinase adaptor protein Ste50 all resulted in phenotypes similar to that of the previously described BMP1 MAP kinase mutant, namely defects in germination, delayed vegetative growth, reduced size of conidia, lack of sclerotia formation and loss of pathogenicity. Mutants lacking Ste12 showed normal germination, but delayed infection as a result of low penetration efficiency. Two differently spliced ste12 transcripts were detected, and both were able to complement the ste12 mutant, except for a defect in sclerotium formation, which was only corrected by the full-sized transcript. Overexpression of the smaller ste12 transcript resulted in delayed germination and strongly reduced infection. Bc-Gas2, a homologue of Magnaporthe grisea Gas2 that is required for appressorial function, was found to be non-essential for growth and infection, but its expression was under the control of both Bmp1 and Ste12. In summary, the role and regulatory connections of the Fus3/Kss1-type MAP kinase cascade in B. cinerea revealed both common and unique properties compared with those of other plant pathogenic fungi, and provide evidence for a regulatory link between the BMP1 MAP kinase cascade and Ste12. PMID:20078780

  1. Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling.

    Science.gov (United States)

    Tsioumpekou, Maria; Papadopoulos, Natalia; Burovic, Fatima; Heldin, Carl-Henrik; Lennartsson, Johan

    2016-09-01

    Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase Cγ and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The co-operation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKCζ interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling. PMID:27339033

  2. A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana.

    Science.gov (United States)

    Haughn, G W; Somerville, C R

    1990-04-01

    A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS. PMID:16667374

  3. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  4. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    Science.gov (United States)

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  5. Regulating Global Sumoylation by a MAP Kinase Hog1 and Its Potential Role in Osmo-Tolerance in Yeast

    OpenAIRE

    Abu Irqeba, Ameair; Li, Yang; Panahi, Mahmoud; Zhu, Ming; Wang, Yuqi

    2014-01-01

    Sumoylation, a post-translational protein modification by small ubiquitin-like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo-response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal yeast cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find th...

  6. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen

    OpenAIRE

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only r...

  7. FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

    OpenAIRE

    Chengkang eZhang; Jianqiang eWang; Hong eTao; Xie eDang; Yang eWang; Miaoping eChen; Zhenzhen eZhai; Wenying eYu; Liping eXu; Won-Bo eShim; Guodong eLu; Zonghua eWang

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only r...

  8. Stimulation of MAP kinase pathways after maternal IL-1β exposure induces fetal lung fluid absorption in guinea pigs

    Directory of Open Access Journals (Sweden)

    Carter Ethan P

    2007-03-01

    Full Text Available Abstract Background We tested the hypothesis that maternal interleukin-1β (IL-1β pretreatment and induction of fetal cortisol synthesis activates MAP kinases and thereby affects lung fluid absorption in preterm guinea pigs. Methods IL-1β was administered subcutaneously daily to timed-pregnant guinea pigs for three days. Fetuses were obtained by abdominal hysterotomy and instilled with isosmolar 5% albumin into the lungs and lung fluid movement was measured over 1 h by mass balance. MAP kinase expression was measured by western blot. Results Lung fluid absorption was induced at 61 days (D gestation and stimulated at 68D gestation by IL-1β. Maternal IL-1β pretreatment upregulated ERK and upstream MEK expression at both 61 and 68D gestation, albeit being much more pronounced at 61D gestation. U0126 instillation completely blocked IL-1β-induced lung fluid absorption as well as IL-1β-induced/stimulated ERK expression. Cortisol synthesis inhibition by metyrapone attenuated ERK expression and lung fluid absorption in IL-1β-pretreated fetal lungs. JNK expression after maternal IL-1β pretreatment remained unaffected at either gestation age. Conclusion These data implicate the ERK MAP kinase pathway as being important for IL-1β induction/stimulation of lung fluid absorption in fetal guinea pigs.

  9. Sequence based polymorphic (SBP marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Sahu Binod B

    2012-01-01

    Full Text Available Abstract Background Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs for any genomic regions. Here a sequence based polymorphic (SBP marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described. Results A ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0 was obtained by applying Illumina's sequencing by synthesis (Solexa technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0 genome sequence identified putative single nucleotide polymorphisms (SNPs throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated. Conclusions The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for

  10. Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene.

    Science.gov (United States)

    Salmon, Jemma; Ward, Sally P; Hanley, Steven J; Leyser, Ottoline; Karp, Angela

    2014-05-01

    Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short-rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time-consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone-related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot-branching traits. Single-nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%-99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.

  11. Activation of the Cph1-dependent MAP kinase signaling pathway induces white-opaque switching in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Bernardo Ramírez-Zavala

    Full Text Available Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white to an elongated cell type (opaque, which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11(ΔN467 efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.

  12. Genome-wide association mapping of time-dependent growth responses to moderate drought stress in Arabidopsis.

    Science.gov (United States)

    Bac-Molenaar, Johanna A; Granier, Christine; Keurentjes, Joost J B; Vreugdenhil, Dick

    2016-01-01

    Large areas of arable land are often confronted with irregular rainfall resulting in limited water availability for part(s) of the growing seasons, which demands research for drought tolerance of plants. Natural variation was observed for biomass accumulation upon controlled moderate drought stress in 324 natural accessions of Arabidopsis. Improved performance under drought stress was correlated with early flowering and lack of vernalization requirement, indicating overlap in the regulatory networks of flowering time and drought response or correlated responses of these traits to natural selection. In addition, plant size was negatively correlated with relative water content (RWC) independent of the absolute water content (WC), indicating a prominent role for soluble compounds. Growth in control and drought conditions was determined over time and was modelled by an exponential function. Genome-wide association (GWA) mapping of temporal plant size data and of model parameters resulted in the detection of six time-dependent quantitative trait loci (QTLs) strongly associated with drought. Most QTLs would not have been identified if plant size was determined at a single time point. Analysis of earlier reported gene expression changes upon drought enabled us to identify for each QTL the most likely candidates.

  13. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    Science.gov (United States)

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS.

  14. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    Science.gov (United States)

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS. PMID:25995471

  15. Induction of Macrophage Function in Human THP-1 Cells is Associated with MAPK Signaling and Activation of MAP3K7 (TAK1 Protein Kinase

    Directory of Open Access Journals (Sweden)

    Erik eRichter

    2016-03-01

    Full Text Available Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cycline-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1 acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

  16. Probabilistic mapping and image clustering for quantitative assessment of fluorescent protein localizations in Arabidopsis guard cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Takumi Higaki, Natsumaro Kutsuna & Seiichiro Hasezawa ### Abstract The protocol reported here describes a method to quantitatively evaluate fluorescently-tagged protein localizations from fluorescent microscopic images with a combination of probabilistic mapping and image clustering. We demonstrate the use of this protocol using kidney-shaped guard cells of plants. ### Introduction Microscopic assessment of protein localizations with fluorescent protein taggin...

  17. STATE OF JNK AND P38 MAP-KINASE SYSTEM IN BLOOD monon uclea r le ucocytes DUR ING INFLAMMATION

    Directory of Open Access Journals (Sweden)

    N. Y. Chasovskih

    2009-01-01

    Full Text Available Abstract. Pogrammed cell death of peripheral blood mononuclear leucocytes from patients with acute inflammatory diseases (non-nosocomial pneumonia, acute appendicitis was investigated under ex vivo conditions, upon cultivation of the cells with selective inhibitors of JNK (SP600125 and р38 МАРК (ML3403. In vitro addition of SP600125 and ML3403 under oxidative stress conditions prevents increase of annexinpositive mononuclear cells numbers, thus suggesting JNK and р38 МАР-kinases to be involved into oxidative mechanisms of apoptosis deregulation. A role of JNK in IL-8 production by mononuclear leucocytes was revealed in cases of acute inflammation. Regulatory effect of JNK and p38 MAP-kinases can be mediated through activation of redox-sensitive apoptogenic signal transduction systems, as well as due to changes in cellular cytokine-producing function.

  18. The Role of MAP Kinase Cascade in Neonatal Brain Response to Hypoxia-Ischemic Insult

    OpenAIRE

    Thei, L. J.

    2014-01-01

    Babies that are born more than 8 weeks premature or those deprived of Oxygen during the perinatal period are susceptible to brain injury, particularly in conjunction with maternal or fetal infection, leading to neurological deficits later in life. Multiple studies have shown that even brief exposure to hypoxic conditions will cause rapid and selective increase in specific mitogen-activated protein kinases including extracellular signal - related kinase 1 and 2 (ERK1/2) and C-Jun N-te...

  19. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins.

    Science.gov (United States)

    Mei, Yu; Jia, Wen-Jing; Chu, Yu-Jia; Xue, Hong-Wei

    2012-03-01

    Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport. PMID:21894193

  20. The Arabidopsis lectin receptor kinase LecRK-V.5 represses stomatal immunity induced by Pseudomonas syringae pv. tomato DC3000.

    Directory of Open Access Journals (Sweden)

    Marie Desclos-Theveniau

    2012-02-01

    Full Text Available Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5 negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO(2 uptake and photosynthesis.

  1. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078.

    Science.gov (United States)

    Tang, Yong; Zhao, Chun-Yan; Tan, Shu-Tang; Xue, Hong-Wei

    2016-08-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5-1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5-1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5-1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5-1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  2. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

    Institute of Scientific and Technical Information of China (English)

    Yu Mei; Wen-Jing Jia; Yu-Jia Chu; Hong-Wei Xue

    2012-01-01

    Phosphatidylinositol monophosphate 5-kinase(PIP5K)catalyzes the synthesis of PI-4,5-bisphosphate(PtdIns(4,5)P2)by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring,and is involved in regulating multiple developmental processes and stress responses.We here report on the functional characterization of Arabidopsis PIP5K2,which is expressed during lateral root initiation and elongation,and whose expression is enhanced by exogenous auxin.The knockout mutant pip5k2 shows reduced lateral root formation,which could be recovered with exogenous auxin,and interestingly,delayed root gravity response that could not be recovered with exogenous auxin.Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2.In addition,analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P2 reduction,which hence results in suppressed cycling of PIN proteins(PIN2 and 3),and delayed redistribution of PIN2 and auxin under gravistimulation in pipSk2 roots.On the contrary,PtdIns(4,5)P2 significantly enhanced the vesicle trafficking and cycling of PIN proteins.These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response,and reveal a critical role of PIP5K2/Ptdlns(4,5)P2 in root development through regulation of PIN proteins,providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response,and new insights into the control of polar auxin transport.

  3. Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome.

    Science.gov (United States)

    Le Goff, Carine; Rogers, Curtis; Le Goff, Wilfried; Pinto, Graziella; Bonnet, Damien; Chrabieh, Maya; Alibeu, Olivier; Nistchke, Patrick; Munnich, Arnold; Picard, Capucine; Cormier-Daire, Valérie

    2016-08-01

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. PMID:27426734

  4. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    Science.gov (United States)

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  5. Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Devadas, Balekudru; Selness, Shaun R.; Xing, Li; Madsen, Heather M.; Marrufo, Laura D.; Shieh, Huey; Messing, Dean M.; Yang, Jerry Z.; Morgan, Heidi M.; Anderson, Gary D.; Webb, Elizabeth G.; Zhang, Jian; Devraj, Rajesh V.; Monahan, Joseph B. (Pfizer)

    2012-02-28

    A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-{alpha} in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

  6. Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

    Science.gov (United States)

    Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

    2016-01-01

    Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

  7. A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1

    OpenAIRE

    Kusari, A B; D. M. Molina; W Sabbagh; Lau, C S; Bardwell, Lee

    2004-01-01

    The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7rn region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially ...

  8. Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.

    OpenAIRE

    Ma, D; Cook, J G; Thorner, J

    1995-01-01

    Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 d...

  9. FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells.

    Directory of Open Access Journals (Sweden)

    Juan Pablo Macagno

    2014-03-01

    Full Text Available Receptor Tyrosine Kinases (RTKs and Focal Adhesion Kinase (FAK regulate multiple signalling pathways, including mitogen-activated protein (MAP kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.

  10. Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.

    OpenAIRE

    Bardwell, L; Cook, J G; E. C. Chang; Cairns, B R; Thorner, J

    1996-01-01

    Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in ...

  11. Mapping the residues of protein kinase CK2 implicated in substrate recognition

    DEFF Research Database (Denmark)

    Sarno, S; Boldyreff, B; Marin, O;

    1995-01-01

    Six mutants of protein kinase CK2 alpha subunit in which basic residues have been mutated into alanines were assayed for their capability to phosphorylate the peptide RRRADDSDDDDD. Two mutants (R228A and R278K279R280A) behaved more or less as alpha wild type and one (H160,166A) was nearly inactive...

  12. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.

    2003-02-01

    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

  13. Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors

    DEFF Research Database (Denmark)

    Vaglio, P; Sarno, S; Marin, O;

    1996-01-01

    The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0...

  14. QSAR Analysis of Some Antagonists for p38 map kinase Using Combination of Principal Component Analysis and Artificial Intelligence.

    Science.gov (United States)

    Doosti, Elham; Shahlaei, Mohsen

    2015-01-01

    Quantitative relationships between structures of a set of p38 map kinase inhibitors and their activities were investigated by principal component regression (PCR) and principal componentartificial neural network (PC-ANN). Latent variables (called components) generated by principal component analysis procedure were applied as the input of developed Quantitative structure- activity relationships (QSAR) models. An exact study of predictability of PCR and PC-ANN showed that the later model has much higher ability to calculate the biological activity of the investigated molecules. Also, experimental and estimated biological activities of compounds used in model development step have indicated a good correlation. Obtained results show that a non-linear model explaining the relationship between the pIC50s and the calculated principal components (that extract from structural descriptors of the studied molecules) is superior than linear model. Some typical figures of merit for QSAR studies explaining the accuracy and predictability of the suggested models were calculated. Therefore, to design novel inhibitors of p38 map kinase with high potency and low undesired effects the developed QSAR models were used to estimate biological pIC50 of the studied compounds.

  15. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

    Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

  16. A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

    Directory of Open Access Journals (Sweden)

    Meng Zhang

    Full Text Available PsSAK1, a mitogen-activated protein (MAP kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b direct germination of sporangia, and c afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

  17. Nuclear Localization of the ERK MAP Kinase Mediated by Drosophila αPS2βPS Integrin and Importin-7

    OpenAIRE

    James, Brian P.; Bunch, Thomas A.; Krishnamoorthy, Srinivasan; Perkins, Lizabeth A.; Brower, Danny L.

    2007-01-01

    The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is n...

  18. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry.

    Science.gov (United States)

    Dangoria, N S; Breau, W C; Anderson, H A; Cishek, D M; Norkin, L C

    1996-09-01

    Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca(2+)-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

  19. Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

    International Nuclear Information System (INIS)

    Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC

  20. Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome.

    OpenAIRE

    Jackson, S A; Cheng, Z; Wang, M L; Goodman, H M; Jiang, J

    2000-01-01

    Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A...

  1. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    Science.gov (United States)

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  2. Corticosterone Regulates pERK1/2 Map Kinase in a Chronic Depression Model

    OpenAIRE

    Gourley, Shannon L.; Wu, Florence J.; Taylor, Jane R.

    2008-01-01

    Neurotransmitter- or neurotrophin-regulated intracellular signaling in the hippocampus is hypothesized to contribute to depression and antidepressant (ADT) efficacy. Extracellular signal-regulated kinase 1/2 (ERK1/2) is downstream of several receptor types and regulates transcriptional activity of many targets; ERK1/2 may thereby influence mood and affect. Using a novel, ADT-sensitive depression model in mice, we show that prior corticosterone exposure decreases motivated behavior, sucrose co...

  3. Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa

    OpenAIRE

    Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna

    2014-01-01

    Mitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrate...

  4. A MAP kinase dependent feedback mechanism controls Rho1 GTPase and actin distribution in yeast.

    Directory of Open Access Journals (Sweden)

    Shuguang Guo

    Full Text Available In the yeast Saccharomyces cerevisiae the guanosine triphosphatase (GTPase Rho1 controls actin polarization and cell wall expansion. When cells are exposed to various environmental stresses that perturb the cell wall, Rho1 activates Pkc1, a mammalian Protein Kinase C homologue, and Mpk1, a mitogen activated protein kinase (MAPK, resulting in actin depolarization and cell wall remodeling. In this study, we demonstrate a novel feedback loop in this Rho1-mediated Pkc1-MAPK pathway that involves regulation of Rom2, the guanine nucleotide exchange factor of Rho1, by Mpk1, the end kinase of the pathway. This previously unrecognized Mpk1-dependent feedback is a critical step in regulating Rho1 function. Activation of this feedback mechanism is responsible for redistribution of Rom2 and cell wall synthesis activity from the bud to cell periphery under stress conditions. It is also required for terminating Rho1 activity toward the Pkc1-MAPK pathway and for repolarizing actin cytoskeleton and restoring growth after the stressed cells become adapted.

  5. Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma.

    Directory of Open Access Journals (Sweden)

    Brian J Capaldo

    Full Text Available Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

  6. The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB

    OpenAIRE

    Felton-Edkins, Zoe A.; Fairley, Jennifer A.; Graham, Emma L.; Johnston, Imogen M.; White, Robert J.; Scott, Pamela H.

    2003-01-01

    RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNALeu genes in cells decrea...

  7. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    International Nuclear Information System (INIS)

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interferon-γ and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1β and TNF-α correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1β and TNF-α, and downstream by MAP kinase signaling pathways and metalloproteinases.

  8. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Menschikowski, Mario, E-mail: Mario.Menschikowski@uniklinikum-dresden.de [Institute of Clinical Chemistry and Laboratory Medicine, Technical University of Dresden, Medical Faculty ' Carl Gustav Carus' , Fetscherstrasse 74, D-01307 Dresden (Germany); Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele [Institute of Clinical Chemistry and Laboratory Medicine, Technical University of Dresden, Medical Faculty ' Carl Gustav Carus' , Fetscherstrasse 74, D-01307 Dresden (Germany)

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  9. Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells

    Science.gov (United States)

    Hamanoue, Makoto; Morioka, Kazuhito; Ohsawa, Ikuroh; Ohsawa, Keiko; Kobayashi, Masaaki; Tsuburaya, Kayo; Akasaka, Yoshikiyo; Mikami, Tetsuo; Ogata, Toru; Takamatsu, Ken

    2016-01-01

    Endogenous neural stem/progenitor cells (NPCs) can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase (p38) is expressed in doublecortin-positive adult NPCs. Experiments using the p38 inhibitor SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we generated a cell-permeable wild-type p38 protein (PTD-p38WT) in which the HIV protein transduction domain (PTD) was fused to the N-terminus of p38. Treatment with PTD-p38WT significantly promoted the random migration of adult NPCs without affecting cell survival or differentiation; this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-p38WT is a novel and useful tool for unraveling the roles of p38, and that this protein provides a reasonable approach for regenerating the injured brain by enhancing NPC migration. PMID:27067799

  10. Inflammatory and catabolic signalling in intervertebral discs: The roles of NF-B and MAP Kinases

    Directory of Open Access Journals (Sweden)

    K Wuertz

    2012-02-01

    Full Text Available Painful intervertebral disc disease is characterised not only by an imbalance between anabolic (i.e., matrix synthesis and catabolic (i.e., matrix degradation processes, but also by inflammatory mechanisms. The increased expression and synthesis of matrix metalloproteinases and inflammatory factors is mediated by specific signal transduction, in particular the nuclear factor-kappaB (NF-kB and mitogen-activated protein kinase (MAPK-mediated pathways. NF-kB and MAPK have been identified as the master regulators of inflammation and catabolism in several musculoskeletal disorders (e.g., osteoarthritis, and recently growing evidence supports the importance of these signalling pathways in painful disc disease. With continuing research exploiting in vitro and in vivo model systems to elucidate the roles of these pathways in disc degeneration, it may be possible in the near future to specifically target these major inflammatory / catabolic signalling pathways to treat painful degenerative disc disease. In this perspective, we aim to summarise the current state of knowledge concerning the inflammatory and catabolic molecular pathways of intervertebral disc disease (IDD, with a detailed description of NF-kB and MAP kinase-mediated signal transduction in disc cells. Furthermore, we will discuss the emerging novel molecular treatment modalities for IDD using pharmacological inhibitors targeting these pathways.

  11. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    Science.gov (United States)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  12. A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

    Science.gov (United States)

    Kusari, Anasua B; Molina, Douglas M; Sabbagh, Walid; Lau, Chang S; Bardwell, Lee

    2004-01-19

    The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans. PMID:14734536

  13. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Science.gov (United States)

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells. PMID:26406906

  14. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Science.gov (United States)

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  15. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes.

    Directory of Open Access Journals (Sweden)

    Katrin Witte

    Full Text Available Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630 on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.

  16. Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

    DEFF Research Database (Denmark)

    Cayla, M.; Rachidi, N.; Leclercq, O.;

    2014-01-01

    Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even...... though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage...... at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10...

  17. Mapping moves on Arabidopsis

    OpenAIRE

    Kloth, K.J.

    2016-01-01

                    Aphids are pest insects in a wide variety of crops. They penetrate the host plant in order to feed on the sugar-rich phloem sap. Apart from the removal of nutrients from the host, they can transmit plant viruses. To control aphid populations and virus transmission in agriculture, insecticides are applied on a large scale with often negative effects on biodiversity. Improvement of crop resistance to aphids would be a more sustainable pes...

  18. The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway

    NARCIS (Netherlands)

    Birkenkamp, KU; Tuyt, LML; Lummen, C; Wierenga, LTJ; Kruijer, W; Vellenga, E

    2000-01-01

    1 In the present study we investigated a possible role for the p38 mitogen-activated protein (MAP) kinase pathway in mediating nuclear factor-kappa B (NF-kappa B) transcriptional activity in the erythroleukaemic cell line TF-1. 2 TF-1 cells stimulated with the phosphatase inhibitor okadaic acid (OA)

  19. Synergistic effect of vasoactive intestinal peptides on TNF-alpha-induced IL-6 synthesis in osteoblasts: amplification of p44/p42 MAP kinase activation.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Mizutani, Jun; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Minamitani, Chiho; Kato, Kenji; Kozawa, Osamu; Otsuka, Takanobu

    2010-05-01

    We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of vasoactive intestinal peptide (VIP) on TNF-alpha-induced IL-6 synthesis in these cells. VIP, which by itself slightly stimulated IL-6 synthesis, synergistically enhanced the TNF-alpha-induced IL-6 synthesis in MC3T3-E1 cells. The synergistic effect of VIP on the TNF-alpha-induced IL-6 synthesis was concentration-dependent in the range between 1 and 70 nM. We previously reported that VIP stimulated cAMP production in MC3T3-E1 cells. Forskolin, a direct activator of adenylyl cyclase, or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP), a plasma membrane-permeable cAMP analogue, markedly enhanced the TNF-alpha-induced IL-6 synthesis as well as VIP. VIP markedly up-regulated the TNF-alpha-induced p44/p42 MAP kinase phosphorylation. The Akt phosphorylation stimulated by TNF-alpha was only slightly affected by VIP. PD98059, a specific inhibitor of MEK1/2, significantly suppressed the enhancement of TNF-alpha-induced IL-6 synthesis by VIP. The synergistic effect of a combination of VIP and TNF-alpha on the phosphorylation of p44/p42 MAP kinase was diminished by H-89, an inhibitor of cAMP-dependent protein kinase. These results strongly suggest that VIP synergistically enhances TNF-alpha-stimulated IL-6 synthesis via up-regulating p44/p42 MAP kinase through the adenylyl cyclase-cAMP system in osteoblasts.

  20. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    Science.gov (United States)

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR RLK) genetic…

  1. UVB-irradiated human keratinocytes and interleukin-1αindirectly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradiated dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-yong; BI Zhi-gang

    2006-01-01

    Background Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1α on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins)mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.Methods Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1α. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).Results Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1α increased MAP kinase activity and c-Jun mRNA expression,IL-1 α also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1 α increased MMP-1 production in UVA-irradiated fibroblasts.Conclusions UVB-irradiated keratinocytes and IL-1α indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

  2. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

    OpenAIRE

    Bunone, G; Briand, P A; Miksicek, R J; Picard, D.

    1996-01-01

    The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary...

  3. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    Science.gov (United States)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  4. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress.

    Science.gov (United States)

    Zou, Jun-Jie; Li, Xi-Dong; Ratnasekera, Disna; Wang, Cun; Liu, Wen-Xin; Song, Lian-Fen; Zhang, Wen-Zheng; Wu, Wei-Hua

    2015-05-01

    Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)- and Ca(2+)-mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H2O2-, and Ca(2+)-induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H2O2 compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca(2+) inhibition of inward K(+) currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity. PMID:25966761

  5. The LysM Receptor-Like Kinase LysM RLK1 Is Required to Activate Defense and Abiotic-Stress Responses Induced by Overexpression of Fungal Chitinases in Arabidopsis Plants

    Institute of Scientific and Technical Information of China (English)

    Yariv Brotman; Ada Viterbo; Udi Landau; Smadar Pnini; Jan Lisec; Salma Balazadeh; Bernd Mueller-Roeber; Aviah Zilberstein; Lothar Willmitzer; Ilan Chet

    2012-01-01

    Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene,known to play a critical role in signaling defense responses induced by exogenous chitin.Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity,heavy-metal stresses,and Botrytis cinerea infection.Resistant lines,overexpressing fungal chitinases at different levels,were outcrossed to lysm rlk1 mutants.Independent homozygous hybrids lost resistance to biotic and abiotic stresses,despite enhanced chitinase activity.Expression analysis of 270 stress-related genes,including those induced by reactive oxygen species (ROS) and chitin,revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation.These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.

  6. Mapping of Tonoplast Intrinsic Proteins in Maturing and Germinating Arabidopsis Seeds Reveals Dual Localization of Embryonic TIPS to the Tonoplast and Plasma Membrane

    Institute of Scientific and Technical Information of China (English)

    Stefano Gattolin; Mathias Sorieul; Lorenzo Frigerio

    2011-01-01

    We have mapped the expression of the tonoplast intrinsic protein (TIP) gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms (TIP1;1, TIP2;1,and TIP2;2) have distinct patterns of expression in maternal tissues (outer integument and placento-chalazal region). Two isoforms, TIP3;1 and the previously uncharacterized TIP3;2, are the only detectable TIPS in embryos during seed maturation and the early stages of seed germination. Throughout these developmental stages, both isoforms co-locate to the tonoplast of the protein storage vacuoles, but also appear to label the plasma membrane. Plasma membrane labeling is specific to TIP3;1 and TIP3;2, is independent of the position of the fluorescent protein tag, and appears to be specific to early seed maturation and early germination stages. We discuss these results in the context of the predicted distribution of aquaporins in Arabidopsis seeds.

  7. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    Science.gov (United States)

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  8. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    Science.gov (United States)

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  9. Therapeutic potential of p38 MAP kinase inhibition in the management of cardiovascular disease.

    Science.gov (United States)

    Fisk, Marie; Gajendragadkar, Parag R; Mäki-Petäjä, Kaisa M; Wilkinson, Ian B; Cheriyan, Joseph

    2014-06-01

    p38 mitogen-activated protein kinases (p38 MAPKs) are key signalling molecules that regulate cellular behavior in response to environmental stresses. They regulate pro-inflammatory cytokines and therefore p38 MAPKs are implicated in the pathogenesis of many inflammatory-driven conditions, including atherosclerosis. Therapeutic inhibition of p38 MAPKs to attenuate inflammation has been the focus of comprehensive research in the last 2 decades, following the discovery of p38α as the molecular target of pyrindinyl imidazole compounds, which suppress the cytokines tumor necrosis factor-α and interleukin-1. The potential of p38 MAPK inhibitors was initially explored within archetypal inflammatory conditions such as rheumatoid arthritis and Crohn's disease, but early studies demonstrated poor clinical efficacy and unacceptable side effects. Subsequent clinical trials evaluating different p38 MAPK inhibitor compounds in disease models such as chronic obstructive pulmonary disease (COPD) and atherosclerosis have shown potential clinical efficacy. This review aims to provide succinct background information regarding the p38 MAPK signaling pathway, a focus of p38 MAPKs in disease, and a brief summary of relevant pre-clinical studies. An update of human clinical trial experience encompassing a clinically orientated approach, dedicated to cardiovascular disease follows. It provides a current perspective of the therapeutic potential of p38 MAPK inhibitors in the cardiovascular domain, including safety, tolerability, and pharmacokinetics.

  10. Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses.

    Science.gov (United States)

    Igbaria, Aeid; Lev, Sophie; Rose, Mark S; Lee, Bee Na; Hadar, Ruthi; Degani, Ofir; Horwitz, Benjamin A

    2008-06-01

    Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways. PMID:18473669

  11. Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1)

    NARCIS (Netherlands)

    Russinova, E.T.; Borst, J.W.; Kwaaitaal, M.A.C.J.; Yanhai Yin, Y.; Caño-Delgrado, A.; Chory, J.; Vries, de S.C.

    2004-01-01

    In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 [AtSERK3]). Genetic, biochemical, and yeast (Sac

  12. Topical alpha-selective p38 MAP kinase inhibition reduces acute skin inflammation in guinea pig

    Directory of Open Access Journals (Sweden)

    Satyanarayana Medicherla

    2010-02-01

    Full Text Available Satyanarayana Medicherla, Jing Ying Ma, Mamtha Reddy, Irina Esikova, Irene Kerr, Fabiola Movius, Linda S Higgins, Andrew A ProtterScios Inc, Fremont, CA , USAAbstract: Certain skin pathologies, including psoriasis, are thought to be immune-mediated inflammatory diseases. Available literature clearly indicates the involvement of inflammatory cells (neutrophils, T cells, and macrophages, their cytokines, and the p38 mitogen-activated protein kinase (MAPK signaling pathway in the pathophysiology of psoriasis. Neutrophils play an important role in the formation of acute inflammatory changes in psoriasis. Acute inflammation or acute flares in psoriasis remain poorly addressed in clinical medicine. In this communication, we first establish a simple and reproducible model for studying neutrophil-mediated acute skin inflammation. Using the hairless guinea pig, due to the similarity of skin architecture to that of human, acute inflammation was induced with an intradermal injection of 50 μg/mL lipopolysaccharide (LPS in 50 μL solution. Myeloperoxidase (MPO activity was measured by MPO-positive neutrophils and shown to increase for 24-hours post-injection. Simultaneously, the level of phosphorylated p38 MAPK was documented for 48-hours post-LPS injection in the skin. Next, we used this model to examine the therapeutic potential of an α-selective p38 MAPK inhibitor, SCIO-469. A comparison of topical application of SCIO-469 at 5 mg/mL or 15 mg/mL to vehicle revealed that SCIO-469 dose-dependently reduces acute skin inflammation and that this effect is statistically significant at the higher dose. Further examination of tissues that received this dose also revealed statistically significant reduction of MPO activity, phosphorylated p38 MAPK, interleukin-6, and cyclooxygenase-2. These data suggest that the α-selective p38 MAPK inhibitor, SCIO-469, acts as a topical anti-inflammatory agent via the p38 MAPK pathway to reduce neutrophil induced acute

  13. The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma

    LENUS (Irish Health Repository)

    Keld, Richard

    2010-12-09

    Abstract Background Many members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers. Results Here, we have studied the expression of the PEA3 subfamily members PEA3\\/ETV4 and ER81\\/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas. Conclusions This study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

  14. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. PMID:24491552

  15. [Roles of the cross talk between MAP kinases and Keap1-Nrf2-ARE signaling pathways in chronic obstructive pulmonary disease].

    Science.gov (United States)

    Wang, Shu-Jun; Chen, Ya-Jun; Wang, Shan-Shan; Wang, Dian-Lei; Wang, Chen-Yin; Yang, Li-Li; Chen, Jin-Pei

    2015-02-01

    Chronic obstructive pulmonary disease (COPD), a common preventable and treatable disease, is characterized by airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways. Its main pathological manifestations include airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. Recent research suggests that MAP kinases and Keap1-Nrf2-ARE signaling pathway are involved in the pathological process of inflammation and oxidative stress. This review explores the potential role of the cross talk of these signaling pathways in airway inflammation, mucus hypersecretion, oxidative stress and apoptotic epithelial cells. To clarify the roles of cross talk between MAP kinases and Keap1-Nrf2-ARE signaling pathway, we also focus on the drugs related to that in the treatment of COPD, and it provides ideas for more drug research in the treatment of COPD.

  16. Arabidopsis CDS blastp result: AK105724 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105724 001-201-G07 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bisph...osphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

  17. Arabidopsis CDS blastp result: AK072243 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072243 J023003N10 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bispho...sphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

  18. Localization of the human stress responsive MAP kinase-like CSAIDs binding protein (CSBP) gene to chromosome 6p21.3/21.2

    Energy Technology Data Exchange (ETDEWEB)

    McDonnell, P.C.; Young, P.R.; DiLella, A.G. [SmithKline Beecham Pharmaceuticals, King of Prussia, PA (United States)] [and others

    1995-09-01

    The proinflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) play a pivotal role in the initiation of inflammatory responses. Soluble protein antagonists of IL-1 and TNF, such as IL-1ra, sTNFR-Fc fusion, and monoclonal antibodies to TNF have proven to be effective at blocking acute and chronic responses in a number of animal models of inflammatory diseases such as rheumatoid arthritis, septic shock, and inflammatory bowel disease. Consequently, there has been considerable interest in discovering compounds that could inhibit the production of these cytokines and might therefore become treatments. Recently, a structurally related series of pyridinyl imidazoles was found to block IL-1 and TNF production from LPS-stimulated human monocytes and to ameliorate inflammatory diseases significantly in vivo, leading to their being named CSAIDs (cytokine suppressive anti-inflammatory drugs). The protein target of these compounds, termed CSBP (CSAID binding protein), was discovered to be a new member of the MAP kinase family of serine-threonine protein kinases whose kinase activity is activated by LPS in human monocytes. Independently, the same kinase, or its rodent homologues, was found to respond also to chemical, thermal, and osmotic stress and IL-1 treatment. Inhibition of this kinase correlated with reduction in inflammatory cytokine production from LPS-activated monocytes. 15 refs., 1 fig.

  19. Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

    OpenAIRE

    Burghoorn, Jan; Dekkers, Martijn P. J.; Rademakers, Suzanne; De Jong, Ton; Willemsen, Rob; Jansen, Gert

    2007-01-01

    In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in ...

  20. The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola.

    Science.gov (United States)

    Cho, Yangrae; Cramer, Robert A; Kim, Kwang-Hyung; Davis, Josh; Mitchell, Thomas K; Figuli, Patricia; Pryor, Barry M; Lemasters, Emily; Lawrence, Christopher B

    2007-06-01

    Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway. PMID:17280842

  1. Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP markers: homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes

    Directory of Open Access Journals (Sweden)

    Gupta Vibha

    2008-03-01

    Full Text Available Abstract Background Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome and A. thaliana and analyzed the arrangement of 24 (previously described genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes. Results IP markers were highly efficient and generated easily discernable polymorphisms on agarose gels. Comparative analysis of the segmental organization of the A and B genomes of B. juncea (present study with the A and B genomes of B. napus and B. nigra respectively (described earlier, revealed a high degree of colinearity suggesting minimal macro-level changes after polyploidization. The ancestral block arrangements that remained unaltered during evolution and the karyotype rearrangements that originated in the Oleracea lineage after its divergence from Rapa lineage were identified. Genomic rearrangements leading to the gain or loss of one chromosome each between the A-B and A-C lineages were deciphered. Complete homoeology in terms of block organization was found between three linkage groups (LG each for the A-B and A-C genomes. Based on the homoeology shared between the A, B and C genomes, a new nomenclature for the B genome LGs was assigned to establish uniformity in the international Brassica LG nomenclature code. Conclusion IP markers were highly effective in generating comparative relationships between Arabidopsis and various Brassica species. Comparative genomics between the three Brassica lineages established the major rearrangements

  2. A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

    Energy Technology Data Exchange (ETDEWEB)

    Nishina, Atsuyoshi, E-mail: nishina@yone.ac.jp [Yonezawa Women' s Junior College, 6-15-1 Tohrimachi, Yonezawa, Yamagata 992-0025 (Japan); Kimura, Hirokazu; Kozawa, Kunihisa [Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki, Maebashi, Gunma 371-0052 (Japan); Sommen, Geoffroy [Lonza Braine SA, Chaussee de Tubize 297, B-1420 Braine l' Alleud (Belgium); Nakamura, Takao [Department of Biomedical Information Engineering, Graduate School of Medical Science, Yamagata University, Yamagata 990-9585 (Japan); Heimgartner, Heinz [University of Zuerich, Institut of Organic Chemistry, Winterthurerstrasse 190, CH-8057 Zuerich (Switzerland); Koketsu, Mamoru [Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan); Furukawa, Shoei [Laboratory of Molecular Biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585 (Japan)

    2011-12-15

    Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) at 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to

  3. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Aniek van der Vaart

    2015-12-01

    Full Text Available Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL, resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis.

  4. Microglial Signaling in Chronic Pain with a Special Focus on Caspase 6, p38 MAP Kinase, and Sex Dependence.

    Science.gov (United States)

    Berta, T; Qadri, Y J; Chen, G; Ji, R R

    2016-09-01

    Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some

  5. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans.

    Science.gov (United States)

    van der Vaart, Aniek; Rademakers, Suzanne; Jansen, Gert

    2015-12-01

    Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis.

  6. AthaMap web tools for the analysis of transcriptional and posttranscriptional regulation of gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Hehl, Reinhard; Bülow, Lorenz

    2014-01-01

    The AthaMap database provides a map of verified and predicted transcription factor (TF) and small RNA-binding sites for the A. thaliana genome. The database can be used for bioinformatic predictions of putative regulatory sites. Several online web tools are available that address specific questions. Starting with the identification of transcription factor-binding sites (TFBS) in any gene of interest, colocalizing TFBS can be identified as well as common TFBS in a set of user-provided genes. Furthermore, genes can be identified that are potentially targeted by specific transcription factors or small inhibitory RNAs. This chapter provides detailed information on how each AthaMap web tool can be used online. Examples on how this database is used to address questions in circadian and diurnal regulation are given. Furthermore, complementary databases and databases that go beyond questions addressed with AthaMap are discussed. PMID:24792049

  7. 拟南芥PKS5激酶磷酸化ABI5参与植物ABA响应%PKS5 Kinase is Involved in ABA Response through Phosphorylating ABI5 in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    赵菲佚; 焦成瑾; 陈荃; 王太术; 田春芳; 高雅梅

    2015-01-01

    植物激素脱落酸(abscisic acid, ABA)在植物生长、发育及环境胁迫中起着重要的作用。本研究发现拟南芥PKSes (SOS2-like protein kinases)蛋白激酶家族成员PKS5(SOS2-like protein kinase 5)参与植物ABA响应。PKS5功能获得性点突变体pks5-3与pks5-4表现出对ABA的敏感表型。在外源ABA处理下, pks5-3与pks5-4种子萌发率降低,幼苗生长矮小、黄化。体外磷酸化测试显示, PKS5特异磷酸化ABA响应元件ABI5(ABA-insensitive 5) N末端多肽(1~211 aa)。qRT-PCR分析表明pks5-3与pks5-4突变体中ABI5下游ABA响应基因RAB18(RESPONSIVE TO ABA18)与EM6(LATE EMBRYOGENESIS ABUNDANT 6)表达均发生改变。这些研究结果表明,拟南芥PKS5通过磷酸化ABI5的N末端参与植物ABA响应过程。%The phytohormone abscisic acid (ABA) plays an essential role in plant growth and development as well as abiotic stress responses. In this study, we found that PKS5 (SOS2-like protein kinase 5), a family mem-ber of PKSes (SOS2-like protein kinases), involved in ABA response inArabidopsis.PKS5 gain-of-function point mutantspks5-3andpks5-4 exhibited hypersensitive to ABA in the phenotypic test. Additionally, seed ger-minations ofpks5-3andpks5-4 decreased and seedlings of them showed stunted growth and leaf chlorotic symptoms under exogenous ABA treatment.In vitro phosphorylation assay indicated that PKS5 speciifcally phosphorylates the ABA-responsive component ABI5 (ABA-insensitive 5) N terminus fragment range from 1 to 211 amino acids. Moreover, the relative expression levels ofRAB18 andEM6, which are the downstream ABA-responsive genes of ABI5, signiifcantly altered inpks5-3andpks5-4 mutants compared with the wild-type plants by quantitative real-time PCR using gene-speciifc primers. Taken together, the results of this study revealed that PKS5 involves in ABA response via phosphorylating the N-terminus of ABI5 in Arabidopsis.

  8. Inducible and targeted deletion of the ERK5 MAP kinase in adult neurogenic regions impairs adult neurogenesis in the olfactory bulb and several forms of olfactory behavior.

    Directory of Open Access Journals (Sweden)

    Yung-Wei Pan

    Full Text Available Although adult-born neurons in the subventricular zone (SVZ and olfactory bulb (OB have been extensively characterized at the cellular level, their functional impact on olfactory behavior is still highly controversial with many conflicting results reported in the literature. Furthermore, signaling mechanisms regulating adult SVZ/OB neurogenesis are not well defined. Here we report that inducible and targeted deletion of erk5, a MAP kinase selectively expressed in the adult neurogenic regions of the adult brain, impairs adult neurogenesis in the SVZ and OB of transgenic mice. Although erk5 deletion had no effect on olfactory discrimination among discrete odorants in the habituation/dishabituation assay, it reduced short-term olfactory memory as well as detection sensitivity to odorants and pheromones including those evoking aggression and fear. Furthermore, these mice show impaired acquisition of odor-cued associative olfactory learning, a novel phenotype that had not been previously linked to adult neurogenesis. These data suggest that ERK5 MAP kinase is a critical kinase signaling pathway regulating adult neurogenesis in the SVZ/OB, and provide strong evidence supporting a functional role for adult neurogenesis in several distinct forms of olfactory behavior.

  9. Genome wide association mapping in Arabidopsis thaliana identifies novel genes involved in linking allyl glucosinolate to altered biomass and defense

    Directory of Open Access Journals (Sweden)

    Marta Francisco

    2016-07-01

    Full Text Available A key limitation in modern biology is the ability to rapidly identify genes underlying newly identified complex phenotypes. Genome wide association studies (GWAS have become an increasingly important approach for dissecting natural variation by associating phenotypes with genotypes at a genome wide level. Recent work is showing that the Arabidopsis thaliana defense metabolite, allyl glucosinolate (GSL, may provide direct feedback regulation, linking defense metabolism outputs to the growth and defense responses of the plant. However, there is still a need to identify genes that underlie this process. To start developing a deeper understanding of the mechanism(s that modulate the ability of exogenous allyl GSL to alter growth and defense, we measured changes in plant biomass and defense metabolites in a collection of natural 96 A. thaliana accessions fed with 50 µM of allyl GSL. Exogenous allyl GSL was introduced exclusively to the roots and the compound transported to the leaf leading to a wide range of heritable effects upon plant biomass and endogenous GSL accumulation. Using natural variation we conducted GWAS to identify a number of new genes which potentially control allyl responses in various plant processes. This is one of the first instances in which this approach has been successfully utilized to begin dissecting a novel phenotype to the underlying molecular/polygenic basis.

  10. p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

    NARCIS (Netherlands)

    Gosens, Reinoud; Dueck, Gordon; Gerthoffer, William T; Unruh, Helmut; Zaagsma, Johan; Meurs, Herman; Halayko, Andrew J

    2007-01-01

    Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, ho

  11. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway

    DEFF Research Database (Denmark)

    Migliaccio, E; Mele, S; Salcini, A E;

    1997-01-01

    66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms...... on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.......Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have...

  12. Ca(2+ permeable AMPA receptor induced long-term potentiation requires PI3/MAP kinases but not Ca/CaM-dependent kinase II.

    Directory of Open Access Journals (Sweden)

    Suhail Asrar

    Full Text Available Ca(2+ influx via GluR2-lacking Ca(2+-permeable AMPA glutamate receptors (CP-AMPARs can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP through a number of different induction protocols, including high-frequency stimulation (HFS and theta-burst stimulation (TBS. This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca(2+ ions through calcium chelator (BAPTA studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII, the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase (LY294002 and wortmannin or the MAPK cascade (PD98059 and U0126 significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces

  13. Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.

    Directory of Open Access Journals (Sweden)

    Mathieu Cayla

    2014-09-01

    Full Text Available Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10. Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

  14. Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Fuchs Angelika

    2007-03-01

    Full Text Available Abstract Background Receptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function. Results We approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis

  15. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    Science.gov (United States)

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

  16. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    Science.gov (United States)

    Gruber, Sabine; Zeilinger, Susanne

    2014-01-01

    Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

  17. Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

    Science.gov (United States)

    Adderley, Shaquria P; Lawrence, Curtis; Madonia, Eyong; Olubadewo, Joseph O; Breslin, Jerome W

    2015-07-01

    The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity. PMID:25948734

  18. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    Science.gov (United States)

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  19. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    Science.gov (United States)

    Gruber, Sabine; Zeilinger, Susanne

    2014-01-01

    Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12. PMID:25356841

  20. The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

    Directory of Open Access Journals (Sweden)

    Sabine Gruber

    Full Text Available Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

  1. The Calcineurin B-Like Calcium Sensors CBL1 and CBL9 Together with Their Interacting Protein Kinase CIPK26 Regulate the Arabidopsis NADPH Oxidase RBOHF

    Institute of Scientific and Technical Information of China (English)

    Maria Magdalena Drerup; Kathrin Schlücking; Kenji Hashimoto; Prabha Manishankar; Leonie Steinhorst; Kazuyuki Kuchitsu; J(o)rg Kudla

    2013-01-01

    Stimulus-specific accumulation of second messengers like reactive oxygen species (ROS) and Ca2+ are central to many signaling and regulation processes in plants.However,mechanisms that govern the reciprocal interrelation of Ca2+ and ROS signaling are only beginning to emerge.NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS while Calcineurin B-like (CBL) Ca2+ sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca2+-signaling processes.In this study,we identify direct functional interactions between both signaling systems.We report that the CBL-interacting protein kinase ClPK26 specifically interacts with the N-terminal domain of RBOHF in yeast two-hybrid analyses and with the full-length RBOHF protein in plant cells.In addition,CIPK26 phosphorylates RBOHF in vitro and co-expression of either CBL1 or CBL9 with CIPK26 strongly enhances ROS production by RBOHF in HEK293T cells.Together,these findings identify a direct interconnection between CBL-ClPK-mediated Ca2+ signaling and ROS signaling in plants and provide evidence for a synergistic activation of the NADPH oxidase RBOHF by direct Ca2+-binding to its EF-hands and Ca2+-induced phosphorylation by CBL1/9-ClPK26 complexes.

  2. An extracellular subtilase switch for immune priming in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Vicente Ramírez

    Full Text Available In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.

  3. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen.

    Science.gov (United States)

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane. PMID:26500635

  4. FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

    Directory of Open Access Journals (Sweden)

    Chengkang eZhang

    2015-10-01

    Full Text Available Fusarium verticillioides (formerly F. moniliforme is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

  5. Saucerneol F, a New Lignan Isolated from Saururus chinensis, Attenuates Degranulation via Phospholipase Cγ 1 Inhibition and Eicosanoid Generation by Suppressing MAP Kinases in Mast Cells.

    Science.gov (United States)

    Lu, Yue; Son, Jong-Keun; Chang, Hyeun Wook

    2012-11-01

    During our on-going studies to identify bioactive compounds in medicinal herbs, we found that saucerneol F (SF), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis), showed in vitro anti-inflammatory activity. In this study, we examined the effects of SF on the generation of 5-lipoxygenase (5-LO) dependent leukotriene C4 (LTC4), cyclooxygenase-2 (COX-2) dependent prostaglandin D2 (PGD2), and on phospholipase Cγ1 (PLCγ1)-mediated degranulation in SCF-induced mouse bone marrow-derived mast cells (BMMCs). SF inhibited eicosanoid (PGD2 and LTC4) generation and degranulation dose-dependently. To identify the molecular mechanisms underlying the inhibition of eicosanoid generation and degranulation by SF, we examined the effects of SF on the phosphorylation of PLCγ1, intracellular Ca(2+) influx, the translocation of cytosolic phospholipase A2 (cPLA2) and 5-LO, and on the phosphorylation of MAP kinases (MAPKs). SF was found to reduce intracellular Ca(2+) influx by inhibiting PLCγ1 phosphorylation and suppressing the nuclear translocations of cPLA2 and 5-LO via the phosphorylations of MAPKs, including extracellular signal-regulated protein kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Taken together, these results suggest that SF may be useful for regulating mast cell-mediated inflammatory responses by inhibiting degranulation and eicosanoid generation. PMID:24009845

  6. Involvement of OST1 Protein Kinase and PYR/PYL/RCAR Receptors in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Guard Cells.

    Science.gov (United States)

    Yin, Ye; Adachi, Yuji; Nakamura, Yoshimasa; Munemasa, Shintaro; Mori, Izumi C; Murata, Yoshiyuki

    2016-08-01

    Methyl jasmonate (MeJA) induces stomatal closure. It has been shown that stomata of many ABA-insensitive mutants are also insensitive to MeJA, and a low amount of ABA is a prerequisite for the MeJA response. However, the molecular mechanisms of the interaction between ABA and MeJA signaling remain to be elucidated. Here we studied the interplay of signaling of the two hormones in guard cells using the quadruple ABA receptor mutant pyr1 pyl1 pyl2 pyl4 and ABA-activated protein kinase mutants ost1-2 and srk2e. In the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, nitric oxide (NO) production, cytosolic alkalization and plasma membrane Ca(2+)-permeable current (ICa) activation were not impaired. At the same time, the inactivation of the inward-rectifying K(+) current was impaired. In contrast to the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, NO production and cytosolic alkalization were impaired in ost1-2 and srk2e as well as in aba2-2, the ABA-deficient mutant. The activation of ICa was also impaired in srk2e. Collectively, these results indicated that OST1 was essential for MeJA-induced stomatal closure, while PYR1, PYL1, PYL2 and PYL4 ABA receptors were not sufficient factors. MeJA did not appear to activate OST1 kinase activity. This implies that OST1 mediates MeJA signaling through an undetectable level of activity or a non-enzymatic action. MeJA induced the expression of an ABA synthesis gene, NCED3, and increased ABA contents only modestly. Taken together with previous reports, this study suggests that MeJA signaling in guard cells is primed by ABA and is not brought about through the pathway mediated by PYR1, PYL1 PYL2 and PYL4. PMID:27354421

  7. Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

    Science.gov (United States)

    Zhu, Shun; Travis, Sue M; Elcock, Adrian H

    2013-07-01

    A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant

  8. Specific MAP-kinase blockade protects against renal damage in homozygous TGR(mRen2)27 rats

    NARCIS (Netherlands)

    de Borst, MH; Navis, G; de Boer, RA; Vis, LM; van Gilst, WH; van Goor, H

    2003-01-01

    Angiotensin II (AngII) plays an important role in renal damage by acting on hemodynamics, cell-growth, proliferation, and fibrosis, mainly by effects on the AngII type 1 (AT,) receptor. The AT, receptor activates several intracellular signaling molecules such as mitogen-activated protein kinases ext

  9. Mechanical stress triggers cardiomyocyte autophagy through angiotensin II type 1 receptor-mediated p38MAP kinase independently of angiotensin II.

    Directory of Open Access Journals (Sweden)

    Li Lin

    Full Text Available Angiotensin II (Ang II type 1 (AT1 receptor is known to mediate a variety of physiological actions of Ang II including autophagy. However, the role of AT1 receptor in cardiomyocyte autophagy triggered by mechanical stress still remains elusive. The aim of this study was therefore to examine whether and how AT1 receptor participates in cardiomyocyte autophagy induced by mechanical stresses. A 48-hour mechanical stretch and a 4-week transverse aorta constriction (TAC were imposed to cultured cardiomyocytes of neonatal rats and adult male C57B/L6 mice, respectively, to induce cardiomyocyte hypertrophy prior to the assessment of cardiomyocyte autophagy using LC3b-II. Losartan, an AT1 receptor blocker, but not PD123319, the AT2 inhibitor, was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Moreover, inhibition of p38MAP kinase attenuated not only mechanical stretch-induced cardiomyocyte hypertrophy but also autophagy. To the contrary, inhibition of ERK and JNK suppressed cardiac hypertrophy but not autophagy. Intriguingly, mechanical stretch-induced autophagy was significantly inhibited by Losartan in the absence of Ang II. Taken together, our results indicate that mechanical stress triggers cardiomyocyte autophagy through AT1 receptor-mediated activation of p38MAP kinase independently of Ang II.

  10. Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

    Science.gov (United States)

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-01-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

  11. A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae.

    Science.gov (United States)

    Moriwaki, Akihiro; Kihara, Junichi; Mori, Chie; Arase, Sakae

    2007-01-01

    We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae. PMID:16546358

  12. Bakuchiol suppresses proliferation of skin cancer cells by directly targeting Hck, Blk, and p38 MAP kinase.

    Science.gov (United States)

    Kim, Jong-Eun; Kim, Jae Hwan; Lee, Younghyun; Yang, Hee; Heo, Yong-Seok; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2016-03-22

    Bakuchiol is a meroterpene present in the medicinal plant Psoralea corylifolia, which has been traditionally used in China, India, Japan and Korea for the treatment of premature ejaculation, knee pain, alopecia spermatorrhea, enuresis, backache, pollakiuria, vitiligo, callus, and psoriasis. Here, we report the chemopreventive properties of bakuchiol, which acts by inhibiting epidermal growth factor (EGF)-induced neoplastic cell transformation. Bakuchiol also decreased viability and inhibited anchorage-independent growth of A431 human epithelial carcinoma cells. Bakuchiol reduced A431 xenograft tumor growth in an in vivo mouse model. Using kinase profiling, we identified Hck, Blk and p38 mitogen activated protein kinase (MAPK) as targets of bakuchiol, which directly bound to each kinase in an ATP-competitive manner. Bakuchiol also inhibited EGF-induced signaling pathways downstream of Hck, Blk and p38 MAPK, including the MEK/ERKs, p38 MAPK/MSK1 and AKT/p70S6K pathways. This report is the first mechanistic study identifying molecular targets for the anticancer activity of bakuchiol and our findings indicate that bakuchiol exhibits potent anticancer activity by targeting Hck, Blk and p38 MAPK. PMID:26910280

  13. Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

    Science.gov (United States)

    Li, Geoffrey C; Srivastava, Atul K; Kim, Jonggul; Taylor, Susan S; Veglia, Gianluigi

    2015-07-01

    Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold. PMID:26030372

  14. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    Science.gov (United States)

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis. PMID:27457979

  15. Exploiting Natural Variation in Arabidopsis

    NARCIS (Netherlands)

    Molenaar, J.A.; Keurentjes, J.J.B.

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana . This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of

  16. Exploiting natural variation in Arabidopsis

    NARCIS (Netherlands)

    J.A. Molenaar; J.J.B. Keurentjes

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana. This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of g

  17. Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

    Energy Technology Data Exchange (ETDEWEB)

    Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Joseph, Binoy [Spinal Cord and Brain Injury Research Center and Department of Physiology, University of Kentucky, Lexington, KY 40536-0509 (United States); Hitron, John Andrew; Wang, Lei [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Kim, Donghern [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Yin, Yuanqin [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Cancer Institute, The First Affiliated Hospital, China Medical University, Shenyang (China); Roy, Ram Vinod [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Lu, Jian [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Zhang, Zhuo [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Wang, Yitao [State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau (China); and others

    2014-10-01

    Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE{sub 2} and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE{sub 2} production. • C3G

  18. Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

    International Nuclear Information System (INIS)

    Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE2 production. • C3G inhibited UVB

  19. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Science.gov (United States)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  20. Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases

    Science.gov (United States)

    Derbré, Frédéric; Droguet, Mickaël; Léon, Karelle; Troadec, Samuel; Pennec, Jean-Pierre; Giroux-Metges, Marie-Agnès; Rannou, Fabrice

    2016-01-01

    Purpose Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. Methods The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. Results MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill’s model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1–36.0) and 39.3% (95% CI 37.0–41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. Conclusions Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization. PMID:27383612

  1. Arabidopsis CDS blastp result: AK241519 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241519 J065170E12 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 3e-23 ...

  2. Arabidopsis CDS blastp result: AK242651 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242651 J090026B08 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-16 ...

  3. Arabidopsis CDS blastp result: AK243050 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243050 J100011E04 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 2e-24 ...

  4. Arabidopsis CDS blastp result: AK242271 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242271 J075187A19 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 4e-17 ...

  5. Arabidopsis CDS blastp result: AK240655 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240655 J023135E11 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-40 ...

  6. Arabidopsis CDS blastp result: AK242638 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242638 J090023J02 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-29 ...

  7. Arabidopsis CDS blastp result: AK242681 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242681 J090032N04 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 8e-38 ...

  8. Arabidopsis CDS blastp result: AK288923 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288923 J090081P06 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-59 ...

  9. Arabidopsis CDS blastp result: AK243187 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243187 J100039E11 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 4e-24 ...

  10. Arabidopsis CDS blastp result: AK111785 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111785 J023089N11 At5g62310.1 incomplete root hair elongation (IRE) / protein kin...ase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 0.0 ...

  11. Arabidopsis CDS blastp result: AK288095 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288095 J075191E21 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 9e-31 ...

  12. Arabidopsis CDS blastp result: AK242859 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242859 J090073L24 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 2e-21 ...

  13. Arabidopsis CDS blastp result: AK242717 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242717 J090043H19 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-23 ...

  14. Arabidopsis CDS blastp result: AK287631 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287631 J065073J24 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 2e-35 ...

  15. Arabidopsis CDS blastp result: AK242733 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242733 J090047O22 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 2e-24 ...

  16. Arabidopsis CDS blastp result: AK242758 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242758 J090051H03 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 1e-59 ...

  17. Arabidopsis CDS blastp result: AK243656 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243656 J100088L22 At5g62310.1 68418.m07822 incomplete root hair elongation (IRE) .../ protein kinase, putative nearly identical to IRE (incomplete root hair elongation) [Arabidopsis thaliana] gi|6729346|dbj|BAA89783 6e-29 ...

  18. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  19. Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of pavlovian fear conditioning.

    Science.gov (United States)

    Schafe, G E; Atkins, C M; Swank, M W; Bauer, E P; Sweatt, J D; LeDoux, J E

    2000-11-01

    Although much has been learned about the neurobiological mechanisms underlying Pavlovian fear conditioning at the systems and cellular levels, relatively little is known about the molecular mechanisms underlying fear memory consolidation. The present experiments evaluated the role of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade in the amygdala during Pavlovian fear conditioning. We first show that ERK/MAPK is transiently activated-phosphorylated in the amygdala, specifically the lateral nucleus (LA), at 60 min, but not 15, 30, or 180 min, after conditioning, and that this activation is attributable to paired presentations of tone and shock rather than to nonassociative auditory stimulation, foot shock sensitization, or unpaired tone-shock presentations. We next show that infusions of U0126, an inhibitor of ERK/MAPK activation, aimed at the LA, dose-dependently impair long-term memory of Pavlovian fear conditioning but leaves short-term memory intact. Finally, we show that bath application of U0126 impairs long-term potentiation in the LA in vitro. Collectively, these results demonstrate that ERK/MAPK activation is necessary for both memory consolidation of Pavlovian fear conditioning and synaptic plasticity in the amygdala.

  20. Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A new MAPK gene, ZmSIMK1 (Zea mays L. salt-induced mitogen-activated protein kinase 1), is isolated from a maize cDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of encoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 cDNA into pET-42a(+), and transformed into E. coli strain BL21(DE3). A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1mmol/L IPTG.

  1. IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components.

    Science.gov (United States)

    Ariel, Amiram; Novick, Daniela; Rubinstein, Menachem; Dinarello, Charles A; Lider, Ofer; Hershkoviz, Rami

    2002-07-01

    Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, beta(1) integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-gamma (INF-gamma) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-gamma production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites. PMID:12101280

  2. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    Science.gov (United States)

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  3. The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.

    Science.gov (United States)

    Good, Matthew; Tang, Grace; Singleton, Julie; Reményi, Attila; Lim, Wendell A

    2009-03-20

    The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3's activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7. PMID:19303851

  4. 22(R)-hydroxycholesterol induces HuR-dependent MAP kinase phosphatase-1 expression via mGluR5-mediated Ca(2+)/PKCα signaling.

    Science.gov (United States)

    Kim, Hyunmi; Woo, Joo Hong; Lee, Jee Hoon; Joe, Eun-Hye; Jou, Ilo

    2016-08-01

    MAP kinase phosphatase (MKP)-1 plays a pivotal role in controlling MAP kinase (MAPK)-dependent (patho) physiological processes. Although MKP-1 gene expression is tightly regulated at multiple levels, the underlying mechanistic details remain largely unknown. In this study, we demonstrate that MKP-1 expression is regulated at the post-transcriptional level by 22(R)-hydroxycholesterol [22(R)-HC] through a novel mechanism. 22(R)-HC induces Hu antigen R (HuR) phosphorylation, cytoplasmic translocation and binding to MKP-1 mRNA, resulting in stabilization of MKP-1 mRNA. The resulting increase in MKP-1 leads to suppression of JNK-mediated inflammatory responses in brain astrocytes. We further demonstrate that 22(R)-HC-induced phosphorylation of nuclear HuR is mediated by PKCα, which is activated in the cytosol by increases in intracellular Ca(2+) levels mediated by the phospholipase C/inositol 1,4,5-triphosphate receptor (PLC/IP3R) pathway and translocates from cytoplasm to nucleus. In addition, pharmacological interventions reveal that metabotropic glutamate receptor5 (mGluR5) is responsible for the increases in intracellular Ca(2+) that underlie these actions of 22(R)-HC. Collectively, our findings identify a novel anti-inflammatory mechanism of 22(R)-HC, which acts through PKCα-mediated cytoplasmic shuttling of HuR to post-transcriptionally regulate MKP-1 expression. These findings provide an experimental basis for the development of a RNA-targeted therapeutic agent to control MAPK-dependent inflammatory responses. PMID:27206966

  5. Gamma-linolenic and stearidonic acids are required for basal immunity in Caenorhabditis elegans through their effects on p38 MAP kinase activity.

    Directory of Open Access Journals (Sweden)

    Madhumitha Nandakumar

    2008-11-01

    Full Text Available Polyunsaturated fatty acids (PUFAs form a class of essential micronutrients that play a vital role in development, cardiovascular health, and immunity. The influence of lipids on the immune response is both complex and diverse, with multiple studies pointing to the beneficial effects of long-chain fatty acids in immunity. However, the mechanisms through which PUFAs modulate innate immunity and the effects of PUFA deficiencies on innate immune functions remain to be clarified. Using the Caenorhabditis elegans-Pseudomonas aeruginosa host-pathogen system, we present genetic evidence that a Delta6-desaturase FAT-3, through its two 18-carbon products--gamma-linolenic acid (GLA, 18:3n6 and stearidonic acid (SDA, 18:4n3, but not the 20-carbon PUFAs arachidonic acid (AA, 20:4n6 and eicosapentaenoic acid (EPA, 20:5n3--is required for basal innate immunity in vivo. Deficiencies in GLA and SDA result in increased susceptibility to bacterial infection, which is associated with reduced basal expression of a number of immune-specific genes--including spp-1, lys-7, and lys-2--that encode antimicrobial peptides. GLA and SDA are required to maintain basal activity of the p38 MAP kinase pathway, which plays important roles in protecting metazoan animals from infections and oxidative stress. Transcriptional and functional analyses of fat-3-regulated genes revealed that fat-3 is required in the intestine to regulate the expression of infection- and stress-response genes, and that distinct sets of genes are specifically required for immune function and oxidative stress response. Our study thus uncovers a mechanism by which these 18-carbon PUFAs affect basal innate immune function and, consequently, the ability of an organism to defend itself against bacterial infections. The conservation of p38 MAP kinase signaling in both stress and immune responses further encourages exploring the function of GLA and SDA in humans.

  6. Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

    International Nuclear Information System (INIS)

    Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 μM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A4 (LTA4) hydrolase and leukotriene C4 (LTC4) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA4 hydrolase and LTC4 synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

  7. Pharmacological profile of AW-814141, a novel, potent, selective and orally active inhibitor of p38 MAP kinase

    DEFF Research Database (Denmark)

    Chopra, Puneet; Kulkarni, Onkar; Gupta, Shashank;

    2010-01-01

    The p38 mitogen activated protein kinase (MAPK) is a key signaling molecule that plays a crucial role in the progression of various inflammatory diseases such as rheumatoid arthritis (RA), asthma and chronic obstructive pulmonary disease. The objective of the present study was to evaluate the anti...... and it displays promising in vitro and in vivo anti-inflammatory activities and can be used for the treatment of rheumatoid arthritis....... and collagen-induced arthritis model (CIA), AW-814141 dose dependently inhibited paw swelling. In different in vivo efficacy models, efficacy of AW-814141 was found to be better as compared to the reference compounds (Vx-745 and BIRB-796). This study demonstrated that AW-814141 is a novel p38 MAPK inhibitor...

  8. p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

    Science.gov (United States)

    Hori, Takeshi; Moore, Rick

    2016-01-01

    Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38. PMID:27074912

  9. Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

    Science.gov (United States)

    Rispail, Nicolas; Soanes, Darren M; Ant, Cemile; Czajkowski, Robert; Grünler, Anke; Huguet, Romain; Perez-Nadales, Elena; Poli, Anna; Sartorel, Elodie; Valiante, Vito; Yang, Meng; Beffa, Roland; Brakhage, Axel A; Gow, Neil A R; Kahmann, Regine; Lebrun, Marc-Henri; Lenasi, Helena; Perez-Martin, José; Talbot, Nicholas J; Wendland, Jürgen; Di Pietro, Antonio

    2009-04-01

    Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches. PMID:19570501

  10. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae. PMID:27193947

  11. P38 MAP kinase inhibitors as potential therapeutics for the treatment of joint degeneration and pain associated with osteoarthritis

    Directory of Open Access Journals (Sweden)

    Taiwo Yetunde O

    2008-12-01

    Full Text Available Abstract Background Evaluate the potential role of p38 inhibitors for the treatment of osteoarthritis using an animal model of joint degeneration (iodoacetate-induced arthritis and a pain model (Hargraeves assay. Methods P38 kinase activity was evaluated in a kinase assay by measuring the amount of phosphorylated substrate ATF2 using a phosphoATF2 (Thr71 specific primary antibody and an alkaline phosphate coupled secondary antibody and measuring the OD at 405 nm. TNFα and IL-1β secretion from LPS stimulated THP-1 monocytic cells and human peripheral blood mononuclear cells were measured by ELISA. Rats treated with vehicle or p38 inhibitor were injected intra-articularly in one knee with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The effect of p38 inhibitors on hyperalgesia was evaluated in rats given an intraplantar injection of carrageenan and 4 h later the paw withdrawal time to a radiant heat source was measured. Results SB-203580 and VX-745 are both potent inhibitors of p38 with IC50s of 136 ± 64 nM and 35 ± 14 nM (mean ± S.D., respectively. Similarly, SB-203580 and VX-745 potently inhibited TNF release from LPS stimulated human THP-1 cells with IC50s of 72 ± 15 nM; and 29 ± 14 nM (mean ± S.D. respectively. TNF release from LPS stimulated human peripheral blood mononuclear cells was inhibited with IC50s 16 ± 6 nM and 14 ± 8 nM, (mean ± S.D. for SB-203580 and VX-745 and IL-1 was inhibited with IC50s of 20 ± 8 nM and 15 ± 4 nM (mean ± S.D., respectively. SB-203580 and VX-745 administered orally at a dose of 50 mg/kg resulted in the significant (p Conclusion SB203580 and VX-745 demonstrated attenuation of both cartilage degeneration and pain in animal models and suggest that p38 inhibitors may be a useful approach for the treatment of osteoarthritis.

  12. Shock Waves Increase T-cell Proliferation or IL-2 Expression by Activating p38 MAP Kinase

    Institute of Scientific and Technical Information of China (English)

    Tie-Cheng YU; Yi LIU; Yan TAN; Yanfang JIANG; Xueqing ZHENG; Xinxiang XU

    2004-01-01

    Shock waves were elicited by transient pressure disturbances, which could be used to treat musculoskeletal disorders. In present studies, we i. nvestigated whether the low-density shock waves (LDSWs), which are able to damage plasma membrane without impairing the vimentin or other organelles, might augment T-cell proliferation as well as IL-2 expression, and if mitogen activated protein kinase p38 (p38 MAPK)might be an underlying mechanism through which the LDSWs enhanced T-cell function. We found that the LDSWs increased activation of p38 MAPK in Jurkat T cells. The LDSWs alone didn't result in the T-cell proliferation and IL-2 expression. However, in combination with other stimuli, LDSWs could augment the T-cell proliferation and IL-2 expression. Inhibition of p38 MAPK using SB203580 reduced the stimulatory effects of the LDSWs, which indicated that the LDSWs enhanced IL-2 expression through a mechanism that involved p38 MAPK activation. We concluded that the p38 MAPK activation played a key role in the regulation of T cell function by the LDSWs.

  13. Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

    Institute of Scientific and Technical Information of China (English)

    Liu Q; Chen T; Chen H; Zhang M; Li N; Lu Z; Ma P; Cao X

    2004-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F. , has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part,to its apoptosis-inducing effects on DCs.

  14. Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway.

    Directory of Open Access Journals (Sweden)

    Milene Carmes Vallejo

    Full Text Available In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.

  15. Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

    Science.gov (United States)

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Grollman, Arthur P; Rosenquist, Thomas

    2015-01-01

    Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells. PMID:24792323

  16. Regulation of IκBα expression involves both NF-κB and the MAP kinase signaling pathways

    Directory of Open Access Journals (Sweden)

    Sambucetti Lidia C

    2005-10-01

    Full Text Available Abstract IκBα is an inhibitor of the nuclear transcription factor NF-κB. Binding of IκBα to NF-κB inactivates the transcriptional activity of NF-κB. Expression of IκBα itself is regulated by NF-κB, which provides auto-regulation of this signaling pathway. Here we present a mouse model for monitoring in vivo IκBα expression by imaging IκBα-luc transgenic mice for IκBα promoter driven luciferase activity. We demonstrated a rapid and systemic induction of IκBα expression in the transgenic mice following treatment with LPS. The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain. The luciferase induction in the liver correlated with increased IκBα mRNA level. Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity. The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity. Analysis of IκBα mRNA in the liver tissue showed a surprising increase of the IκBα mRNA after bortezomib and SB203580 treatments, which could be due to increased IκBα mRNA stability. Our data demonstrate that regulation of IκBα expression involves both the NF-κB and the p38 signaling pathways. The IκBα-luc transgenic mice are useful for analyzing IκBα expression and the NF-κB transcriptional activity in vivo.

  17. Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress

    Science.gov (United States)

    O’Meara, Teresa R.; Valaei, Seyedeh Fereshteh; Diezmann, Stephanie; Cowen, Leah E.

    2016-01-01

    Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90’s functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90

  18. Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-κB pathway

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    Wang Bao-Wei

    2010-03-01

    Full Text Available Abstract Background Toll-like receptor 4 (TLR4 plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. Methods Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected. Results Cyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-α antibody, and p38α MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-κB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-κB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-α antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-α and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-α antibody, or p38α siRNA 30 min before Ang II and TNF-α stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-α. Conclusions Cyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF

  19. Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin

    Energy Technology Data Exchange (ETDEWEB)

    Divya, Sasidharan Padmaja; Wang, Xin; Pratheeshkumar, Poyil; Son, Young-Ok; Roy, Ram Vinod [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Kim, Donghern; Dai, Jin [Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Hitron, John Andrew; Wang, Lei [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Asha, Padmaja [National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Cochin (India); Shi, Xianglin [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Zhang, Zhuo, E-mail: zhuo.zhang@uky.edu [Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States)

    2015-04-01

    Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm{sup 2}) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E{sub 2} (PGE{sub 2}), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways. - Highlights: • Blackberry extract inhibits UVB-induced glutathione depletion.

  20. The role of EGFR/ERK/ELK-1 MAP kinase pathway in the underlying damage to diabetic rat skin

    Directory of Open Access Journals (Sweden)

    Xinhong Ge

    2013-01-01

    Full Text Available Background: Diabetes mellitus (DM is a highly prevalent disease. Atrophy and spontaneous ulcers are the most common cutaneous manifestation of diabetic dermopathy (DD. Before spontaneous ulcers, we believe there is an underlying damage stage although the mechanism is unknown. Aims: To explore the expression of extracellular signal-regulated kinase1/2 (ERK1/2, its correlated upstream protein epidermal growth factor receptor (EGFR and its downstream transcription factor E twenty-six (ETS-like 1(ELK-1in the damage of the diabetic rat skin, and to explore the role of ERK1/2 on the recessive damage to diabetic rat skin. Materials and Methods: Eighty Sprague-Dawley (SD rats weighing 260-300 g were randomly divided into control and streptozotocin (STZ-induced diabetes groups. After 0.5, 2, 4, and 8 weeks, the shaved skin specimens from the back of rats in both groups were collected to observe the histological characteristics of the skin, to measure the thickness of the epidermis and the dermis, and to observe the ultrastructure. Immunohistochemistry (IHC and Western blot techniques were used to detect the expression and activation of ERK1/2, EGFR, ELK-1 in the skin of the rats. Results: There are ultrastructural changes in the DM skin. With the continuance of the diabetes course, the thicknesses of the epidermis and dermis decreased, and the expression of phospho-ERK1/2 (P-ERK1/2, EGFR, and ELK-1 was decreased gradually in the back skin of the diabetes rats. It was significantly lower in 4 and 8 week DM than that of the normal control ( P < 0.05. The expression of P-EGFR and P-ERK1/2 in the back skin of the diabetes rats was positively correlated ( r = 0.572 P < 0.05, and the positive correlation was also obtained between P-ERK1/2 and P-ELK-1 ( r = 0.715, P < 0.05. Conclusion: The phenomenon of recessive damage exists in the skin of diabetes rats, which probably may relate to the weakness of the signal transduction: P-EGFR → ERK1/2 → ELK-1.

  1. Immunomodulation by the Pseudomonas syringae HopZ type III effector family in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Jennifer D Lewis

    Full Text Available Pseudomonas syringae employs a type III secretion system to inject 20-30 different type III effector (T3SE proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family members and characterized them for immune suppression phenotypes. We show that all of the HopZ family members can actively suppress various facets of Arabidopsis immunity in a catalytic residue-dependent manner. HopZ family members can differentially suppress the activation of mitogen-activated protein (MAP kinase cascades or the production of reactive oxygen species, whereas all members can promote the growth of non-virulent P. syringae. Localization studies show that four of the HopZ family members containing predicted myristoylation sites are localized to the vicinity of the plasma membrane while HopZ3 which lacks the myristoylation site is at least partially nuclear localized, suggesting diversification of immunosuppressive mechanisms. Overall, we demonstrate that despite significant evolutionary diversification, all HopZ family members can suppress immunity in Arabidopsis.

  2. Are free energy calculations useful in practice? A comparison with rapid scoring functions for the p38 MAP kinase protein system.

    Science.gov (United States)

    Pearlman, D A; Charifson, P S

    2001-10-11

    Precise thermodynamic integration free energy simulations have been applied to a congeneric series of 16 inhibitors to the p38 MAP kinase protein for which the experimental binding data (IC(50)) is known. The relative free energy of binding for each compound has been determined. For comparison, the same series of compounds have also scored using the best rapid scoring functions used in database screening. From the results of these calculations, we find (1) that precise free energy simulations allow predictions that are reliable and in good agreement with experiment; (2) that predictions of lower reliability, but still in good qualitative agreement with experiment, can be obtained using the OWFEG free energy grid method, at a much lower computational cost; (3) and that other methods, not based on free energy simulations yield results in much poorer agreement with experiment. A new predictive index, which measures the reliability of a prediction method in the context of normal use, is defined and calculated for each scoring method. Predictive indices of 0.84, 0.56, 0.04, -0.05, and 0.25 are calculated for thermodynamic integration, OWFEG, ChemScore, PLPScore, and Dock Energy Score, respectively, where +1.0 is perfect correct prediction, -1.0 is perfect incorrect prediction, and 0.0 is random.

  3. Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans.

    Science.gov (United States)

    Burghoorn, Jan; Dekkers, Martijn P J; Rademakers, Suzanne; de Jong, Ton; Willemsen, Rob; Jansen, Gert

    2007-04-24

    In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. PMID:17420466

  4. Involvement of the H1 histamine receptor, p38 MAP kinase, MLCK, and Rho/ROCK in histamine-induced endothelial barrier dysfunction

    Science.gov (United States)

    Adderley, Shaquria P.; Zhang, Xun E.; Breslin, Jerome W.

    2015-01-01

    Objective The mechanisms by which histamine increases microvascular permeability remain poorly understood. We tested the hypothesis that H1 receptor activation disrupts the endothelial barrier and investigated potential downstream signals. Methods We used confluent endothelial cell (EC) monolayers, assessing transendothelial electrical resistance (TER) as an index of barrier function. Human umbilical vein EC (HUVEC), cardiac microvascular EC (HCMEC), and dermal microvascular EC (HDMEC) were compared. Receptor expression was investigated using Western blotting, immunofluorescence (IF) confocal microscopy and RT-PCR. Receptor function and downstream signaling pathways were tested using pharmacologic antagonists and inhibitors, respectively. Results We identified H1-H4 receptors on all three EC types. H1 antagonists did not affect basal TER but prevented the histamine-induced decrease in TER. Blockade of H2 or H3 attenuated the histamine response only in HDMEC, while inhibition of H4 attenuated the response only in HUVEC. Combined inhibition of both PKC and PI3K caused exaggerated histamine-induced barrier dysfunction in HDMEC, whereas inhibition of p38 MAP kinase attenuated the histamine response in all three EC types. Inhibition of RhoA, ROCK, or MLCK also prevented the histamine-induced decrease in TER in HDMEC. Conclusion The data suggest that multiple signaling pathways contribute to histamine-induced endothelial barrier dysfunction via the H1 receptor. PMID:25582918

  5. Isoflavonoid-Rich Flemingia macrophylla Extract Attenuates UVB-Induced Skin Damage by Scavenging Reactive Oxygen Species and Inhibiting MAP Kinase and MMP Expression

    Directory of Open Access Journals (Sweden)

    Hsiu-Mei Chiang

    2013-01-01

    Full Text Available In this study, we investigated the antioxidant activity and anti-photoaging properties of an extract of Flemingia macrophylla, a plant rich in isoflavonoid content. Pretreatment of fibroblasts with Flemingia macrophylla extract (FME inhibited elastase activity, promoted the protein expression of type I procollagen, and attenuated the phosphorylation of mitogen-activated protein (MAP kinase and the protein expression of matrix-metalloproteinase- (MMP- 1, 3, and 9. The IC50 values were 2.1 μg/mL for DPPH radical scavenging ability, 366.8 μg/mL for superoxide anion scavenging ability, 178.9 μg/mL for hydrogen peroxide scavenging ability, and 230.9 μg/mL for hydroxyl radical scavenging ability. Also, exposure of erythrocytes to various concentrations of FME (50–500 μg/mL resulted in a dose- and time-dependent inhibition of AAPH-induced hemolysis. In human fibroblasts, FME at 10 μg/mL was shown to be a potent scavenger of UV-induced reactive oxygen species (ROS. The antioxidant and anti-photoaging properties of FME make it an ideal anti-intrinsic aging and anti-photoaging agent.

  6. The FUS3/KSS1-type MAP kinase gene FPK1 is involved in hyphal growth, conidiation and plant infection of Fusarium proliferatum.

    Science.gov (United States)

    Zhao, Pei-Bao; Ren, Ai-Zhi; Li, Duo-Chuan

    2011-01-01

    Fusarium proliferatum is an important pathogen of maize that is responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade, F. proliferatum has caused several severe epidemics of maize seedling blight in many areas of China, which led to significant losses in maize. To understand the molecular mechanisms in the fungal developmental regulation and pathogenicity, we isolated and characterized the FPK1 gene (GenBank accession No. HQ844224) encoding a MAP kinase homolog of FUS3/KSS1 in yeast. The gene includes a 1,242-bp DNA sequence from ATG to TAA, with a coding region of 1,068 bp, 3 introns (58 bp, 56 bp and 60 bp) and a predicted protein of 355 aa.The mutant ΔFPK1, which has a disruption of the FPK1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and spore germination, as well as deviant germ tube outgrowth. When the strain was inoculated in susceptible maize varieties, the infection of the mutant ΔFPK1 was delayed, and the infection efficiency was reduced compared to the wild-type strain. Complementation of the disruptions within the FPK1 open reading frame restored wild-type levels of conidiation, growth rate and virulence to maize seedlings. Our results indicated that the FPK1 gene functioned in hyphal growth, conidiation, spore germination and virulence in F. proliferatum. PMID:22286038

  7. Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor.

    Science.gov (United States)

    Yamamoto, Katsuyoshi; Tatebayashi, Kazuo; Tanaka, Keiichiro; Saito, Haruo

    2010-10-01

    Membrane localization of the Ste11 MAPKKK is essential for activation of both the filamentous growth/invasive growth (FG/IG) MAP kinase (MAPK) pathway and the SHO1 branch of the osmoregulatory HOG MAPK pathway, and is mediated by binding of the Ste50 scaffold protein to the Opy2 membrane anchor. We found that Opy2 has two major (CR-A and CR-B), and one minor (CR-D), binding sites for Ste50. CR-A binds Ste50 constitutively and can transmit signals to both the Hog1 and Fus3/Kss1 MAPKs. CR-B, in contrast, binds Ste50 only when Opy2 is phosphorylated by Yck1/Yck2 under glucose-rich conditions and transmits the signal preferentially to the Hog1 MAPK. Ste50 phosphorylation by activated Hog1/Fus3/Kss1 MAPKs downregulates the HOG MAPK pathway by dissociating Ste50 from Opy2. Furthermore, Ste50 phosphorylation, together with MAPK-specific protein phosphatases, reduces the basal activity of the HOG and the mating MAPK pathways. Thus, dynamic regulation of Ste50-Opy2 interaction fine-tunes the MAPK signaling network. PMID:20932477

  8. Comparative analysis of drought resistance genes in Arabidopsis and rice

    NARCIS (Netherlands)

    Trijatmiko, K.R.

    2005-01-01

    Keywords: rice, Arabidopsis, drought, genetic mapping,microarray, transcription factor, AP2/ERF, SHINE, wax, stomata, comparative genetics, activation tagging, Ac/Ds, En/IThis thesis describes the use of genomics information and tools from Arabidopsis and

  9. Pseudomonas syringae Effector HopF2 Suppresses Arabidopsis Immunity by Targeting BAK1

    Science.gov (United States)

    Zhou, Jinggeng; Wu, Shujing; Chen, Xin; Liu, Chenglong; Sheen, Jen; Shan, Libo; He, Ping

    2014-01-01

    Summary Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe-associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of MAMP-activated MAP kinase (MPK) cascades. Besides blocking MKK5 (MPK kinase 5) activation in the MEKK1/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 or two other P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild-type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to further support that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells. PMID:24237140

  10. Cardiac myosin binding protein C and MAP-kinase activating death domain-containing gene polymorphisms and diastolic heart failure.

    Directory of Open Access Journals (Sweden)

    Cho-Kai Wu

    Full Text Available OBJECTIVE: Myosin binding protein C (MYBPC3 plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3 gene polymorphisms and diastolic heart failure (DHF in a human case-control study. METHODS: A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥ 5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD structure of the MYBPC3 gene. RESULTS: In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031. The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004 for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013, corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89; permuted p = 0.029. CONCLUSIONS: We identified a SNP (rs2290149 among the tagging SNP set that was significantly associated with early DHF in a Chinese population.

  11. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity

    Science.gov (United States)

    The Homo sapiens and Arabidopsis thaliana genomes are believed to encode >500 and >1,000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Mass spectrometry (MS)-based approaches have been integral to the large-scale mapp...

  12. 拟南芥蛋白激酶 SnRK2.6的原核表达、纯化及活性分析%Prokaryotic Expression,Purification and Activity Analysis of Protein Kinase SnRK2·6 in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    杨凤博; 王鲜萍; 李坤

    2014-01-01

    从哥伦比亚生态型拟南芥(Arabidopsis thaliana Columbia 0)中克隆 SnRK2·6[SNF1(su-crose non-fermenting-1)-related protein kinase 2·6]的完整编码序列(coding sequence,CDS),构建该基因的原核表达载体,将其转化 BL21(DE3),经表达纯化得到 SnRK2·6蛋白。激酶活性分析发现,原核表达纯化的 SnRK2·6有自磷酸化和磷酸化 MBP(myelin basin protein)的活性,为后续试验分析 SnRK2·6的功能奠定基础。%The whole coding sequence of SnRK2·6 [SNF1 (sucrose non-fermenting-1 )-related protein kinase 2·6]was cloned from Arabidopsis thaliana ecotype Columbia 0.The prokaryotic expression vector of the gene was constructed and transformed into Escherichia coli strain BL21 (DE3)to obtain SnRK2·6 protein by induction and purification.By activity analysis,the purified SnRK2·6 had both autophosphorylation activity and phosphorylation activity of the myelin basic protein(MBP)substrate.This study lays the foundation for further study of the function of SnRK2·6.

  13. Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

    Science.gov (United States)

    Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

    2013-07-26

    RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

  14. p38 MAP kinase-mediated NMDA receptor-dependent suppression of hippocampal hypersynchronicity in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Ittner, Arne A; Gladbach, Amadeus; Bertz, Josefine; Suh, Lisa S; Ittner, Lars M

    2014-01-01

    Hypersynchronicity of neuronal brain circuits is a feature of Alzheimer's disease (AD). Mouse models of AD expressing mutated forms of the amyloid-β precursor protein (APP), a central protein involved in AD pathology, show cortical hypersynchronicity. We studied hippocampal circuitry in APP23 transgenic mice using telemetric electroencephalography (EEG), at the age of onset of memory deficits. APP23 mice display spontaneous hypersynchronicity in the hippocampus including epileptiform spike trains. Furthermore, spectral contributions of hippocampal theta and gamma oscillations are compromised in APP23 mice, compared to non-transgenic controls. Using cross-frequency coupling analysis, we show that hippocampal gamma amplitude modulation by theta phase is markedly impaired in APP23 mice. Hippocampal hypersynchronicity and waveforms are differentially modulated by injection of riluzole and the non-competitive N-methyl-D-aspartate (NMDA) receptor inhibitor MK801, suggesting specific involvement of voltage-gated sodium channels and NMDA receptors in hypersynchronicity thresholds in APP23 mice. Furthermore, APP23 mice show marked activation of p38 mitogen-activated protein (MAP) kinase in hippocampus, and injection of MK801 but not riluzole reduces activation of p38 in the hippocampus. A p38 inhibitor induces hypersynchronicity in APP23 mice to a similar extent as MK801, thus supporting suppression of hypersynchronicity involves NMDA receptors-mediated p38 activity. In summary, we characterize components of hippocampal hypersynchronicity, waveform patterns and cross-frequency coupling in the APP23 mouse model by pharmacological modulation, furthering the understanding of epileptiform brain activity in AD.

  15. Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

    Science.gov (United States)

    Violet, Pierre-Christian; Billon-Denis, Emmanuelle; Robin, Philippe

    2012-11-01

    The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

  16. Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase1[C][W][OA

    Science.gov (United States)

    Bi, Dongling; Cheng, Yu Ti; Li, Xin; Zhang, Yuelin

    2010-01-01

    Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases. PMID:20508139

  17. Phosphorylation of ERK/MAP Kinase Is Required for Long-Term Potentiation in Anatomically Restricted Regions of the Lateral Amygdala in Vivo

    Science.gov (United States)

    Schafe, Glenn E.; Swank, Michael W.; Rodriguez, Sarina M.; Debiec, Jacek; Doyere, Valerie

    2008-01-01

    We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term…

  18. Tobacco-specific Carcinogen 4-(Methylnitrosoamino)-1-(3-pyridyl )-1-butanone(NNK) Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Hmnan Mammary Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Cigarette smoking is correlated with the development of various cancers. 4 - (Methylnitrosoamino) -1 - ( 3 -pyridyl) -1-butanone (NNK) is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.

  19. ‘MicroRNA Targets’, a new AthaMap web-tool for genome-wide identification of miRNA targets in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bülow Lorenz

    2012-07-01

    Full Text Available Background The AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation. Methods To identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome. Results Putative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, ‘MicroRNA Targets’, was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new ‘Small RNA Targets’ web-tool. Conclusions The integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/

  20. In vivo Evaluation of Two Thiazolidin-4-one Derivatives in High Sucrose Diet Fed Pre-diabetic Mice and Their Modulatory Effect on AMPK, Akt and p38 MAP Kinase in L6 Cells

    Science.gov (United States)

    Mudgal, Jayesh; Shetty, Priya; Reddy, Neetinkumar D.; Akhila, H. S.; Gourishetti, Karthik; Mathew, Geetha; Nayak, Pawan G.; Kumar, Nitesh; Kishore, Anoop; Kutty, Nampurath G.; Nandakumar, Krishnadas; Shenoy, Rekha R.; Rao, Chamallamudi M.; Joseph, Alex

    2016-01-01

    We had previously demonstrated the anti-diabetic potential and pancreatic protection of two thiazolidin-4-one derivatives containing nicotinamide moiety (NAT-1 and NAT-2) in STZ-induced diabetic mice. However, due to the limitations of the STZ model, we decided to undertake a detailed evaluation of anti-diabetic potential of the molecules on a high sucrose diet (HSD) fed diabetic mouse model. Further, in vitro mechanistic studies on the phosphorylation of AMPK, Akt and p38 MAP kinase in L6 myotubes and anti-inflammatory studies in RAW264.7 mouse monocyte macrophage cells were performed. 15 months of HSD induced fasting hyperglycaemia and impaired glucose tolerance in mice. Treatment with NAT-1 and NAT-2 (100 mg/kg) for 45 days significantly improved the glucose tolerance and lowered fasting blood glucose levels compared to untreated control. An improvement in the elevated triglycerides and total cholesterol levels, and favorable rise in HDL cholesterol were also observed with test drug treatment. Also, no major changes were observed in the liver (albumin, AST and ALT) and kidney (creatinine and urea) parameters. This was further confirmed in their respective histology profiles which revealed no gross morphological changes. In L6 cells, significant phosphorylation of Akt and p38 MAP kinase proteins were observed with 100 μM of NAT-1 and NAT-2 with no significant changes in phosphorylation of AMPK. The molecules failed to exhibit anti-inflammatory activity as observed by their effect on the generation of ROS and nitrite, and nuclear levels of NF-κB in LPS-stimulated RAW264.7 cells. In summary, the molecules activated Akt and p38 MAP kinase which could have partly contributed to their anti-hyperglycaemic and hypolipidemic activities in vivo. PMID:27790148

  1. Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones, and overexpression of PtMKK4 improves the drought tolerance of poplar.

    Science.gov (United States)

    Wang, Lei; Su, Hongyan; Han, Liya; Wang, Chuanqi; Sun, Yanlin; Liu, Fenghong

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H2O2 and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.

  2. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.; Hood, Molly M.; Lord, John W.; Lu, Wei-Ping; Miller, David F.; Patt, William C.; Smith, Bryan D.; Vogeti, Lakshminarayana; Kaufman, Michael D.; Petillo, Peter A.; Wise, Scott C.; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L. (Deciphera); (Emerald); (Cocrystal)

    2012-01-20

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  3. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

    Science.gov (United States)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

    2010-10-01

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  4. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Lohscheider, Jens N; Friso, Giulia; van Wijk, Klaas J

    2016-06-01

    Plastoglobules (PGs) are plastid lipid-protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  5. Proteomic Analysis of Two Brassica napus Differing in the Oil Contents and Map Alignment between the DEPs with Arabidopsis and B. rapa

    Institute of Scientific and Technical Information of China (English)

    Maoteng Li; Lu Gan; Xiaodong Wang; Dianrong Li; Hao Wang; Chunhua Fu; Longjiang Yu

    2012-01-01

    Rapeseed (Brassica napus) is one of the most important oilseed crops in the world.Improve the oil content is the main object for breeders at present.Oil contents is an important and complex metabolic trait of B.napus,in our previous studies,about 50 materials with oil content over 50% were obtained.Lots of studies revealed that some factors,such as seed coat,embryo,and endosperm that were all correlated with oil conten,but the studies focus on the proteomic studies on seed oil content were few reported.It is generally considered that the protein content is negatively correlated with oil content,which was further verified with QTL analysis for protein and oil content,Total and oil body protein in mature seeds of two B.napus cultivars with the oil content of 36.49% and 55.19% were analsized by using 2-DE technique.154 differentially expressed proteins (DEPs) have been successfully identified by using MALDI-TOF-MS method.The DEPs were mainly involved in Storage protein,Carbohydrate pathway protein,Cell Growth/Division protein,and Defense/Disease protein.Significant difference of cell growth/division protein both detected in total and oil body protein may be correlated to the size of oil body in B.napus with different oil content.The genome alignment analysis showed that among the 378 key genes,24 genes were underlying the N block,36 genes were underlying the U block and 17 genes were belong to the C block,then the 77 genes were used as candidate genes to do in silico mapping in A4 and C3 chromosomes of TN linkage map.For example,compared with the QTL mapping results and candidate genes,6 of the 24 genes underlying N block of A4 chromosome were mapped on the QTL qA4-1 confidence interval,including AT3G56350,AT3G57620,PGL34,AT3G58450,BGLU27 and BGLU30,and 5 genes were mapped on the QTL qOCDA-2 confidence interval,including AT3G52470,ATELP,MDAR1,FBA8 and AT3G53040.

  6. GmSARK启动子驱动IPT基因在拟南芥中的表达研究%Heterologous Expression of Isopentenyl Transferase(Ipt) Gene Directed by Senescence Associated Receptor Protein Kinase(SARK) Promoter in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    霍巍巍; 闫璇玲; 孙晓文; 李海燕

    2012-01-01

    [Objective] This study aimed to investigate the expression of IPT gene directed by GmSARK promoter in Arabidopsis. [ Method] IPT gene and GmSARK promoter were respectively cloned to construct plant expression vector and transform Arabidopsis. T, transgenic plant lines were detected by PPT(phosphinothricin) herbicide selection and treated under drought and dark conditions for semi-quantitive RT-PCR analysis. [ Result] GmSARK and IPT fusion gene was successfully cloned and the plant expression vector p3301 -GmSAHK-IPT was constructed. RT-PCR analysis showed that the target gene was expressed in T1 transgenic plant lines at the mRNA level. [ Conclusion] This study laid the foundation for further investigating the roles of IPT gene directed by GmSARK promoter in plant stress resistance.%[目的]对GmSARK启动子驱动IPT基因在拟南芥中的表达进行研究.[方法]分别克隆IPT基因及GmSARK基因启动子,构建它们的植物表达栽体并进行拟南芥的转化,利用PPT(phosphinothricin)除草剂筛选,检测T1代转基因植株;对T1代转基因植株进行黑暗避光和干旱处理后进行半定量RT-PCR分析.[结果]成功克隆得到了IPT基因及GmSARK基因,并构建了它们的p3301 -GmSARK-IPT植物表达载体;对T代转基因植株的RT-PCR表明,目的基因mRNA水平上有所表达.[结论]为进一步研究GmSARK启动子驱动的IPT基因在抗逆中的作用奠定了基础.

  7. Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM

    DEFF Research Database (Denmark)

    Hansen, L; Arden, K C; Rasmussen, S B;

    1997-01-01

    Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active ...

  8. The NO-cGMP-PKG Signaling Pathway Regulates Synaptic Plasticity and Fear Memory Consolidation in the Lateral Amygdala via Activation of ERK/MAP Kinase

    Science.gov (United States)

    Ota, Kristie T.; Pierre, Vicki J.; Ploski, Jonathan E.; Queen, Kaila; Schafe, Glenn E.

    2008-01-01

    Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and…

  9. Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

    Directory of Open Access Journals (Sweden)

    Soltis Pamela S

    2006-02-01

    Full Text Available Abstract Background The glycogen synthase kinase 3 (GSK3/SHAGGY-like kinases (GSKs are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences. Results We found that the structure of GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences. Conclusion Our results indicate that (1 the plant-specific GSK gene lineage was established early in the history of green plants, (2 plant GSKs began to diversify prior to the origin of extant seed plants, (3 three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4 diversification into four major clades (as initially reported in Arabidopsis occurred either just prior to the origin of the angiosperms or very early in angiosperm history.

  10. Pseudomonas aeruginosa pyocyanin activates NRF2-ARE-mediated transcriptional response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP kinase signaling in pulmonary epithelial cells.

    Science.gov (United States)

    Xu, Ying; Duan, Chaohui; Kuang, Zhizhou; Hao, Yonghua; Jeffries, Jayme L; Lau, Gee W

    2013-01-01

    The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.

  11. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  12. Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

    Directory of Open Access Journals (Sweden)

    Peter Dinér

    Full Text Available The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG pathway is a conserved mitogen-activated protein kinase (MAPK signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

  13. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation

    International Nuclear Information System (INIS)

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca2+]i increases which involved the mobilization of intracellular Ca2+ stored in the endoplasmic reticulum and Ca2+ influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent paroxetine-induced [Ca2+]i increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca2+-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation

  14. Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development

    Science.gov (United States)

    Cervantes, Emilio; Tocino, Angel

    2005-01-01

    Structurally, ethylene is the simplest phytohormone and regulates multiple aspects of plant growth and development. Its effects are mediated by a signal transduction cascade involving receptors, MAP kinases and transcription factors. Many morphological effects of ethylene in plant development, including root size, have been previously described. In this article a combined geometric and algebraic approach has been used to analyse the shape and the curvature in the root apex of Arabidopsis seedlings. The process requires the fitting of Bezier curves that reproduce the root apex shape, and the calculation of the corresponding curvatures. The application of the method has allowed us to identify significant differences in the root curvatures of ethylene insensitive mutants (ein2-1 and etr1-1) with respect to the wild-type Columbia.

  15. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    , no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-specific expression of CK-2 at the mRNA and at the protein level has also been given attention. The fact that the enzyme activity is surprisingly high in brain and low in heart and lung may be indicative of involvement of CK-2 in processes other than proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)...

  16. The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens

    DEFF Research Database (Denmark)

    Roux, Milena Edna; Schwessinger, Benjamin; Albrecht, Catherine;

    2011-01-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs...... and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae...... and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important...

  17. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development.

    Science.gov (United States)

    Dean, Derek M; Maroja, Luana S; Cottrill, Sarah; Bomkamp, Brent E; Westervelt, Kathleen A; Deitcher, David L

    2016-02-01

    Inositol 1,4,5-trisphosphate (IP3) regulates a host of biological processes from egg activation to cell death. When IP3-specific receptors (IP3Rs) bind to IP3, they release calcium from the ER into the cytoplasm, triggering a variety of cell type- and developmental stage-specific responses. Alternatively, inositol polyphosphate kinases can phosphorylate IP3; this limits IP3R activation by reducing IP3 levels, and also generates new signaling molecules altogether. These divergent pathways draw from the same IP3 pool yet cause very different cellular responses. Therefore, controlling the relative rates of IP3R activation vs. phosphorylation of IP3 is essential for proper cell functioning. Establishing a model system that sensitively reports the net output of IP3 signaling is crucial for identifying the controlling genes. Here we report that mutant alleles of wavy (wy), a classic locus of the fruit fly Drosophila melanogaster, map to IP3 3-kinase 2 (IP3K2), a member of the inositol polyphosphate kinase gene family. Mutations in wy disrupt wing structure in a highly specific pattern. RNAi experiments using GAL4 and GAL80(ts) indicated that IP3K2 function is required in the wing discs of early pupae for normal wing development. Gradations in the severity of the wy phenotype provide high-resolution readouts of IP3K2 function and of overall IP3 signaling, giving this system strong potential as a model for further study of the IP3 signaling network. In proof of concept, a dominant modifier screen revealed that mutations in IP3R strongly suppress the wy phenotype, suggesting that the wy phenotype results from reduced IP4 levels, and/or excessive IP3R signaling. PMID:26613949

  18. Inhibitory effects of pentosan polysulfate sodium on MAP-kinase pathway and NF-κB nuclear translocation in canine chondrocytes in vitro.

    Science.gov (United States)

    Sunaga, Takafumi; Oh, Namgil; Hosoya, Kenji; Takagi, Satoshi; Okumura, Masahiro

    2012-06-01

    Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.

  19. The ethylene signal transduction pathway in Arabidopsis

    Science.gov (United States)

    Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

  20. Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a Clinical p38[alpha] MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunjian; Lin, James; Wrobleski, Stephen T.; Lin, Shuqun; Hynes, Jr., John; Wu, Hong; Dyckman, Alaric J.; Li, Tianle; Wityak, John; Gillooly, Kathleen M.; Pitt, Sidney; Shen, Ding Ren; Zhang, Rosemary F.; McIntyre, Kim W.; Salter-Cid, Luisa; Shuster, David J.; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Sack, John S.; Kiefer, Susan E.; Kish, Kevin F.; Newitt, John A.; McKinnon, Murray; Dodd, John H.; Barrish, Joel C.; Schieven, Gary L.; Leftheris, Katerina (BMS)

    2013-11-20

    The discovery and characterization of 7k (BMS-582949), a highly selective p38{alpha} MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38{alpha} inhibitor. Unlike alkyl and other cycloalkyls, the sp{sup 2} character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38{alpha} enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38{alpha} was confirmed by X-ray crystallographic analysis.

  1. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    Science.gov (United States)

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  2. Fluoride-induced IL-8 release in human epithelial lung cells: Relationship to EGF-receptor-, SRC- and MAP-kinase activation

    International Nuclear Information System (INIS)

    Exposure of human epithelial lung cells to fluorides is known to induce a marked increase in the release of interleukin (IL)-8, a chemokine involved in neutrophil recruitment. In the present study, the involvement of mitogen-activating protein kinases (MAPKs), the role of upstream activation of Src family kinases (SFKs), epidermal growth factor receptor (EGFR) activation and the interrelationships between these pathways in fluoride-induced IL-8 were examined in a human epithelial lung cell line (A549). Sodium fluoride strongly activated MAPK, in particular JNK1/2 and p38. The ERK1/2-inhibitor PD98059, the p38-inhibitor SB202190 and the JNK1/2-inhibitor SP600125 partially inhibited the fluoride-induced IL-8 response. Combinations of these inhibitors reduced the responses nearly to basal levels. Treatment with siRNA against JNK2 also reduced the IL-8 response to fluoride. Furthermore, fluoride activated SFKs, which was abolished by the SFK-inhibitor PP2. PP2 substantially inhibited the increased levels of IL-8, and partially reduced the fluoride-induced activation of ERK1/2, p38 and JNK1/2. Fluoride exposure also led to a phosphorylation of the EGFR, that was partially inhibited by PP2. AG1478, an EGFR-inhibitor, partially reduced the fluoride-induced IL-8 response and the phosphorylation of JNK1/2 and ERK1/2, but less the phosphorylation of p38. The effects of AG1478 were less than that of PP2. In conclusion, our findings suggest that the fluoride-induced IL-8 release involves the combined activation of ERK1/2, JNK1/2 and p38, and that the phosphorylation of these kinases, and in particular JNK1/2 and ERK1/2, partly, is mediated via a SFK-dependent EGFR-linked pathway. SFK-dependent, but EGFR-independent mechanisms seem important, and especially for phosphorylation of p38

  3. The Role of Mitogen-Activated Protein (MAP Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana.

    Directory of Open Access Journals (Sweden)

    Yueqiang Leng

    Full Text Available Mitogen-activated protein kinases (MAPKs have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3 and one MAPK kinase kinase (MAPKKK gene (CsSTE11 were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal

  4. The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana.

    Science.gov (United States)

    Leng, Yueqiang; Zhong, Shaobin

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under

  5. TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis.

    Science.gov (United States)

    Li, Linxuan; Song, Yun; Wang, Kai; Dong, Pan; Zhang, Xueyan; Li, Fuguang; Li, Zhengguo; Ren, Maozhi

    2015-01-01

    Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, T O R - i nhibitor i n sensitive- 1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 μM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.

  6. Isoquercitrin suppresses the expression of histamine and pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells.

    Science.gov (United States)

    Li, Li; Zhang, Xiao-Hui; Liu, Guang-Rong; Liu, Chang; Dong, Yin-Mao

    2016-06-01

    Mast cells and basophils are multifunctional effector cells that contain abundant secretory granules in their cytoplasm. Both cell types are involved in a variety of inflammatory and immune events, producing an array of inflammatory mediators, such as cytokines. The aim of the study was to examine whether isoquercitrin modulates allergic and inflammatory reactions in the human basophilic KU812 cells and to elucidate its influence on the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation. The KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus the calcium ionophore A23187 (PMACI). The inhibitory effects of isoquercitrin on the productions of histamine and pro-inflammatory cytokines in the stimulated KU812 cells were measured using cytokine-specific enzyme-linked immunosorbent (ELISA) assays. Western blotting analysis was used to assess the effects of isoquercitrin on the MAPKs and NF-κB protein levels. Our results indicated that the isoquercitrin treatment of PMACI-stimulated KU812 cells significantly reduced the production of histamine and the pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, IL-1β, and tumor necrosis factor (TNF)-α. The treated cells exhibited decreased phosphorylation of extracellular signal-regulated kinase (ERK), revealing the role of ERK MAPK in isoquercitrin-mediated allergy inhibition. Furthermore, isoquercitrin suppressed the PMACI-mediated activation of NF-κB in the human basophil cells. In conclusion, the results from the present study provide insights into the potential therapeutic use of isoquercitrin for the treatment of inflammatory and allergic reactions. PMID:27473957

  7. Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

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    Lee Suk-Keun

    2007-09-01

    Full Text Available Abstract Background Interleukin-8 (IL-8 is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK and malignant human oral keratinocytes (HN12 cells with deferoxamine (DFO. Methods IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. Electrophoretic mobility shift assays was used to determine NF-κB binding activity. Phosphorylation and degradation of the I-κB were analyized by Western blot. Results IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1β to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IκB, and activation of NF-κB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione, and by pyrrolidine dithiocarbamate, an inhibitor of NF-κB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(PH oxidase. Conclusion This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

  8. Isoquercitrin suppresses the expression of histamine and pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells

    Institute of Scientific and Technical Information of China (English)

    LI Li; ZHANG Xiao-Hui; LIU Guang-Rong; LIU Chang; DONG Yin-Mao

    2016-01-01

    Mast cells and basophils are multifunctional effector cells that contain abundant secretory granules in their cytoplasm.Both cell types are involved in a variety of inflammatory and immune events,producing an array of inflammatory mediators,such as cytokines.The aim of the study was to examine whether isoquercitrin modulates allergic and inflammatory reactions in the human basophilic KU812 cells and to elucidate its influence on the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation.The KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus the calcium ionophore A23187 (PMACI).The inhibitory effects of isoquercitrin on the productions of histamine and pro-inflammatory cytokines in the stimulated KU812 cells were measured using cytokine-specific enzyme-linked immunosorbent (ELISA) assays.Western blotting analysis was used to assess the effects of isoquercitrin on the MAPKs and NF-κB protein levels.Our results indicated that the isoquercitrin treatment of PMACI-stimulated KU812 cells significantly reduced the production of histamine and the pro-inflammatory cytokines,such as interleukin (IL)-6,IL-8,IL-1β,and tumor necrosis factor (TNF)-α.The treated cells exhibited decreased phosphorylation of extracellular signal-regulated kinase (ERK),revealing the role of ERK MAPK in isoquercitrin-mediated allergy inhibition.Furthermore,isoquercitrin suppressed the PMACI-mediated activation of NF-κB in the human basophil cells.In conclusion,the results from the present study provide insights into the potential therapeutic use of isoquercitrin for the treatment of inflammatory and allergic reactions.

  9. Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

    International Nuclear Information System (INIS)

    Interleukin-8 (IL-8) is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK) and malignant human oral keratinocytes (HN12) cells with deferoxamine (DFO). IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Electrophoretic mobility shift assays was used to determine NF-κB binding activity. Phosphorylation and degradation of the I-κB were analyized by Western blot. IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1β to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IκB, and activation of NF-κB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione), and by pyrrolidine dithiocarbamate, an inhibitor of NF-κB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(P)H oxidase. This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions

  10. Mitogen-activated protein kinases in atherosclerosis

    Directory of Open Access Journals (Sweden)

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  11. Roles for farnesol and ABA in Arabidopsis flower development

    OpenAIRE

    Fitzpatrick, A. Heather; Shrestha, Nisha; Bhandari, Jayaram; Crowell, Dring N

    2011-01-01

    The Arabidopsis FOLK (At5g58560) gene encodes farnesol kinase, which phosphorylates farnesol to farnesyl phosphate. Loss-of-function mutations in the FOLK gene are associated with enhanced sensitivity to abscisic acid (ABA), suggesting that FOLK negatively regulates ABA signaling. Moreover, folk flowers develop supernumerary carpels under water stress, providing evidence for a molecular link between farnesol metabolism, abiotic stress signaling and flower development. Here, we show that farne...

  12. Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

    Directory of Open Access Journals (Sweden)

    Deyholos Michael K

    2006-10-01

    Full Text Available Abstract Background Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks. Results We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or down-regulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like; signalling molecules (e.g. PERK kinases, MLO-like receptors, carbohydrate active enzymes (e.g. XTH18, transcription factors (e.g. members of ZIM, WRKY, NAC, and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1. We verified the NaCl-inducible expression of selected transcription factors and other genes by qRT-PCR. Conclusion Micorarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent

  13. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

    Full Text Available Hee-Jeong Im,1–4 Andrew D Sharrocks,5 Xia Lin,6 Dongyao Yan,1 Jaesung Kim,1 Andre J van Wijnen,7 Robert A Hipskind81Departments of Biochemistry, 2Internal Medicine, 3Section of Rheumatology, Orthopedic Surgery, 4Rush University Medical Center, and Department of Bioengineering; University of Illinois at Chicago, IL USA; 5Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, UK; 6Michael D DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA; 7Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 8Institute De Genetique Moleculaire de Montpellier, FranceAbstract: Degradation of the extracellular matrix (ECM by matrix metalloproteinases (MMPs and release of basic fibroblast growth factor (bFGF are principal aspects of the pathology of osteoarthritis (OA. ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation

  14. N-Formyl peptides drive mitochondrial damage associated molecular pattern induced neutrophil activation through ERK1/2 and P38 MAP kinase signalling pathways.

    Science.gov (United States)

    Hazeldine, Jon; Hampson, Peter; Opoku, Francis Adusei; Foster, Mark; Lord, Janet M

    2015-01-01

    Traumatic injury results in a systemic inflammatory response syndrome (SIRS), a phenomenon characterised by the release of pro-inflammatory cytokines into the circulation and immune cell activation. Released from necrotic cells as a result of tissue damage, damage associated molecular patterns (DAMPs) are thought to initiate the SIRS response by activating circulating immune cells through surface expressed pathogen recognition receptors. Neutrophils, the most abundant leucocyte in human circulation, are heavily implicated in the initial immune response to traumatic injury and have been shown to elicit a robust functional response to DAMP stimulation. Here, we confirm that mitochondrial DAMPs (mtDAMPs) are potent activators of human neutrophils and show for the first time that signalling through the mitogen-activated-protein-kinases p38 and extracellular-signal-related-kinase 1/2 (ERK1/2) is essential for this response. At 40 and/or 100 μg/ml, mtDAMPs activated human neutrophils, indicated by a significant reduction in the surface expression of L-selectin, and triggered a number of functional responses from both resting and tumour necrosis factor-α primed neutrophils, which included reactive oxygen species (ROS) generation, degranulation, secretion of interleukin-8 and activation of p38 and ERK1/2 MAPKs. Pre-treatment of neutrophils with Cyclosporin H, a selective inhibitor of formyl peptide receptor-1 (FPR-1), significantly inhibited mtDAMP-induced L-selectin shedding as well as p38 and ERK1/2 activation, suggesting that N-formyl peptides are the main constituents driving mtDAMP-induced neutrophil activation. Indeed, no evidence of L-selectin shedding or p38 and ERK1/2 activation was observed in neutrophils challenged with mitochondrial DNA alone. Interestingly, pharmacological inhibition of p38 or ERK1/2 either alone or in combination significantly inhibited L-selectin shedding and IL-8 secretion by mtDAMP-challenged neutrophils, revealing for the first time

  15. Protein Kinase D family kinases

    OpenAIRE

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lo...

  16. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Science.gov (United States)

    Kong, Xiangpei; Lv, Wei; Zhang, Dan; Jiang, Shanshan; Zhang, Shizhong; Li, Dequan

    2013-01-01

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  17. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Directory of Open Access Journals (Sweden)

    Xiangpei Kong

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  18. Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation.

    Science.gov (United States)

    Agrawal, Ganesh K; Agrawal, Shyam K; Shibato, Junko; Iwahashi, Hitoshi; Rakwal, Randeep

    2003-01-17

    We report isolation of two novel rice (Oryza sativa L.) mitogen-activated protein kinases (MAPKs), OsMSRMK3 (multiple stress responsive) and OsWJUMK1 (wound- and JA-uninducible) that most likely exist as single copy genes in its genome. OsMSRMK3 and OsWJUMK1 encode 369 and 569 amino acid polypeptides having the MAPK family signature and phosphorylation activation motifs TEY and TDY, respectively. Steady state mRNA analyses of these MAPKs with constitutive expression in leaves of two-week-old seedlings revealed that OsMSRMK3 was up-regulated upon wounding (by cut), jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid, hydrogen peroxide (H(2)O(2)), protein phosphatase inhibitors, chitosan, high salt/sugar, and heavy metals, whereas OsWJUMK1 not induced by either wounding, JA or SA, showed up-regulation only by H(2)O(2), heavy metals, and cold stress (12 degrees C). Moreover, these MAPKs were developmentally regulated. These results strongly suggest a role for OsMSRMK3 and OsWJUMK1 in both stress-signalling pathways and development in rice. PMID:12507518

  19. Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Department of Laboratory Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Li, Juan [Department of Laboratory Medicine, Tianjin Medical University, 300070 Tianjin (China); Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China)

    2010-05-28

    Estrogen receptor alpha (ER{alpha}), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ER{alpha} activity and has been applied in breast cancer treatment. TAM-bound ER{alpha} associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ER{alpha} mediated signaling. We show that activated MAPK represses interaction of TAM-bound ER{alpha} with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ER{alpha} to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ER{alpha} activity via enhanced recruitment of SMRT, leading to reduced expression of ER{alpha} target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ER{alpha}, suggesting corepressor mediates inhibition of ER{alpha} transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

  20. Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

    Science.gov (United States)

    Zeng, Huawei; Botnen, James H; Briske-Anderson, Mary

    2010-01-01

    The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75-300 micromol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

  1. Novel Function of Serine Protease HTRA1 in Inhibiting Adipogenic Differentiation of Human Mesenchymal Stem Cells via MAP Kinase-Mediated MMP Upregulation.

    Science.gov (United States)

    Tiaden, André N; Bahrenberg, Gregor; Mirsaidi, Ali; Glanz, Stephan; Blüher, Matthias; Richards, Peter J

    2016-06-01

    Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614. PMID:26864869

  2. Biochemical and pharmacological assessment of MAP-kinase signaling along pain pathways in experimental rodent models: a potential tool for the discovery of novel antinociceptive therapeutics.

    Science.gov (United States)

    Edelmayer, Rebecca M; Brederson, Jill-Desiree; Jarvis, Michael F; Bitner, Robert S

    2014-02-01

    Injury to the peripheral or central nervous system can induce changes within the nervous tissues that promote a state of sensitization that may underlie conditions of pathological chronic pain. A key biochemical event in the initiation and maintenance of peripheral and central neuronal sensitization associated with chronic pain is the phosphorylation and subsequent activation of mitogen-activated protein kinases (MAPKs) and immediate early gene transcription factors, in particular cAMP-response element binding protein (CREB). In this commentary we review the preclinical data that describe anatomical and mechanistic aspects of nociceptive-induced signaling along nociceptive pathways including peripheral cutaneous axons, the dorsal root ganglia, spinal cord dorsal horn and cerebral cortex. In addition to the regional manifestation of nociceptive signaling, investigations have attempted to elucidate the cellular origin of biochemical nociceptive processing in which communication, i.e. cross-talk between neurons and glia is viewed as an essential component of pathogenic pain development. Here, we outline a research strategy by which nociceptive-induced cellular signaling in experimental pain models, specifically MAPK and CREB phosphorylation can be utilized to provide mechanistic insight into drug-target interaction along the nociceptive pathways. We describe a series of studies using nociceptive inflammatory and neuropathic pain models to investigate the effects of known pain therapeutics on nociceptive-induced biochemical signaling and present this as a complementary research strategy for assessing antinociceptive activity useful in the preclinical development of novel pain therapeutics.

  3. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice.

    Science.gov (United States)

    Rao, Kudupudi Prabhakara; Richa, Tambi; Kumar, Kundan; Raghuram, Badmi; Sinha, Alok Krishna

    2010-06-01

    Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in plant growth and development. In the sequenced Arabidopsis genome 80 MAPKKKs were identified and currently being analysed for its role in different stress. In rice, economically important monocot cereal crop only five MAPKKKs were identified so far. In this study using computational analysis of sequenced rice genome we have identified 75 MAPKKKs. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analyses of MAPKKKs from rice and Arabidopsis have classified them into three subgroups, which include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Further expression analysis of the MAPKKKs in MPSS database revealed that their transcripts were differentially regulated in various stress and tissue-specific libraries.

  4. A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology

    Science.gov (United States)

    While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made towards identifying their individual client proteins. Herein we describe use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase...

  5. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  6. Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomato.

    Science.gov (United States)

    Shen, Hui; Zhong, Xiangbin; Zhao, Fangfang; Wang, Yanmei; Yan, Bingxiao; Li, Qun; Chen, Genyun; Mao, Bizeng; Wang, Jianjun; Li, Yangsheng; Xiao, Guoying; He, Yuke; Xiao, Han; Li, Jianming; He, Zuhua

    2015-09-01

    The detrimental effects of global warming on crop productivity threaten to reduce the world's food supply. Although plant responses to changes in temperature have been studied, genetic modification of crops to improve thermotolerance has had little success to date. Here we demonstrate that overexpression of the Arabidopsis thaliana receptor-like kinase ERECTA (ER) in Arabidopsis, rice and tomato confers thermotolerance independent of water loss and that Arabidopsis er mutants are hypersensitive to heat. A loss-of-function mutation of a rice ER homolog and reduced expression of a tomato ER allele decreased thermotolerance of both species. Transgenic tomato and rice lines overexpressing Arabidopsis ER showed improved heat tolerance in the greenhouse and in field tests at multiple locations in China during several seasons. Moreover, ER-overexpressing transgenic Arabidopsis, tomato and rice plants had increased biomass. Our findings could contribute to engineering or breeding thermotolerant crops with no growth penalty. PMID:26280413

  7. Role of the HaHOG1 MAP kinase in response of the conifer root and butt rot pathogen (heterobasidion annosum to osmotic and oxidative stress [corrected].

    Directory of Open Access Journals (Sweden)

    Tommaso Raffaello

    Full Text Available The basidiomycete Heterobasidion annosum (Fr. Bref. s.l. is a filamentous white rot fungus, considered to be the most economically important pathogen of conifer trees. Despite the severity of the tree infection, very little is known about the molecular and biochemical aspects related to adaptation to abiotic stresses. In this study, the osmotic and oxidative tolerance as well as the role of the HaHOG1 Mitogen Activated Protein Kinase (MAPK gene were investigated. The transcript levels of the yeast orthologues GPD1, HSP78, STL1, GRE2 and the ATPase pumps ENA1, PMR1, PMC1 known to have an important role in osmotolerance were also quantified under salt osmotic conditions. The HaHOG1 gene was used for a heterologous expression and functional study in the Saccharomyces cerevisiae Δhog1 strain. Moreover, the phosphorylation level of HaHog1p was studied under salt osmotic and oxidative stress. The result showed that H. annosum displayed a decreased growth when exposed to an increased concentration of osmotic and oxidative stressors. GPD1, HSP78, STL1 and GRE2 showed an induction already at 10 min after exposure to salt stress. Among the ATPase pumps studied, PMC1 was highly induced when the fungus was exposed to 0.2 M CaCl₂ for 60 min. The heterologous expression of the HaHOG1 sequence in yeast confirmed that the gene is able to restore the osmotolerance and oxidative tolerance in the S. cerevisiae hog1Δ mutant strain. The HaHog1p was strongly phosphorylated in the presence of NaCl, KCl, H₂O₂ but not in the presence of CaCl₂ and MgCl₂. The GFP-HaHog1p fusion protein accumulated in the nuclei of the S. cerevisiae hog1Δ cells when exposed to high osmotic conditions but not under oxidative stress. These results provide the first insights about the response of H. annosum to osmotic and oxidative stress and elucidate the role of the HaHOG1 gene in such conditions.

  8. Skepinone-L, a Novel Potent and Highly Selective Inhibitor of p38 MAP Kinase, Effectively Impairs Platelet Activation and Thrombus Formation

    Directory of Open Access Journals (Sweden)

    Oliver Borst

    2013-06-01

    Full Text Available Background/Aims: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK, on platelet activation and thrombus formation. Methods: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca2+ concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber. Results: Skepinone-L (1 μM virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml, thrombin (5 mU/ml or thromboxane A2 analogue U-46619 (1 μM. Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca2+ signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2. Skepinone-L further markedly blunted thrombus formation under low (500-s and high (1700-s arterial shear rates. Conclusions: The present study discloses

  9. The Cek1‑mediated MAP kinase pathway regulates exposure of α‑1,2 and β‑1,2‑mannosides in the cell wall of Candida albicans modulating immune recognition.

    Science.gov (United States)

    Román, E; Correia, I; Salazin, A; Fradin, C; Jouault, T; Poulain, D; Liu, F-T; Pla, J

    2016-07-01

    The Cek1 MAP kinase (MAPK) mediates vegetative growth and cell wall biogenesis in the fungal pathogen Candida albicans. Alterations in the fungal cell wall caused by a defective Cek1‑mediated signaling pathway leads to increased β‑1,3‑glucan exposure influencing dectin‑1 fungal recognition by immune cells. We show here that cek1 cells also display an increased exposure of α‑1,2 and β‑1,2‑mannosides (α‑M and β‑M), a phenotype shared by strains defective in the activating MAPKK Hst7, suggesting a general defect in cell wall assembly. cek1 cells display walls with loosely bound material as revealed by transmission electron microscopy and are sensitive to tunicamycin, an inhibitor of N‑glycosylation. Transcriptomal analysis of tunicamycin treated cells revealed a differential pattern between cek1 and wild type cells which involved mainly cell wall and stress related genes. Mapping α‑M and β‑M epitopes in the mannoproteins of different cell wall fractions (CWMP) revealed an important shift in the molecular weight of the mannan derived from mutants defective in this MAPK pathway. We have also assessed the role of galectin‑3, a member of a β‑galactoside‑binding protein family shown to bind to and kill C. albicans through β‑M recognition, in the infection caused by cek1 mutants. Increased binding of cek1 to murine macrophages was shown to be partially blocked by lactose. Galectin-3(-/-) mice showed increased resistance to fungal infection, although galectin-3 did not account for the reduced virulence of cek1 mutants in a mouse model of systemic infection. All these data support a role for the Cek1‑mediated pathway in fungal cell wall maintenance, virulence and antifungal discovery. PMID:27191378

  10. A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals.

    Science.gov (United States)

    Kaur, Ravneet; Singh, Kashmir; Singh, Jaswinder

    2013-06-01

    Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in -1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions. PMID:23443578

  11. Suppressor Screens in Arabidopsis.

    Science.gov (United States)

    Li, Xin; Zhang, Yuelin

    2016-01-01

    Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses. PMID:26577776

  12. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    Science.gov (United States)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  13. PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle.

    Science.gov (United States)

    Dong, Lily Q; Liu, Feng

    2005-08-01

    Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses. PMID:16014356

  14. Glutamate functions in stomatal closure in Arabidopsis and fava bean.

    Science.gov (United States)

    Yoshida, Riichiro; Mori, Izumi C; Kamizono, Nobuto; Shichiri, Yudai; Shimatani, Tetsuo; Miyata, Fumika; Honda, Kenji; Iwai, Sumio

    2016-01-01

    Guard cells are indispensable for higher plants because they control gas exchange and water balance to maintain photosynthetic activity. The signaling processes that govern their movement are controlled by several factors, such as abscisic acid (ABA), blue light, pathogen-associated molecular patterns (PAMPs), and carbon dioxide. Herein, we demonstrated that the amino acid glutamate (Glu), a well-known mammalian neurotransmitter, functions as a novel signaling molecule in stomatal closure in both Arabidopsis and fava bean (Vicia faba L.). Pharmacological and electrophysiological analyses provided important clues for the participation of Glu-receptors, Ca(2+), and protein phosphorylation during the signaling process. Genetic analyses using Arabidopsis ABA-deficient (aba2-1) and ABA-insensitive (abi1-1 and abi2-1) mutants showed that ABA is not required for Glu signaling. However, loss-of-function of the Arabidopsis gene encoding Slow Anion Channel-Associated 1 (SLAC1) and Calcium-Dependent Protein Kinase 6 (CPK6) impaired the Glu response. Moreover, T-DNA knockout mutations of the Arabidopsis Glu receptor-like gene (GLR), GLR3.5, lost their sensitivity to Glu-dependent stomatal closure. Our results strongly support functional Glu-signaling in stomatal closure and the crucial roles of GLRs in this signaling process. PMID:26586261

  15. Arabidopsis in Wageningen

    OpenAIRE

    Koornneef, M

    2013-01-01

    Arabidopsis thaliana is the plant species that in the past 25 years has developed into the major model species in plant biology research. This was due to its properties such as short generation time, its small genome and its easiness to be transformed. Wageningen University has played an important role in the development of this model, based on interdisciplinary collaborations using genetics as a major tool to investigate aspects of physiology, development, plant-microbe interactions and evol...

  16. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    Science.gov (United States)

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  17. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj

    2009-01-01

    As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase...

  18. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Jørgen; Seidelin, Jakob Benedict;

    2011-01-01

    of cellular activities and have been implicated in the pathogenesis of several diseases, including inflammatory bowel disease (IBD). This review summarizes recent findings on the regulatory mechanism of MAPK signaling pathways, focusing on nuclear targets and their role in IBD. Finally, it summarizes how...... these signaling pathways have been exploited for the development of therapeutics and discuss the current knowledge of potential MAPK inhibitors and their anti-inflammatory effects in clinical trials related to IBD....

  19. CK (Creatine Kinase) Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Creatine Kinase Share this page: Was this page helpful? Also known as: CK; Total CK; Creatine Phosphokinase; CPK Formal name: Creatine Kinase Related tests: ...

  20. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  1. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole;

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  2. Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast

    DEFF Research Database (Denmark)

    Kleinow, T.; Bhalerao, R.; Breuer, F.;

    2000-01-01

    Yeast Snf4 is a prototype of activating gamma-subunits of conserved Snf1/AMPK-related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast....... By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc...

  3. Quantitative trait loci for floral morphology in Arabidopsis thaliana.

    OpenAIRE

    Juenger, T; Purugganan, M.; Mackay, T F

    2000-01-01

    A central question in biology is how genes control the expression of quantitative variation. We used statistical methods to estimate genetic variation in eight Arabidopsis thaliana floral characters (fresh flower mass, petal length, petal width, sepal length, sepal width, long stamen length, short stamen length, and pistil length) in a cosmopolitan sample of 15 ecotypes. In addition, we used genome-wide quantitative trait locus (QTL) mapping to evaluate the genetic basis of variation in these...

  4. Identification of Target Ligands of CORYNE in Arabidopsis by Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Heng Zhao; Shuzhen Li; Jiping Sheng; Lin Shen; Yuhui Yang; Bin Yao

    2011-01-01

    CORYNE (CRN) plays important roles in stem cell division and differentiation of shoot apical meristem (SAM) in Arabidopsis thaliana. The cytoplasmic kinase domain of CRN has been cloned and expressed in Escherichia coil, and further purified by two consecutive steps of affinity chromatography. By using this purified CRN as a ligand, a 12-mer random-peptide library was used to determine the specific amino acid sequences binding with the recombinant CRN molecule. After four rounds of biopanning, positive phage clones were isolated and sequenced, and further tested by enzyme linked immunosorbent assay for their binding ability and specificity. Two positive clones that specifically bind to the intracellular protein kinase domain of CRN have been identified. Alignment of these peptides and the kinase-associated protein phosphatase (KAPP) shows high similarity, indicating that KAPP might interact with the cytoplasmic kinase domain of CRN and negatively regulate the CLV signal. Our current study would be helpful to better understand the CLV3 signal pathway.

  5. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  6. Post-transcriptional regulation of ethylene perception and signaling in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Schaller, George Eric

    2014-03-19

    The simple gas ethylene functions as an endogenous regulator of plant growth and development, and modulates such energy relevant processes as photosynthesis and biomass accumulation. Ethylene is perceived in the plant Arabidopsis by a five-member family of receptors related to bacterial histidine kinases. Our data support a general model in which the receptors exist as parts of larger protein complexes. Our goals have been to (1) characterize physical interactions among members of the signaling complex; (2) the role of histidine-kinase transphosphorylation in signaling by the complex; and (3) the role of a novel family of proteins that regulate signal output by the receptors.

  7. Trichoderma volatiles effecting Arabidopsis

    DEFF Research Database (Denmark)

    Ramadan, Metwaly; Gigolashvili, Tamara; Grosskinsky, Dominik Kilian;

    2015-01-01

    Trichoderma species are present in many ecosystems and some strains have the ability to reduce the severity of plant diseases by activating various defense pathways via specific biologically active signaling molecules. Hence we investigated the effects of low molecular weight volatile compounds...... of Trichoderma asperellum IsmT5 on Arabidopsis thaliana. During co-cultivation of T. asperellum IsmT5 without physical contact to A. thaliana we observed smaller but vital and robust plants. The exposed plants exhibit increased trichome numbers, accumulation of defense-related compounds such as H2O2, anthocyanin......, camalexin, and increased expression of defense-related genes. We conclude that A. thaliana perceives the Trichoderma volatiles as stress compounds and subsequently initiates multilayered adaptations including activation of signaling cascades to withstand this environmental influence. The prominent headspace...

  8. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Sayed, M; Kim, S O; Salh, B S;

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities....... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

  9. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    Science.gov (United States)

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  10. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael;

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea...... that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell....

  11. Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

    Institute of Scientific and Technical Information of China (English)

    Ting-Ting Li; Hong Qiao; Hui-Xin Tong; Tian-Wei Zhuang; Tong-Tong Wang

    2016-01-01

    Background:A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) gene for type 2 diabetes mellitus (T2DM) within the German population.Among the variants,five single nucleotide polymorphisms (SNPs) of MAP4K4 (rs1003376,rs11674694,rs2236935,rs2236936,and rs6543087) showed significant association with T2DM or diabetes-related quantitative traits.We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population.Methods:Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects,using the SNPscanTM method.All subjects were recruited from the Second Affiliated Hospital,Harbin Medical University from October 2010 to September 2013.We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis,and the association between the five SNPs and metabolic traits in the subgroups.Results:Of the five variants,SNP rs2236935T/C was significantly associated with T2DM in this study population (odds ratio =1.293;95% confidence interval:1.034-1.619,P =0.025).In addition,among the controls,rs 1003376 was significantly associated with an increased body mass index (P =0.045) and homeostatic model assessment-insulin resistance (P =0.037).Conclusions:MAP4K4 gene is associated with T2DM in a Chinese Han population,and MAP4K4 gene variants may contribute to the risk toward the development of T2DM.

  12. Arabidopsis Kinesins HINKEL and TETRASPORE Act Redundantly to Control Cell Plate Expansion during Cytokinesis in the Male Garnetophyte

    Institute of Scientific and Technical Information of China (English)

    Sung-Aeong Oh; Valérie Bourdon; Madhumita Das'Pal; Hugh Dickinson; David Twell

    2008-01-01

    Asymmetric cell division at pollen mitosis I(PMI)is required to specify the differentiaI fate of the daughter vegetative and generative cells.Cytokinesis at PMI displays specialized features,and it has been suggested that there might be distinct molecular pathways underpinning different modes of cytokinesis in plants.Activation of the NACKPQR MAP kinase signaling pathway,which is essentiaI for somatic cell cytokinesis in tobacco,depends upon the NACK1and NACK2 kinesin-related proteins.Their Arabidopsis orthologs.HINKEL(HIK)and TETRAsPORE(TES).were reported to be essential for cytokinesis in somatic cells and in microsporoctes.respectively.More recently,HIK and TES were shown to have a functionally redundant role in female gametophytic cvtokinesis.We report here that HIK and TES are co-expressed in microspores and developing pollen,and,through analysis of microspore and pollen development in double heterozygote mutants.the occurrence of cell plate expansion defects during cytokinesis at PMI.The data demonstrate a functionally redundant role for HIK and TES in cell plate expansion during male gametophytic cytokinesis.extending the concept that different modes of cytokinesis are executed by a common signaling pathway,but reinforcing the individuality of gametophytic cytokinesis in its requirement for either TES or HIK.

  13. Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

    Science.gov (United States)

    Hao, Nan; Yildirim, Necmettin; Nagiec, Michal J; Parnell, Stephen C; Errede, Beverly; Dohlman, Henrik G; Elston, Timothy C

    2012-10-01

    Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity. PMID:22875986

  14. Protein kinase A signalling in Schistosoma mansoni cercariae and schistosomules.

    Science.gov (United States)

    Hirst, Natasha L; Lawton, Scott P; Walker, Anthony J

    2016-06-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24h in vitro cultured somules of the blood parasite, Schistosoma mansoni, which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal 'stem' cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu(31),Pro(34)]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni, many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. PMID:26777870

  15. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  16. POPCORN Functions in the Auxin Pathway to Regulate Embryonic Body Plan and Meristem Organization in Arabidopsis

    NARCIS (Netherlands)

    Xiang, D.Q.; Yang, H.; Venglat, P.; Cao, Y.G.; Wen, R.; Ren, M.Z.; Stone, S.; Wang, E.; Wang, H.; Xiao, W.; Weijers, D.; Berleth, T.; Laux, T.; Selvaraj, G.; Datla, R.

    2011-01-01

    The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN

  17. Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development

    NARCIS (Netherlands)

    Coluccio Leskow, Carla; Kamenetzky, Laura; Dominguez, Pia Guadalupe; Díaz Zirpolo, José Antonio; Obata, Toshihiro; Costa, Hernán; Martí, Marcelo; Taboga, Oscar; Keurentjes, Joost; Sulpice, Ronan; Ishihara, Hirofumi; Stitt, Mark; Fernie, Alisdair Robert; Carrari, Fernando

    2016-01-01

    Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-In

  18. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    Science.gov (United States)

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. PMID:27259981

  19. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L;

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  20. Mapping out Map Libraries

    Directory of Open Access Journals (Sweden)

    Ferjan Ormeling

    2008-09-01

    Full Text Available Discussing the requirements for map data quality, map users and their library/archives environment, the paper focuses on the metadata the user would need for a correct and efficient interpretation of the map data. For such a correct interpretation, knowledge of the rules and guidelines according to which the topographers/cartographers work (such as the kind of data categories to be collected, and the degree to which these rules and guidelines were indeed followed are essential. This is not only valid for the old maps stored in our libraries and archives, but perhaps even more so for the new digital files as the format in which we now have to access our geospatial data. As this would be too much to ask from map librarians/curators, some sort of web 2.0 environment is sought where comments about data quality, completeness and up-to-dateness from knowledgeable map users regarding the specific maps or map series studied can be collected and tagged to scanned versions of these maps on the web. In order not to be subject to the same disadvantages as Wikipedia, where the ‘communis opinio’ rather than scholarship, seems to be decisive, some checking by map curators of this tagged map use information would still be needed. Cooperation between map curators and the International Cartographic Association ( ICA map and spatial data use commission to this end is suggested.

  1. BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping.

    Science.gov (United States)

    Vu, Giang T H; Dear, Paul H; Caligari, Peter D S; Wilkinson, Mike J

    2010-02-08

    Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

  2. Design, Implementation and Maintenance of a Model Organism Database for Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Seung Y. Rhee

    2006-04-01

    Full Text Available The Arabidopsis Information Resource (TAIR is a web-based community database for the model plant Arabidopsis thaliana. It provides an integrated view of genes, sequences, proteins, germplasms, clones, metabolic pathways, gene expression, ecotypes, polymorphisms, publications, maps and community information. TAIR is developed and maintained by collaboration between software developers and biologists. Biologists provide specification and use cases for the system, acquire, analyse and curate data, interact with users and test the software. Software developers design, implement and test the database and software. In this review, we briefly describe how TAIR was built and is being maintained.

  3. Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

    Directory of Open Access Journals (Sweden)

    Daniele Repetto

    Full Text Available Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation. p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk, previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.

  4. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Edelhoff, S.; Disteche, C.M. [Univ. of Washington School of Medicine, Seattle, WA (United States); Lai, C. [Scripps Research Inst., LaJolla, CA (United States)

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  5. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  6. Comparative Proteomic Analysis of Arabidopsis Mature Pollen and Germinated Pollen

    Institute of Scientific and Technical Information of China (English)

    Junjie Zou; Lianfen Song; Wenzheng Zhang; Yi Wang; Songlin Ruan; Wei-Hua Wu

    2009-01-01

    Proteomic analysis was applied to generating the map of Arabidopsis mature pollen proteins and analyzing the differentially expressed proteins that are potentially involved in the regulation of Arabidopsis pollen germination. By applying 2-D electrophoresis and silver staining, we resolved 499 and 494 protein spots from protein samples extracted from pollen grains and pollen tubes, respectively. Using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, we identified 189 distinct proteins from 213 protein spots expressed in mature pollen or pollen tubes, and 75 new identified proteins that had not been reported before in research into the Arabidopsis pollen proteome. Comparative analysis revealed that 40 protein spots exhibit reproducible significant changes between mature pollen and pollen tubes. And 21 proteins from 17 downregulated and six upregulated protein spots were identified. Functional category analysis indicated that these differentially expressed proteins mainly involved in signaling, cellular structure, transport, defense/stress responses, transcription, metabolism, and energy production. The patterns of changes at protein level suggested the important roles for energy metabolism-related proteins in pollen tube growth, accompanied by the activation of the stress response pathway and modifications to the cell wall.

  7. The Receptor-Like Cytoplasmic Kinase(OsRL CK) Gene Family in Rice:Organization,Phylogenetic Relationship,and Expressionduring Development and Stress

    Institute of Scientific and Technical Information of China (English)

    Shubha Vij; Jitender Giri; Prasant Kumar Dansana; Sanjay Kapoor; Akhilesh K.Tyagi

    2008-01-01

    Receptor-like cytoplasmic kinases(RLCKs)in plants belong to the super family of receptor-like kinases(RLKs).These proteins show homology to RLKs in kinase domain but Iack the transmembrane domain.Some of the functionally characterized RLCKs from plants have been shown to play roles jn development and stress responses.Previously,149 and 187 RLCK encoding genes were identified from Arabidopsis and rice,respectively.By using HMM-based domain structure and phylogenetic relationships,we have identified 379 OsRLCKs from rice.OsRLCKs are distributed on all 12 chromosomes of rice and some members are located on duplicated chromosomal segments.Several OsRLCKs probably also undergo alternative splicing,some having evidence only in the form of gene models.To understand their possible functions,expression patterns during Iandmark stages of vegetative and reproductive development as welI as abiotic and biotic stress using microarray and MPSS-based data were analyzed.Real-time PCR-based expression profiling for a selected few genes confirmed the outcome of microarray analysis.Differential expression patterns observed for majority of OsRLCKs during development and stress suggest their involvement in diverse functions in rice.Majority of the stress-responsive OsRLCKs were also found to be localized within mapped regions of abiotic stress QTLs.Outcome of this study would help in selecting organ/development stage specific OsRLCK genes/targets for functionaI validation studies.

  8. Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

    Science.gov (United States)

    Kessler, Sharon A; Lindner, Heike; Jones, Daniel S; Grossniklaus, Ueli

    2015-01-01

    The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception. PMID:25490905

  9. Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

    KAUST Repository

    Rayapuram, Naganand

    2014-04-04

    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

  10. Consensus maps of cloned plant cuticle genes

    Institute of Scientific and Technical Information of China (English)

    Eviatar; Nevo

    2010-01-01

    Plant cuticle,which covers the plant surface,consists of waxes and cutins,and is associated with plant drought,cold,and salt resistance.Hitherto,at least 47 genes participating in the formation of plant cuticle have been cloned from Arabidopsis thaliana,Oryza sativa,Zea mays,Ricinus communis,Brassica napus,and Medicago truncatula;and about 85% of them encode proteins sharing above 50% identities with their rice homologous sequences.These cloned cuticle genes were mapped in silico on different chromosomes of rice and Arabidopsis,respectively.The mapping results revealed that plant cuticle genes were not evenly distributed in both genomes.About 40% of the mapped cuticle genes were located on chromosome 1 in Arabidopsis,while 20% of the mapped cuticle genes were located on chromosome 2 but none on chromosome 12 in rice.Some cloned plant cuticle genes have several rice homologous sequences,which might be produced by chromosomal segment duplication.The consensus map of cloned plant cuticle genes will provide important clues for the selection of candidate genes in a positional cloning of an unknown cuticle gene in plants.

  11. Asparagine Metabolic Pathways in Arabidopsis.

    Science.gov (United States)

    Gaufichon, Laure; Rothstein, Steven J; Suzuki, Akira

    2016-04-01

    Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages. PMID:26628609

  12. Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

    International Nuclear Information System (INIS)

    The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity

  13. Arabidopsis thaliana—Aphid Interaction

    OpenAIRE

    Louis, Joe; Singh, Vijay,; Shah, Jyoti

    2012-01-01

    Aphids are important pests of plants that use their stylets to tap into the sieve elements to consume phloem sap. Besides the removal of photosynthates, aphid infestation also alters source-sink patterns. Most aphids also vector viral diseases. In this chapter, we will summarize on recent significant findings in plant-aphid interaction, and how studies involving Arabidopsis thaliana and Myzus persicae (Sülzer), more commonly known as the green peach aphid (GPA), are beginning to provide impor...

  14. Stem cell organization in Arabidopsis

    OpenAIRE

    Wendrich, J.R.

    2016-01-01

    Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a hig...

  15. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V;

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  16. Phosphatidylinositol kinase from rabbit reticulocytes

    International Nuclear Information System (INIS)

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of [32P]ATP and Mg2+. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements

  17. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  18. Arabidopsis CDS blastp result: AK240730 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240730 J043030K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-11 ...

  19. Arabidopsis CDS blastp result: AK288052 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288052 J075151I09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 6e-14 ...

  20. Arabidopsis CDS blastp result: AK240911 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240911 J065037E05 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-22 ...

  1. Arabidopsis CDS blastp result: AK241119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241119 J065094C22 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-13 ...

  2. Arabidopsis CDS blastp result: AK243149 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243149 J100032I21 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 7e-12 ...

  3. Arabidopsis CDS blastp result: AK241581 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241581 J065181K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-15 ...

  4. Arabidopsis CDS blastp result: AK287479 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287479 J043023O14 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 1e-17 ...

  5. Kinase Inhibitors from Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ana Zivanovic

    2011-10-01

    Full Text Available Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included.

  6. An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community

    Science.gov (United States)

    The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee (MASC) and the North American Arabidopsis Steering C...

  7. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  8. Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Adachi, Seiji; Takai, Shinji; Matsushima-Nishiwaki, Rie; Kato, Kenji; Minamitani, Chiho; Niida, Shunpei; Mizutani, Jun; Kozawa, Osamu; Otsuka, Takanobu

    2010-04-01

    We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.

  9. Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function

    DEFF Research Database (Denmark)

    Petersen, Klaus; Qiu, Jin-Long; Lütje, Juri;

    2010-01-01

    Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn...

  10. Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

    Directory of Open Access Journals (Sweden)

    Lee Shu-Hong

    2009-11-01

    Full Text Available Abstract Background Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP, is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments. Results In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr

  11. IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tai Sang Lee; Hyun Ju Song; Ji Hoon Jeong; Young Sil Min; Chang Yell Shin; Uy Dong Sohn

    2006-01-01

    AIM: To examine the pathway related to the IL-1β-induced activation of mitogen-activated protein (MAP)kinases in cat esophageal smooth muscle cells.METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1β. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases.RESULTS: In the primary cultured cells, although IL-1βfailed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP klnase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively.The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level.Phosphokinase C (PKC) was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity.In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds.CONCLUSION: Based on these results, IL-1β-Induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1β.

  12. Natural genetic variation in Arabidopsis for responsiveness to plant growth-promoting rhizobacteria.

    Science.gov (United States)

    Wintermans, Paul C A; Bakker, Peter A H M; Pieterse, Corné M J

    2016-04-01

    The plant growth-promoting rhizobacterium (PGPR) Pseudomonas simiae WCS417r stimulates lateral root formation and increases shoot growth in Arabidopsis thaliana (Arabidopsis). These plant growth-stimulating effects are partly caused by volatile organic compounds (VOCs) produced by the bacterium. Here, we performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis for the ability to profit from rhizobacteria-mediated plant growth-promotion. To this end, 302 Arabidopsis accessions were tested for root architecture characteristics and shoot fresh weight in response to exposure to WCS417r. Although virtually all Arabidopsis accessions tested responded positively to WCS417r, there was a large variation between accessions in the increase in shoot fresh weight, the extra number of lateral roots formed, and the effect on primary root length. Correlation analyses revealed that the bacterially-mediated increase in shoot fresh weight is related to alterations in root architecture. GWA mapping for WCS417r-stimulated changes in root and shoot growth characteristics revealed 10 genetic loci highly associated with the responsiveness of Arabidopsis to the plant growth-promoting activity of WCS417r. Several of the underlying candidate genes have been implicated in important plant growth-related processes. These results demonstrate that plants possess natural genetic variation for the capacity to profit from the plant growth-promoting function of a beneficial rhizobacterium in their rhizosphere. This knowledge is a promising starting point for sustainable breeding strategies for future crops that are better able to maximize profitable functions from their root microbiome.

  13. Natural genetic variation in Arabidopsis for responsiveness to plant growth-promoting rhizobacteria.

    Science.gov (United States)

    Wintermans, Paul C A; Bakker, Peter A H M; Pieterse, Corné M J

    2016-04-01

    The plant growth-promoting rhizobacterium (PGPR) Pseudomonas simiae WCS417r stimulates lateral root formation and increases shoot growth in Arabidopsis thaliana (Arabidopsis). These plant growth-stimulating effects are partly caused by volatile organic compounds (VOCs) produced by the bacterium. Here, we performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis for the ability to profit from rhizobacteria-mediated plant growth-promotion. To this end, 302 Arabidopsis accessions were tested for root architecture characteristics and shoot fresh weight in response to exposure to WCS417r. Although virtually all Arabidopsis accessions tested responded positively to WCS417r, there was a large variation between accessions in the increase in shoot fresh weight, the extra number of lateral roots formed, and the effect on primary root length. Correlation analyses revealed that the bacterially-mediated increase in shoot fresh weight is related to alterations in root architecture. GWA mapping for WCS417r-stimulated changes in root and shoot growth characteristics revealed 10 genetic loci highly associated with the responsiveness of Arabidopsis to the plant growth-promoting activity of WCS417r. Several of the underlying candidate genes have been implicated in important plant growth-related processes. These results demonstrate that plants possess natural genetic variation for the capacity to profit from the plant growth-promoting function of a beneficial rhizobacterium in their rhizosphere. This knowledge is a promising starting point for sustainable breeding strategies for future crops that are better able to maximize profitable functions from their root microbiome. PMID:26830772

  14. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  15. Jasmonate Signal Pathway in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yi Shan; Zhi-Long Wang; Daoxin Xie

    2007-01-01

    Jasmonates (JAs), which include jasmonic acid and its cyclopentane derivatives are synthesized from the octadecanoid pathway and widely distributed throughout the plant kingdom. JAs modulate the expression of numerous genes and mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage. They also affect a variety of processes in many plant developmental processes. The JA signal pathway involves two important events: the biosynthesis of JA and the transduction of JA signal. Several important Arabidopsis mutants in jasmonate signal pathway were described in this review.

  16. Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1

    DEFF Research Database (Denmark)

    Frödin, M; Sekine, N; Roche, E;

    1995-01-01

    converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues...... glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK......-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation...

  17. SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress.

    Science.gov (United States)

    Kulik, Anna; Anielska-Mazur, Anna; Bucholc, Maria; Koen, Emmanuel; Szymanska, Katarzyna; Zmienko, Agnieszka; Krzywinska, Ewa; Wawer, Izabela; McLoughlin, Fionn; Ruszkowski, Dariusz; Figlerowicz, Marek; Testerink, Christa; Sklodowska, Aleksandra; Wendehenne, David; Dobrowolska, Grazyna

    2012-10-01

    Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd(2+) showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd(2+) treatment. Our data show significantly lower Cd(2+)-induced ROS accumulation in the mutants' roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.

  18. Pyruvate Dehydrogenase Kinase 4

    OpenAIRE

    Cadoudal, Thomas; Distel, Emilie; Durant, Sylvie; Fouque, Françoise; Blouin, Jean-Marc; Collinet, Martine; Bortoli, Sylvie; Forest, Claude; Benelli, Chantal

    2008-01-01

    OBJECTIVE—Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled ...

  19. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Planetary maps

    Science.gov (United States)

    ,

    1992-01-01

    An important goal of the USGS planetary mapping program is to systematically map the geology of the Moon, Mars, Venus, and Mercury, and the satellites of the outer planets. These geologic maps are published in the USGS Miscellaneous Investigations (I) Series. Planetary maps on sale at the USGS include shaded-relief maps, topographic maps, geologic maps, and controlled photomosaics. Controlled photomosaics are assembled from two or more photographs or images using a network of points of known latitude and longitude. The images used for most of these planetary maps are electronic images, obtained from orbiting television cameras, various optical-mechanical systems. Photographic film was only used to map Earth's Moon.

  1. Cyclin-dependent kinases.

    Science.gov (United States)

    Malumbres, Marcos

    2014-01-01

    Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  2. Comparative genomics of Gossypium and Arabidopsis: Unraveling the consequences of both ancient and recent polyploidy

    Science.gov (United States)

    Rong, Junkang; Bowers, John E.; Schulze, Stefan R.; Waghmare, Vijay N.; Rogers, Carl J.; Pierce, Gary J.; Zhang, Hua; Estill, James C.; Paterson, Andrew H.

    2005-01-01

    Both ancient and recent polyploidy, together with post-polyploidization loss of many duplicated gene copies, complicates angiosperm comparative genomics. To explore an approach by which these challenges might be mitigated, genetic maps of extant diploid and tetraploid cottons (Gossypium spp.) were used to infer the approximate order of 3016 loci along the chromosomes of their hypothetical common ancestor. The inferred Gossypium gene order corresponded more closely than the original maps did to a similarly inferred ancestral gene order predating an independent paleopolyploidization (α) in Arabidopsis. At least 59% of the cotton map and 53% of the Arabidopsis transcriptome showed correspondence in multilocus gene arrangements based on one or both of two software packages (CrimeStatII, FISH). Genomic regions in which chromosome structural rearrangement has been rapid (obscuring gene order correspondence) have also been subject to greater divergence of individual gene sequences. About 26%-44% of corresponding regions involved multiple Arabidopsis or cotton chromosomes, in some cases consistent with known, more ancient, duplications. The genomic distributions of multiple-locus probes provided early insight into the consequences for chromosome structure of an ancient large-scale duplication in cotton. Inferences that mitigate the consequences of ancient duplications improve leveraging of genomic information for model organisms in the study of more complex genomes. PMID:16109973

  3. Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants:A phylogenomic approach

    Institute of Scientific and Technical Information of China (English)

    Tao Shi; Hongwen Huang; Michael J.Sanderson; Frans E.Tax

    2014-01-01

    Leucine-rich repeat (LRR) receptor-like kinases (RLKs), evolutionarily related LRR receptor-like proteins (RLPs) and receptor-like cytoplasmic kinases (RLCKs) have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata (Arabidopsis), Lotus japonicus, and Medicago truncatula (Legumes), Oryza sativa ssp. japonica, and O. sativa ssp. indica (Rice), we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication(s) in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss potential pitfal s related to functional prediction for genes that have undergone frequent turnover (duplications, losses, and domain architecture changes), and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.

  4. Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte

    Institute of Scientific and Technical Information of China (English)

    HUO Lijun; FAN Hengyu; CHEN Dayuan; SUN Qingyuan

    2004-01-01

    Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.

  5. AtPIN: Arabidopsis thaliana Protein Interaction Network

    Directory of Open Access Journals (Sweden)

    Silva-Filho Marcio C

    2009-12-01

    Full Text Available Abstract Background Protein-protein interactions (PPIs constitute one of the most crucial conditions to sustain life in living organisms. To study PPI in Arabidopsis thaliana we have developed AtPIN, a database and web interface for searching and building interaction networks based on publicly available protein-protein interaction datasets. Description All interactions were divided into experimentally demonstrated or predicted. The PPIs in the AtPIN database present a cellular compartment classification (C3 which divides the PPI into 4 classes according to its interaction evidence and subcellular localization. It has been shown in the literature that a pair of genuine interacting proteins are generally expected to have a common cellular role and proteins that have common interaction partners have a high chance of sharing a common function. In AtPIN, due to its integrative profile, the reliability index for a reported PPI can be postulated in terms of the proportion of interaction partners that two proteins have in common. For this, we implement the Functional Similarity Weight (FSW calculation for all first level interactions present in AtPIN database. In order to identify target proteins of cytosolic glutamyl-tRNA synthetase (Cyt-gluRS (AT5G26710 we combined two approaches, AtPIN search and yeast two-hybrid screening. Interestingly, the proteins glutamine synthetase (AT5G35630, a disease resistance protein (AT3G50950 and a zinc finger protein (AT5G24930, which has been predicted as target proteins for Cyt-gluRS by AtPIN, were also detected in the experimental screening. Conclusions AtPIN is a friendly and easy-to-use tool that aggregates information on Arabidopsis thaliana PPIs, ontology, and sub-cellular localization, and might be a useful and reliable strategy to map protein-protein interactions in Arabidopsis. AtPIN can be accessed at http://bioinfo.esalq.usp.br/atpin.

  6. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    Science.gov (United States)

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  7. Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

    Directory of Open Access Journals (Sweden)

    Sundler Roger

    2006-05-01

    Full Text Available Abstract Background Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cγ2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk. Methods Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1 were used as well as the Btk inhibitor LFM-A13. Results Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cγ2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38 and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type. Conclusion Arachidonate release

  8. Polyploidy in the Arabidopsis genus.

    Science.gov (United States)

    Bomblies, Kirsten; Madlung, Andreas

    2014-06-01

    Whole genome duplication (WGD), which gives rise to polyploids, is a unique type of mutation that duplicates all the genetic material in a genome. WGD provides an evolutionary opportunity by generating abundant genetic "raw material," and has been implicated in diversification, speciation, adaptive radiation, and invasiveness, and has also played an important role in crop breeding. However, WGD at least initially challenges basic biological functions by increasing cell size, altering relationships between cell volume and DNA content, and doubling the number of homologous chromosome copies that must be sorted during cell division. Newly polyploid lineages often have extensive changes in gene regulation, genome structure, and may suffer meiotic or mitotic chromosome mis-segregation. The abundance of species that persist in nature as polyploids shows that these problems are surmountable and/or that advantages of WGD might outweigh drawbacks. The molecularly especially tractable Arabidopsis genus has several ancient polyploidy events in its history and contains several independent more recent polyploids. This genus can thus provide important insights into molecular aspects of polyploid formation, establishment, and genome evolution. The ability to integrate ecological and evolutionary questions with molecular and genetic understanding makes comparative analyses in this genus particularly attractive and holds promise for advancing our general understanding of polyploid biology. Here, we highlight some of the findings from Arabidopsis that have given us insights into the origin and evolution of polyploids. PMID:24788061

  9. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  10. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    Science.gov (United States)

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  11. Re-Evaluation of Reportedly Metal Tolerant Arabidopsis thaliana Accessions

    Science.gov (United States)

    Silva-Guzman, Macarena; Addo-Quaye, Charles; Dilkes, Brian P.

    2016-01-01

    Santa Clara, Limeport, and Berkeley are Arabidopsis thaliana accessions previously identified as diversely metal resistant. Yet these same accessions were determined to be genetically indistinguishable from the metal sensitive Col-0. We robustly tested tolerance for Zn, Ni and Cu, and genetic relatedness by growing these accessions under a range of Ni, Zn and Cu concentrations for three durations in multiple replicates. Neither metal resistance nor variance in growth were detected between them and Col-0. We re-sequenced the genomes of these accessions and all stocks available for each accession. In all cases they were nearly indistinguishable from the standard laboratory accession Col-0. As Santa Clara was allegedly collected from the Jasper Ridge serpentine outcrop in California, USA we investigated the possibility of extant A. thaliana populations adapted to serpentine soils. Botanically vouchered Arabidopsis accessions in the Jepson database were overlaid with soil maps of California. This provided no evidence of A. thaliana collections from serpentine sites in California. Thus, our work demonstrates that the Santa Clara, Berkeley and Limeport accessions are not metal tolerant, not genetically distinct from Col-0, and that there are no known serpentine adapted populations or accessions of A. thaliana. PMID:27467746

  12. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  13. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

    Science.gov (United States)

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-11-17

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  14. TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

    Science.gov (United States)

    Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

    2015-01-01

    Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

  15. Arabidopsis CDS blastp result: AK288065 [KOME

    Lifescience Database Archive (English)

    Full Text Available al to sulfate tansporter Sultr1;3 [Arabidopsis thaliana] GI:10716805; contains Pfam profile PF00916: Sulfate... transporter family; contains Pfam profile PF01740: STAS domain; contains TIGRfam profile TIGR00815: sulfate permease 1e-145 ...

  16. Arabidopsis CDS blastp result: AK061395 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061395 006-305-E02 At2g02180.1 tobamovirus multiplication protein 3 (TOM3) identical to tobamovirus multip...lication protein (TOM3) GI:15425641 from [Arabidopsis thaliana] 1e-125 ...

  17. Arabidopsis CDS blastp result: AK104882 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104882 001-044-H04 At2g02180.1 tobamovirus multiplication protein 3 (TOM3) identical to tobamovirus multip...lication protein (TOM3) GI:15425641 from [Arabidopsis thaliana] 1e-119 ...

  18. Arabidopsis CDS blastp result: AK066854 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066854 J013075C10 At2g02180.1 tobamovirus multiplication protein 3 (TOM3) identical to tobamovirus multipl...ication protein (TOM3) GI:15425641 from [Arabidopsis thaliana] 1e-119 ...

  19. Arabidopsis CDS blastp result: AK101318 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101318 J033034D12 At2g02180.1 tobamovirus multiplication protein 3 (TOM3) identical to tobamovirus multipl...ication protein (TOM3) GI:15425641 from [Arabidopsis thaliana] 1e-125 ...

  20. Arabidopsis CDS blastp result: AK069960 [KOME

    Lifescience Database Archive (English)

    Full Text Available thyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltrans...T1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 5e-60 ...