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Sample records for plant shikimate kinase

  1. Evolutionary diversification of plant shikimate kinase gene duplicates.

    Directory of Open Access Journals (Sweden)

    Geoffrey Fucile

    2008-12-01

    Full Text Available Shikimate kinase (SK; EC 2.7.1.71 catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1 and -2 (SKL2, do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein-protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation.

  2. The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

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    Kenyon Colin P

    2012-08-01

    Full Text Available Abstract Background It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP as a molecular probe with site directed mutagenesis (SDM of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. Results We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK and adenylate kinase 1 (AK1, are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. Conclusions The data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It

  3. Shikimate dehydrogenase from Pinu sylvestris L. needles

    International Nuclear Information System (INIS)

    Osipov, V.I.; Shein, I.V.

    1986-01-01

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP + , but also with NAD + . The values of K/sub m/ for shikimate, when NADP + and NAD + are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed

  4. Understanding the Catalytic Mechanism and the Nature of the Transition State of an Attractive Drug-Target Enzyme (Shikimate Kinase) by Quantum Mechanical/Molecular Mechanical (QM/MM) Studies.

    Science.gov (United States)

    Yao, Jianzhuang; Wang, Xia; Luo, Haixia; Gu, Pengfei

    2017-11-16

    Shikimate kinase (SK) is the fifth bacterial enzyme involved in the shikimate pathway for biosynthesis of life-indispensable components, such as aromatic amino acids. The absence of the shikimate pathway in humans makes SK an attractive target for the rational design of drugs aimed at pathogenesis bacteria, such as Mycobacterium tuberculosis and Helicobacter pylori. However, an effective inhibitor of SK (e.g., a transition-state analogue) is still not available on the market due, at least in part, to a lack of knowledge on the catalytic mechanism and the nature of the rate-limiting transition state. Herein, quantum mechanical/molecular mechanical (QM/MM) reaction coordinate, molecular dynamics (MD), and free-energy simulations have been performed to answer these questions. The results presented herein demonstrate that the phosphoryl-transfer process, which is the rate-limiting step of SK-catalyzed phosphorylation of shikimic acid (SKM), is a concerted one-step reaction proceeding through a loose transition state. The computational results agree well with those of experimental studies, specifically NMR results, X-ray crystal structure observation, and activation free-energy barrier. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Structure of Arabidopsis Dehydroquinate Dhydratase-Shikimate Dehydrogeanse and Implications for Metabolic Channeling in the Shikimate Pathway

    International Nuclear Information System (INIS)

    Singh, S.; Christendat, D.

    2006-01-01

    The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites

  6. Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

    Science.gov (United States)

    Scalabrin, Elisa; Radaelli, Marta; Capodaglio, Gabriele

    2016-06-01

    The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Characterization and isolation of some genes of the shikimate pathway in sensitive and resistant Centaurea jacea plants after ozone exposure

    Energy Technology Data Exchange (ETDEWEB)

    Francini, A. [Dipartimento di Coltivazione e Difesa delle Specie Legnose ' Giovanni Scaramuzzi' , University of Pisa, Via del Borghetto 80, 56124 Pisa (Italy); Nali, C. [Dipartimento di Coltivazione e Difesa delle Specie Legnose ' Giovanni Scaramuzzi' , University of Pisa, Via del Borghetto 80, 56124 Pisa (Italy)], E-mail: cristina.nali@agr.unipi.it; Pellegrini, E.; Lorenzini, G. [Dipartimento di Coltivazione e Difesa delle Specie Legnose ' Giovanni Scaramuzzi' , University of Pisa, Via del Borghetto 80, 56124 Pisa (Italy)

    2008-01-15

    Centaurea jacea has been suggested as a potential bioindicator for ozone, but little is known about its intraspecific variation in sensitivity, especially at molecular level. The effects of ozone (200 ppb, 5 h) on sensitive and resistant lines of Centaurea have been investigated at the end of fumigation. Sensitive plants showed characteristic symptoms of injury in the form of diffuse discoloration stipples on leaves. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for PAL and CHS genes. The northern analysis of PAL showed accumulation of transcript in both lines correlated with a typical increase of PAL activity (+41 and +91% in resistant and sensitive material, respectively, compared to controls). On the contrary, the transcripts of CHS, in resistant and sensitive plants, did not change after treatment. Total phenols were not affected by ozone, while anthocyanins were quickly utilised by resistant clone as antioxidant compounds. - Characterization and isolation of PAL and CHS genes in Centaurea jacea exposed to O{sub 3}.

  8. Protein Kinases in Shaping Plant Architecture.

    Science.gov (United States)

    Wu, Juan; Wang, Bo; Xin, Xiaoyun; Ren, Dongtao

    2018-02-13

    Plant architecture, the three-dimensional organization of the plant body, includes the branching pattern and the size, shape, and position of organs. Plant architecture is genetically controlled and is influenced by environmental conditions. The regulations occur at most of the stages from the first division of the fertilized eggs to the final establishment of plant architecture. Among the various endogenous regulators, protein kinases and their associated signaling pathways have been shown to play important roles in regulating the process of plant architecture establishment. In this review, we summarize recent progress in the understanding of the mechanisms by which plant architecture formation is regulated by protein kinases, especially mitogen-activated protein kinase (MAPK). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. SKPDB: a structural database of shikimate pathway enzymes

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    de Azevedo Walter F

    2010-01-01

    Full Text Available Abstract Background The functional and structural characterisation of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design. The main interest in studying shikimate pathway enzymes involves the fact that they are essential for bacteria but do not occur in humans, making them selective targets for design of drugs that do not directly impact humans. Description The ShiKimate Pathway DataBase (SKPDB is a relational database applied to the study of shikimate pathway enzymes in microorganisms and plants. The current database is updated regularly with the addition of new data; there are currently 8902 enzymes of the shikimate pathway from different sources. The database contains extensive information on each enzyme, including detailed descriptions about sequence, references, and structural and functional studies. All files (primary sequence, atomic coordinates and quality scores are available for downloading. The modeled structures can be viewed using the Jmol program. Conclusions The SKPDB provides a large number of structural models to be used in docking simulations, virtual screening initiatives and drug design. It is freely accessible at http://lsbzix.rc.unesp.br/skpdb/.

  10. Plant PA signaling via diacylglycerol kinase

    NARCIS (Netherlands)

    Arisz, S.A.; Testerink, C.; Munnik, T.

    2009-01-01

    Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential

  11. Diversity, classification and function of the plant protein kinase superfamily

    OpenAIRE

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase r...

  12. Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

    Science.gov (United States)

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2017-01-01

    Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

  13. The pattern of shikimate pathway and phenylpropanoids after inhibition by glyphosate or quinate feeding in pea roots.

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    Zabalza, Ana; Orcaray, Luis; Fernández-Escalada, Manuel; Zulet-González, Ainhoa; Royuela, Mercedes

    2017-09-01

    The shikimate pathway is a metabolic route for the biosynthesis of aromatic amino acids (AAAs) (i.e. phenylalanine, tyrosine, and tryptophan). A key enzyme of shikimate pathway (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS) is the target of the widely used herbicide glyphosate. Quinate is a compound synthesized in plants through a side branch of the shikimate pathway. Glyphosate provokes quinate accumulation and exogenous quinate application to plants shows a potential role of quinate in the toxicity of the herbicide glyphosate. Based on this, we hypothesized that the role of quinate accumulation in the toxicity of the glyphosate would be mediated by a deregulation of the shikimate pathway. In this study the effect of the glyphosate and of the exogenous quinate was evaluated in roots of pea plants by analyzing the time course of a full metabolic map of several metabolites of shikimate and phenylpropanoid pathways. Glyphosate application induced an increase of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, first enzyme of the shikimate pathway) protein and accumulation of metabolites upstream of the enzyme EPSPS. No common effects on the metabolites and regulation of shikimate pathway were detected between quinate and glyphosate treatments, supporting that the importance of quinate in the mode of action of glyphosate is not mediated by a common alteration of the regulation of the shikimate pathway. Contrary to glyphosate, the exogenous quinate supplied was probably incorporated into the main trunk from the branch pathway and accumulated in the final products, such as lignin, concomitant with a decrease in the amount of DAHPS protein. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Diverse role of CBL-interacting protein kinases in plant

    Indian Academy of Sciences (India)

    admin

    Diverse role of CBL-interacting protein kinases in plant. Most of the extracellular and ... to their role in stress signalling. Their role in transport of plant hormone auxin and mechanism of action in stress response shed new light on diverse role of.

  15. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    Science.gov (United States)

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  16. Novel links in the plant TOR kinase signaling network.

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    Xiong, Yan; Sheen, Jen

    2015-12-01

    Nutrient and energy sensing and signaling mechanisms constitute the most ancient and fundamental regulatory networks to control growth and development in all life forms. The target of rapamycin (TOR) protein kinase is modulated by diverse nutrient, energy, hormone and stress inputs and plays a central role in regulating cell proliferation, growth, metabolism and stress responses from yeasts to plants and animals. Recent chemical, genetic, genomic and metabolomic analyses have enabled significant progress toward molecular understanding of the TOR signaling network in multicellular plants. This review discusses the applications of new chemical tools to probe plant TOR functions and highlights recent findings and predictions on TOR-mediate biological processes. Special focus is placed on novel and evolutionarily conserved TOR kinase effectors as positive and negative signaling regulators that control transcription, translation and metabolism to support cell proliferation, growth and maintenance from embryogenesis to senescence in the plant system. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Functional characterization of a p-coumaroyl quinate/shikimate 3’-hydroxylase from potato (Solanum tuberosum)

    Science.gov (United States)

    Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3’-hydroxylase (C3’H) for CGA biosynthesis has not been isolated or characterized i...

  18. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  19. Plant cell wall signalling and receptor-like kinases.

    Science.gov (United States)

    Wolf, Sebastian

    2017-02-15

    Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol.

    Science.gov (United States)

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2014-09-01

    Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (∆aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption.

  1. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  2. Evolutionary adaptations of plant AGC kinases: from light signaling to cell polarity regulation

    Directory of Open Access Journals (Sweden)

    Eike Hendrik Rademacher

    2012-11-01

    Full Text Available Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues internal stimuli can modify the activity or abundance of these proteins at the plasma membrane. One such regulatory mechanism is protein phosphorylation by membrane-associated kinases and phosphatases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the plant AGC kinases that most likely allow for sensing of external stimuli (i.e. light through controlled modification of kinase activity.Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the plasma membrane depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP2A phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth.

  3. Tris[(6S)-6-hy-droxy-4-epi-shikimic acid] monohydrate: an enanti-omerically pure hy-droxy-lated shikimic acid derived from methyl shikimate.

    Science.gov (United States)

    Griesbeck, Axel G; Miara, Claus; Neudörfl, Jörg-M

    2012-11-01

    The title compound, 3C(7)H(10)O(6)·H(2)O, is the enanti-omerically pure product of a multi-step synthesis from the enanti-omerically pure natural shikimic acid. The asymmetric unit contains three mol-ecules of the acid and one mol-ecule of water. The cyclo-hexene rings of the acids have half-chair conformations. The carboxyl-ate, the four hydroxide groups and the additional water mol-ecule form a complex three-dimensional hydrogen-bonding network.

  4. Altered Levels of Aroma and Volatiles by Metabolic Engineering of Shikimate Pathway Genes in Tomato Fruits

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    Vered Tzin

    2015-06-01

    Full Text Available The tomato (Solanum lycopersicum fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9 and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12 were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of

  5. Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

    Science.gov (United States)

    Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

    2011-04-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

  6. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen; Gehring, Christoph A

    2017-01-01

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  7. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  8. The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling

    NARCIS (Netherlands)

    Stegmann, Martin; Monaghan, Jacqueline; Smakowska-Luzan, Elwira; Rovenich, Hanna; Lehner, Anita; Holton, Nicholas; Belkhadir, Youssef; Zipfel, Cyril

    2017-01-01

    In plants, perception of invading pathogens involves cell-surface immune receptor kinases. Here, we report that the Arabidopsis SITE-1 PROTEASE (S1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity. This inhibition is mediated by the malectin-like receptor

  9. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  10. Extraction and Chromatographic Determination of Shikimic Acid in Chinese Conifer Needles with 1-Benzyl-3-methylimidazolium Bromide Ionic Liquid Aqueous Solutions

    Science.gov (United States)

    Chen, Fengli; Hou, Kexin; Li, Shuangyang; Zu, Yuangang; Yang, Lei

    2014-01-01

    An ionic liquids-based ultrasound-assisted extraction (ILUAE) method was successfully developed for extracting shikimic acid from conifer needles. Eleven 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were investigated and 1-benzyl-3-methylimidazolium bromide solution was selected as the solvent. The conditions for ILUAE, including the ionic liquid concentration, ultrasound power, ultrasound time, and liquid-solid ratio, were optimized. The proposed method had good recovery (99.37%–100.11%) and reproducibility (RSD, n = 6; 3.6%). ILUAE was an efficient, rapid, and simple sample preparation technique that showed high reproducibility. Based on the results, a number of plant species, namely, Picea koraiensis, Picea meyeri, Pinus elliottii, and Pinus banksiana, were identified as among the best resources of shikimic acid. PMID:24782942

  11. The shikimate pathway: review of amino acid sequence, function and three-dimensional structures of the enzymes.

    Science.gov (United States)

    Mir, Rafia; Jallu, Shais; Singh, T P

    2015-06-01

    The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.

  12. Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3'-hydroxylase from potato (Solanum tuberosum).

    Science.gov (United States)

    Knollenberg, Benjamin J; Liu, Jingjing; Yu, Shu; Lin, Hong; Tian, Li

    2018-02-05

    Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants

    OpenAIRE

    Liu, Ping-Li; Du, Liang; Huang, Yuan; Gao, Shu-Min; Yu, Meng

    2017-01-01

    Background Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution...

  14. [Shikimic acid inhibits the degranulation and histamine release in RBL-2H3 cells].

    Science.gov (United States)

    Chen, Xianyong; Zheng, Qianqian; Liu, Wei; Yu, Lingling; Wang, Jinling; Li, Shigang

    2017-05-01

    Objective To study the effects of shikimic acid on the proliferation of rat RBL-2H3 cells and the degranulation of the cells induced by C48/80 and its mechanism. Methods MTT assay was performed to measure the proliferation of RBL-2H3 cells treated with 3, 10, 30 μg/mL shikimic acid. Toluidine blue staining was used to observe the degranulation of RBL-2H3 cells. The release of β-hexosaminidase from RBL-2H3 cells treated with 0, 12.5, 25, 50, 80, 100 μg/mL C48/80 was determined by substrate assay. ELISA was used to detect the histamine content in the supernatant of each treated group. Results Shikimic acid at 3, 10, 300 μg/mL had no obvious inhibitory effect on the proliferation of RBL-2H3 cells. There was a dose-effect relationship between the degranulation of RBL-2H3 cells and C48/80 concentration. Shikimic acid inhibited the degranulation of RBL-2H3 cells compared with the positive control group, the β-hexosaminidase release rate and histamine release were significantly reduced in RBL-2H3 cells treated with shikimic acid and C48/80. Conclusion Shikimic acid can inhibit the degranulation of RBL-2H3 cells and reduce histamine release.

  15. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

    Science.gov (United States)

    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  16. SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

    Science.gov (United States)

    Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

    2011-02-04

    SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

  17. Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

    Directory of Open Access Journals (Sweden)

    Xiquan Gao

    2014-03-01

    Full Text Available An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP, which is called PAMP-triggered immunity (PTI. The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI. Calcium (Ca2+ signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response.

  18. Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response.

    Science.gov (United States)

    Wang, Pengcheng; Zhao, Yang; Li, Zhongpeng; Hsu, Chuan-Chih; Liu, Xue; Fu, Liwen; Hou, Yueh-Ju; Du, Yanyan; Xie, Shaojun; Zhang, Chunguang; Gao, Jinghui; Cao, Minjie; Huang, Xiaosan; Zhu, Yingfang; Tang, Kai; Wang, Xingang; Tao, W Andy; Xiong, Yan; Zhu, Jian-Kang

    2018-01-04

    As sessile organisms, plants must adapt to variations in the environment. Environmental stress triggers various responses, including growth inhibition, mediated by the plant hormone abscisic acid (ABA). The mechanisms that integrate stress responses with growth are poorly understood. Here, we discovered that the Target of Rapamycin (TOR) kinase phosphorylates PYL ABA receptors at a conserved serine residue to prevent activation of the stress response in unstressed plants. This phosphorylation disrupts PYL association with ABA and with PP2C phosphatase effectors, leading to inactivation of SnRK2 kinases. Under stress, ABA-activated SnRK2s phosphorylate Raptor, a component of the TOR complex, triggering TOR complex dissociation and inhibition. Thus, TOR signaling represses ABA signaling and stress responses in unstressed conditions, whereas ABA signaling represses TOR signaling and growth during times of stress. Plants utilize this conserved phospho-regulatory feedback mechanism to optimize the balance of growth and stress responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

    Science.gov (United States)

    Gao, Xiquan; Cox, Kevin L.; He, Ping

    2014-01-01

    An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

  20. New method for the rapid extraction of natural products: efficient isolation of shikimic acid from star anise.

    Science.gov (United States)

    Just, Jeremy; Deans, Bianca J; Olivier, Wesley J; Paull, Brett; Bissember, Alex C; Smith, Jason A

    2015-05-15

    A new, practical, rapid, and high-yielding process for the pressurized hot water extraction (PHWE) of multigram quantities of shikimic acid from star anise (Illicium verum) using an unmodified household espresso machine has been developed. This operationally simple and inexpensive method enables the efficient and straightforward isolation of shikimic acid and the facile preparation of a range of its synthetic derivatives.

  1. The shikimic acid: an important metabolite for the Aglianico del Vulture wines

    Directory of Open Access Journals (Sweden)

    Pasquale Tamborra

    2014-12-01

    Full Text Available Shikimic acid is a precursor for the biosynthesis of aromatic amino acids and flavonoids (anthocyanins, tannins and flavonols. In the pharmaceutical industry, it is obtained by extraction of star anise from China, and at a yield of 3-7% it is used for the production of antiviral drug, e.g. oseltamivir. Unlike flavonoids which are only present in the grape skins, shikimic acid is present in the juice together with hydroxycinnamil tartaric acids (caffeic, ferulic and p-coumaric acid. Therefore, their content in white wines may not be negligible and their presence may explain the epidemiological studies that showed a reduced incidence of cardiovascular diseases also in people with moderate white wine consumption. The content of shikimic acid has been used to characterize wines. In southern Italy it has been used to distinguish Aglianico grape, which holds medium-high content, from Negroamaro, Primitivo and Uva di Troia grapes who have rather lower levels. It could be useful also to distinguish Fiano di Avellino (high value from Fiano Minutolo (low value. However, results of a recent work showed that the shikimic acid content decreases significantly during the ripening of the grapes and therefore its content in wine is strongly influenced by the harvest period. Finally, in a recent paper it was highlighted the increase in shikimic acid content at the end of fermentation in an Aglianico del Vulture wine, produced in the area of Rapolla (PZ, Italy municipality during the 2013 harvest. These last experimental results explain why the values of shikimic acid were lower in grapes and surprisingly higher in wines produced in the 2011 and 2012 harvest.

  2. The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover

    DEFF Research Database (Denmark)

    Monaghan, Jacqueline; Matschi, Susanne; Shorinola, Oluwaseyi

    2014-01-01

    Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen...... the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28...

  3. Faba Bean Can Adapt to Chocolate Spot Disease by Pretreatment with Shikimic and Salicylic Acids through Osmotic Adjustment, Solutes Allocation and Leaf Turgidity

    Directory of Open Access Journals (Sweden)

    Heshmat S. Aldesuquy

    2014-03-01

    Full Text Available This study investigated the effect of shikimic and salicylic acids at the concentrations of 0.4 and 0.7 mM, respectively, or their combination as phenolic compounds and Ridomil MZ at the concentration of 250 g/100 L as a fungicide on osmotic pressure (OP, solutes allocation, organic acids, inorganic ions and relative water content were quantified in Vicia faba leaves infected by Botrytis fabae. Pathogen induced noticeable decrease in osmotic pressure, total soluble sugar (TSS and inorganic osmolytes (i.e. Na+, K+, Ca2+, Mg2+ and Cl- while caused obvious increase in proline, total soluble nitrogen (TSN and organic acids (i.e. Keto and citric acids in water extract of the leaf of faba bean plants. Furthermore, pathogen caused marked decrease in relative water content (RWC of infected leaves and as a consequence the saturation water deficit (SWD was increased. Exogenous application of shikimic acid, salicylic acid or their combination could counteract the adverse effects of B. fabae on osmotic adjustment by inducing additional increase in proline, total soluble sugars, total soluble nitrogen and organic acids which in turn increase the osmotic pressure as well as relative water content in leaves of infected plants. Recovery of osmotic adjustment as well as leaf turgidity of infected host by using these chemical inducers may encourage the using of them as protective control means. The results of the present study showed also that the application of chemical inducers such as shikimic and salicylic acids or their interaction increased the resistance of Vicia faba against the chocolate spot disease.

  4. Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

    Science.gov (United States)

    Dorjgotov, Dulguun; Jurca, Manuela E; Fodor-Dunai, Csilla; Szucs, Attila; Otvös, Krisztina; Klement, Eva; Bíró, Judit; Fehér, Attila

    2009-04-02

    Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

  5. An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

    Science.gov (United States)

    Malé, Pierre-Jean G; Turner, Kyle M; Doha, Manjima; Anreiter, Ina; Allen, Aaron M; Sokolowski, Marla B; Frederickson, Megan E

    2017-09-13

    In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism. © 2017 The Author(s).

  6. Preparation of crosslinked poly (acryloyloxyethyltrimethyl ammonium chloride) microsphere and its adsorption and mechanism towards shikimic acid

    Energy Technology Data Exchange (ETDEWEB)

    Men, Jiying, E-mail: menjiying@nuc.edu.cn; Wang, Ruixin; Li, Huan; Li, Xinyan; Yang, Shanshan; Liu, Haisi; Gao, Baojiao

    2017-02-01

    Shikimic acid (SA) is a key raw material for the synthesis of the antiviral drug, but its extraction and separation from plants is still limited. Crosslinked poly (acryloyloxyethyltrimethyl ammonium chloride, DAC) microspheres were synthesized via inverse-phase suspension polymerization. In the synthesizing, N,N′-methylene bisacrylamide (MBA) was used as crosslinker, cyclohexane as dispersed medium and span-60 as dispersants, obtaining CPDAC gel microspheres. The effect of polymerization condition on balling performance and the characteristics of CPDAC were examined. The adsorption properties of CPDAC towards SA were mainly explored and the data of adsorption isotherm were analyzed by using Langmuir, Freundlich, Temkin, Sips and Toth models. Furthermore, the adsorption mechanism was analyzed in depth, and the adsorption thermodynamics was also investigated. The results show that in order to prepare CPDAC, water phase must be added dropwise to oil phase, and the volume ratio of oil-water is more than 2:1. The mean diameter of CPDAC decreases with increasing span-60 and accelerating agitating rate. The strong electrostatic interaction is formed between quaternary ammonium nitrogen of CPDAC and −COO{sup –} of SA. The adsorption kinetic data is fitted well with pseudo-first-order model. The adsorption ability is higher in aqueous water than ethanol, reaching 108 mg/g, and Toth model is more suitable for describing the actual adsorption process. The adsorption of CPDAC towards SA is dependent on the pH value of the medium. The adsorption process is exothermic, the adsorption amount decreases with the increase of temperature, and the process is driven by enthalpy. The adsorption amount decreases with the increase of salinity. The reusability of CPDAC towards SA can keep 86.1% at the sixth cycle. - Highlights: • CPDAC microspheres were synthesized via inverse-phase suspension polymerization. • SA was adsorbed strongly by strong electrostatic interaction.

  7. A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants1

    Science.gov (United States)

    Chico, José Manuel; Raíces, Marcela; Téllez-Iñón, María Teresa; Ulloa, Rita María

    2002-01-01

    A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks. PMID:11788771

  8. Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5'-Phosphosulfate Kinase from Cyanobacteria to Plants.

    Science.gov (United States)

    Herrmann, Jonathan; Nathin, David; Lee, Soon Goo; Sun, Tony; Jez, Joseph M

    2015-10-09

    In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Tris[(6S)-6-hy­droxy-4-epi-shikimic acid] monohydrate: an enanti­omerically pure hy­droxy­lated shikimic acid derived from methyl shikimate

    Science.gov (United States)

    Griesbeck, Axel G.; Miara, Claus; Neudörfl, Jörg-M.

    2012-01-01

    The title compound, 3C7H10O6·H2O, is the enanti­omerically pure product of a multi-step synthesis from the enanti­omerically pure natural shikimic acid. The asymmetric unit contains three mol­ecules of the acid and one mol­ecule of water. The cyclo­hexene rings of the acids have half-chair conformations. The carboxyl­ate, the four hydroxide groups and the additional water mol­ecule form a complex three-dimensional hydrogen-bonding network. PMID:23284468

  10. Crosstalk between phospholipase D and sphingosine kinase in plant stress signaling

    Directory of Open Access Journals (Sweden)

    Xuemin eWang

    2012-03-01

    Full Text Available The activation of phospholipase D (PLD produces phosphatidic acid (PA, whereas sphingosine kinase (SPHK phosphorylates long-chain bases (LCBs to generate LCB-1-phosphates (LCBPs such as phytosphingosine-1-phosphate (phyto-S1P. PA and phyto-S1P have been identified as lipid messengers. Recent studies have shown that PA interacts directly with SPHKs in Arabidopsis, and that the interaction promotes SPHK activity. However, SPHK and phyto-S1P act upstream of PLDα1 and PA in the stomatal response to abscisic acid (ABA. These findings indicate that SPHK/phyto-S1P and PLD/PA are co-dependent in the amplification of lipid messengers, and that crosstalk between the sphingolipid- and phospholipid-mediated signaling pathways may play important roles in plant stress signaling.

  11. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.

    Science.gov (United States)

    Rosebrock, Tracy R; Zeng, Lirong; Brady, Jennifer J; Abramovitch, Robert B; Xiao, Fangming; Martin, Gregory B

    2007-07-19

    Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

  12. A Bacillus subtilis Sensor Kinase Involved in Triggering Biofilm Formation on the Roots of Tomato Plants

    Science.gov (United States)

    Chen, Yun; Cao, Shugeng; Chai, Yunrong; Clardy, Jon; Kolter, Roberto; Guo, Jian-hua; Losick, Richard

    2012-01-01

    SUMMARY The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, l-malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signaling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation. PMID:22716461

  13. Toward a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom.

    Science.gov (United States)

    Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and eudicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as Mak-homologous kinases. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

  14. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection

    NARCIS (Netherlands)

    Liebrand, T.W.H.; Berg, van den G.C.M.; Zhang, Z.; Smit, P.; Cordewener, J.H.G.; America, A.H.P.; Sklenar, J.; Jones, A.M.E.; Tameling, W.I.L.; Robatzek, S.; Thomma, B.P.H.J.; Joosten, M.H.A.J.

    2013-01-01

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain

  15. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    Energy Technology Data Exchange (ETDEWEB)

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  16. Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

    Science.gov (United States)

    García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

    2017-08-01

    Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Towards a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom

    Directory of Open Access Journals (Sweden)

    Philipp eJanitza

    2012-12-01

    Full Text Available The mitogen-activated protein kinase (MAPK pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and dicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as MHKs. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

  18. Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

    Science.gov (United States)

    McCue, Kent F.; Conn, Eric E.

    1990-01-01

    Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

  19. Phenotypic analyses of Arabidopsis T-DNA insertion lines and expression profiling reveal that multiple L-type lectin receptor kinases are involved in plant immunity

    NARCIS (Netherlands)

    Wang, Y.; Bouwmeester, K.; Beseh, P.; Shan, W.; Govers, F.

    2014-01-01

    L-type lectin receptor kinases (LecRKs) are membrane-spanning receptor-like kinases with putative roles in biotic and abiotic stress responses and in plant development. In Arabidopsis, 45 LecRKs were identified but their functions are largely unknown. Here, a systematic functional analysis was

  20. 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

  1. Shikimic acid production in Escherichia coli: From classical metabolic engineering strategies to omics applied to improve its production

    Directory of Open Access Journals (Sweden)

    Juan Andrés Martínez

    2015-09-01

    Full Text Available Shikimic acid (SA is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF, an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp., yielding up to 17% of SA (dry basis content. The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several intuitive metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc, in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA producing strains, as the perspective of omics analysis has focused on further strain improvement

  2. The Plant Leucine-Rich Repeat Receptor-Like Kinase PSY1R from Head to Toe

    DEFF Research Database (Denmark)

    Oehlenschlæger, Christian Berg

    PSY1R belongs to the family of plant leucine-rich repeat receptor-like kinases that play important roles in processes such as growth regulation and plant immunity response. PSY1R was proposed to be the receptor of the plant peptide hormone PSY1 which promotes cell expansion. PSY1R was furthermore...... is activated. This work provides the first study of the direct interaction between PSY1R and the peptide ligand PSY1. The binding was evaluated both for full length PSY1R expressed in plants and for the isolated extracellular domain expressed in insect cells. PSY1 binds to the extracellular domain of PSY1R...... shown to phosphorylate and regulate the activity of the plasma membrane localized H+-ATPase, AHA2. While the mechanism of PSY1R-mediated AHA2 phosphorylation has previously been studied in detail, little is known about how PSY1R binds PSY1 peptide ligand and how the intracellular PSY1R kinase domain...

  3. Elucidation of Type of Molecular Interactions for the Imprinting of Shikimic Acid

    International Nuclear Information System (INIS)

    Beng, Y.C.; Nor Azah Yusof

    2016-01-01

    During the preparation of shikimic acid imprinted polymer, the effect of rebinding solution water content on the rebinding performance of shikimic acid (SA) imprinted polymer was studied. Result showed that the highest affinity toward SA was in a 100 % aqueous rebinding solution, suggesting that the interactions occurred between SA and the polymer were ionic and/ or hydrophobic in nature. Further study on the effect of rebinding solution pH to the SA imprinted polymer rebinding performance showed that the performance of the SA imprinted polymer depended on the rebinding solution pH and the highest affinity occurred at pH 4.0; this confirmed that ionic interaction played an important role in the rebinding of SA. Taking into account the deductions made from the combined results of the rebinding solution water content and effect of pH, together with observation of the red shift in the SA spectral during ultraviolet-visible spectroscopic titration experiments, suggest that the specificity shown by SA imprinted polymer in the aqueous solution was due to both ionic and π-π hydrophobic interactions. (author)

  4. Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture

    Directory of Open Access Journals (Sweden)

    Sonia eGazzarrini

    2014-04-01

    Full Text Available Carbohydrates, or sugars, regulate various aspects of plant growth through modulation of cell division and expansion. Besides playing essential roles as sources of energy for growth and as structural components of cells, carbohydrates also regulate the timing of expression of developmental programs. The disaccharide trehalose is used as an energy source, as a storage and transport molecule for glucose, and as a stress-responsive compound important for cellular protection during stress in all kingdoms. Trehalose, however, is found in very low amounts in most plants, pointing to a signaling over metabolic role for this non-reducing disaccharide. In the last decade, trehalose-6-phosphate (T6P, an intermediate in trehalose metabolism, has been shown to regulate embryonic and vegetative development, flowering time, meristem determinacy and cell fate specification in plants. T6P acts as a global regulator of metabolism and transcription promoting plant growth and triggering developmental phase transitions in response to sugar availability. Among the T6P targets are members of the Sucrose-non-fermenting1-Related Kinase1 (SnRK1 family, which are sensors of energy availability and inhibit plant growth and development during metabolic stress to maintain energy homeostasis. In this review, we will discuss the opposite roles of the sugar metabolite T6P and the SnRK1 kinases in the regulation of developmental phase transitions in response to carbohydrate levels. We will focus on how these two global regulators of metabolic processes integrate environmental cues and interact with hormonal signaling pathways to modulate plant development.

  5. Differential Regulation of Two-Tiered Plant Immunity and Sexual Reproduction by ANXUR Receptor-Like Kinases.

    Science.gov (United States)

    Mang, Hyunggon; Feng, Baomin; Hu, Zhangjian; Boisson-Dernier, Aurélien; Franck, Christina M; Meng, Xiangzong; Huang, Yanyan; Zhou, Jinggeng; Xu, Guangyuan; Wang, Taotao; Shan, Libo; He, Ping

    2017-12-01

    Plants have evolved two tiers of immune receptors to detect infections: cell surface-resident pattern recognition receptors (PRRs) that sense microbial signatures and intracellular nucleotide binding domain leucine-rich repeat (NLR) proteins that recognize pathogen effectors. How PRRs and NLRs interconnect and activate the specific and overlapping plant immune responses remains elusive. A genetic screen for components controlling plant immunity identified ANXUR1 (ANX1), a malectin-like domain-containing receptor-like kinase, together with its homolog ANX2, as important negative regulators of both PRR- and NLR-mediated immunity in Arabidopsis thaliana ANX1 constitutively associates with the bacterial flagellin receptor FLAGELLIN-SENSING2 (FLS2) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). Perception of flagellin by FLS2 promotes ANX1 association with BAK1, thereby interfering with FLS2-BAK1 complex formation to attenuate PRR signaling. In addition, ANX1 complexes with the NLR proteins RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2) and RESISTANCE TO P. SYRINGAE PV MACULICOLA1. ANX1 promotes RPS2 degradation and attenuates RPS2-mediated cell death. Surprisingly, a mutation that affects ANX1 function in plant immunity does not disrupt its function in controlling pollen tube growth during fertilization. Our study thus reveals a molecular link between PRR and NLR protein complexes that both associate with cell surface-resident ANX1 and uncovers uncoupled functions of ANX1 and ANX2 during plant immunity and sexual reproduction. © 2017 American Society of Plant Biologists. All rights reserved.

  6. Glutamate kinase as a potential biomarker of heavy metal stress in plants

    Czech Academy of Sciences Publication Activity Database

    Pavlíková, D.; Pavlík, Milan; Staszková, L.; Motyka, Václav; Száková, J.; Tlustoš, P.; Balík, J.

    2008-01-01

    Roč. 70, č. 2 (2008), s. 223-230 ISSN 0147-6513 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cadmium * Chronic stress * Glutamate kinase [E.C.2.7.2.11] * Proline Subject RIV: GA - Agricultural Economics Impact factor: 2.590, year: 2008

  7. 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...

  8. Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

    Directory of Open Access Journals (Sweden)

    Nils J. H. Averesch

    2018-03-01

    Full Text Available The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Reconstruction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

  9. Anticancer screening of medicinal plant phytochemicals against Cyclin-Dependent Kinase-2 (CDK2: An in-silico approach

    Directory of Open Access Journals (Sweden)

    Wajahat Khan

    2017-08-01

    Full Text Available Background: Cyclin-Dependent Kinase-2 (CDK2 is a member of serine/threonine protein kinases family and plays an important role in regulation of various eukaryotic cell division events. Over-expression of CDK2 during cell cycle may lead to several cellular functional aberrations including diverse types of cancers (lung cancer, primary colorectal carcinoma, ovarian cancer, melanoma and pancreatic carcinoma in humans. Medicinal plants phytochemicals which have anticancer potential can be used as an alternative drug resource. Methods: This study was designed to find out anticancer phytochemicals from medicinal plants which could inhibit CDK2 with the help of molecular docking technique. Molecular Operating Environment (MOE v2009 software was used to dock 2300 phytochemicals in this study. Results: The outcome of this study shows that four phytochemicals Kushenol T, Remangiflavanone B, Neocalyxins A and Elenoside showed the lowest S-score (-17.83, -17.57, -17.26, -17.17 respectively and binds strongly with all eight active residues Tyr15, Lys33, Ileu52, Lys56, Leu78, phe80, Asp145 and Phe146 of CDK2 binding site. These phytochemicals could successfully inhibit the CDK2. Conclusion: These phytochemicals can be considered as potential anticancer agents and used in drug development against CDK2. We anticipate that this study would pave way for phytochemical based novel small molecules as more efficacious and selective anti-cancer therapeutic compounds.

  10. Sesquiterpenes, flavonoids, shikimic acid derivatives and pyrrolizidine alkaloids from Senecio kingii Hook.

    Science.gov (United States)

    Ruiz-Vásquez, Liliana; Reina, Matías; López-Rodríguez, M; Giménez, Cristina; Cabrera, Raimundo; Cuadra, Pedro; Fajardo, Víctor; González-Coloma, Azucena

    2015-09-01

    Twenty-four compounds including eleven eremophilanolides (1-11), one eremophilane (13), five shikimic acid derivatives (14-18), six flavonoids (19-24), and the macrocyclic unsaturated pyrrolizidine alkaloid integerrimine (25) were isolated from Senecio kingii, an endemic species from the Magallanes Region (Chile). Compounds 3, 5, 6, 8-11 and 13-18 have not been previously reported as natural products. Their molecular structures were determined by NMR spectroscopic analysis and comparison with published NMR data. An X-ray-analysis of compound 3 has been performed. Their insecticidal and antifungal activities were tested, being compound 3 the strongest insect antifeedant. Compounds 6, 9 and 18 were moderate antifungals. Published by Elsevier Ltd.

  11. Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

    Science.gov (United States)

    Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

    2013-01-01

    In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

  12. Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5′-Phosphosulfate Kinase from Cyanobacteria to Plants*

    Science.gov (United States)

    Herrmann, Jonathan; Nathin, David; Lee, Soon Goo; Sun, Tony; Jez, Joseph M.

    2015-01-01

    In plants, adenosine 5′-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants. PMID:26294763

  13. Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tong, Zongyong; Li, Heng; Zhang, Rongxue; Ma, Lei; Dong, Jiangli; Wang, Tao

    2015-10-01

    Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    Science.gov (United States)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  15. Knowing your friends and foes--plant receptor-like kinases as initiators of symbiosis or defence.

    Science.gov (United States)

    Antolín-Llovera, Meritxell; Petutsching, Elena Kristin; Ried, Martina Katharina; Lipka, Volker; Nürnberger, Thorsten; Robatzek, Silke; Parniske, Martin

    2014-12-01

    The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane-localized receptor complexes. A critical step in their activation is ligand-induced homo- or hetero-oligomerization of leucine-rich repeat (LRR)- and/or lysin motif (LysM) receptor-like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen-associated molecular patterns (PAMPs), including the bacterial flagellin-derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont-derived (lipo)-chitooligosaccharides. The structurally related chitin-oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM-RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin-like domain (MLD)-LRR-RLK Symbiosis Receptor-like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR- and LysM-mediated signalling, the involvement of MLD-LRR-RLKs in symbiosis and defence, and the role of endocytosis in RLK function. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  16. A Direct, Biomass-Based Synthesis of Benzoic Acid: Formic Acid-Mediated Deoxygenation of the Glucose-Derived Materials Quinic Acid and Shikimic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Arceo, Elena; Ellman, Jonathan; Bergman, Robert

    2010-05-03

    An alternative biomass-based route to benzoic acid from the renewable starting materials quinic acid and shikimic acid is described. Benzoic acid is obtained selectively using a highly efficient, one-step formic acid-mediated deoxygenation method.

  17. Plant Aurora kinases play a role in maintenance of primary meristems and control of endoreduplication

    Czech Academy of Sciences Publication Activity Database

    Petrovská, Beáta; Cenklová, Věra; Pochylová, Žaneta; Kourová, Hana; Doskočilová, Anna; Plíhal, Ondřej; Binarová, Lenka; Binarová, Pavla

    2012-01-01

    Roč. 193, č. 3 (2012), s. 590-604 ISSN 0028-646X R&D Projects: GA ČR(CZ) GA204/07/1169; GA ČR GP204/09/P155; GA ČR(CZ) GD204/09/H084; GA MŠk(CZ) LC06034; GA MŠk LC545; GA AV ČR IAA500200719 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50200510 Keywords : Arabidopsis thaliana * AtTPX2 (targeting protein for Xklp2) * Aurora kinases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.736, year: 2012

  18. Toward a Comprehensive Phylogenetic Reconstruction of the Evolutionary History of Mitogen-Activated Protein Kinases in the Plant Kingdom

    OpenAIRE

    Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of...

  19. Hexose kinases and their role in sugar-sensing and plant development

    Directory of Open Access Journals (Sweden)

    David eGranot

    2013-03-01

    Full Text Available Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs and fructokinases (FRKs. Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes - sensing sugar levels independent of their catalytic activity and also controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits.

  20. AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca(2+) increase.

    Science.gov (United States)

    Francia, Doriana; Chiltz, Annick; Lo Schiavo, Fiorella; Pugin, Alain; Bonfante, Paola; Cardinale, Francesca

    2011-09-01

    The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca(2+) in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca(2+)-channel blocker (La(3+)) showed that in these cells, MAPK activation was dependent on the cytosolic Ca(2+) increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca(2+) transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  1. Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity.

    Science.gov (United States)

    Postma, Jelle; Liebrand, Thomas W H; Bi, Guozhi; Evrard, Alexandre; Bye, Ruby R; Mbengue, Malick; Kuhn, Hannah; Joosten, Matthieu H A J; Robatzek, Silke

    2016-04-01

    The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  2. Novel comprehensive multidimensional liquid chromatography approach for elucidation of the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 strain.

    Science.gov (United States)

    Cacciola, Francesco; Mangraviti, Domenica; Rigano, Francesca; Donato, Paola; Dugo, Paola; Mondello, Luigi; Cortes, Hernan J

    2018-06-01

    Shikimic acid is a intermediate of aromatic amino acid biosynthesis and the preferred starting material for production of the most commonly prescribed anti-influenza drug, Tamiflu. Its six-membered carbocyclic ring is adorned with several chiral centers and various functionalities, making shikimic acid a valuable chiral synthon. When microbially-produced, in addition to shikimic acid, numerous other metabolites are exported out of the cytoplasm and accumulate in the culture medium. This extracellular matrix of metabolites is referred to as the microbosphere. Due to the high sample complexity, in this study, the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 was analyzed by liquid chromatography and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection. GC analysis of the trimethylsilyl derivatives was also carried out in order to support the elucidation of the selected metabolites in the microbosphere. The elucidation of the metabolic fraction of this bacterial strain might be of valid aid for improving, through genetic changes, the concentration and yield of shikimic acid synthesized from glucose. Graphical abstract.

  3. Comparative study on the turnover of quinic- and shikimic acid and of its derivatives in needles of Norway spruce, Picea abies (L.) Karst., affected by 'Waldsterben' syndrome

    International Nuclear Information System (INIS)

    Dittrich, P.; Senser, M.; Frielinghaus, J.

    1989-01-01

    A comparative study on the incorporation of 14 C into quinic- and shikimic acid in spruce needles was carried out with four different syndroms of Picea abies (L.) Karst: a tree from high altitudes of the Bayerischer Wald with the syndrom of 'acute yellowing', a light-sensitive mutant with yellowish needles (Picea abies Karst. finedonensis), a tree exhibiting 'Lametta' syndrome with a translucent crown and twigs hanging down like tinsel, and a healthy tree for control. The needles of these trees were labelled via photosynthetic fixation of 14 CO 2 in June, and the fate of labelled compounds was monitored over 125 days. In the youngest needles incorporation rates of quinic and shikimic acid reached a dominating level of about 60% of the label of soluble metabolites, which underlines the decisive role of both acids during needle development. The yellow mutant 'finedonensis' and the tree with 'Lametta' syndrome, which exhibit rather identical turnover kinetics of quinate and shikimate, show lower rates and reach, in particular in one- and two-year-old needles, only about 50% of the incorporation rates of the control spruce. The tree with the syndrome of 'acute yellowing' exhibits a significant reduction of incorporation already in the youngest needles; the older needles of this tree virtually suspend metabolism of both acids all together, though apparently high but dormant pools of shikimate and quinate are present. The degree of label incorporation into shikimate and quinate may possibly serve as a measure of needle damage. Exclusively in the yellow mutant a novel spruce constituent, 3-0-p-coumaroylquinic acid, could be detected; a related compound, 3-0-p-caffeoylshikimic acid was identified in the needles of the 'acute yellowing' tree. (orig./MG) [de

  4. dependent/calmodulin- stimulated protein kinase from moss

    Indian Academy of Sciences (India)

    Unknown

    stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

  5. Effect of chlorate, molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures.

    Science.gov (United States)

    Oliver, Christy E; Beier, Ross C; Hume, Michael E; Horrocks, Shane M; Casey, Thomas A; Caton, Joel S; Nisbet, David J; Smith, David J; Krueger, Nathan A; Anderson, Robin C

    2010-04-01

    Experiments were conducted to determine factors that affect sensitivity of Salmonella enterica serovar Typhimurium to sodium chlorate (5mM). In our first experiment, cultures grown without chlorate grew more rapidly than those with chlorate. An extended lag before logarithmic growth was observed in anaerobic but not aerobic cultures containing chlorate. Chlorate inhibition of growth during aerobic culture began later than that observed in anaerobic cultures but persisted once inhibition was apparent. Conversely, anaerobic cultures appeared to adapt to chlorate after approximately 10h of incubation, exhibiting rapid compensatory growth. In anaerobic chlorate-containing cultures, 20% of total viable counts were resistant to chlorate by 6h and had propagated to 100% resistance (>10(9)CFU mL(-1)) by 24h. In the aerobic chlorate-containing cultures, 12.9% of colonies had detectable resistance to chlorate by 6h, but only 1% retained detectable resistance at 24h, likely because these cultures had opportunity to respire on oxygen and were thus not enriched via the selective pressure of chlorate. In another study, treatment with shikimic acid (0.34 mM), molybdate (1mM) or their combination had little effect on aerobic or anaerobic growth of Salmonella in the absence of added chlorate. As observed in our earlier study, chlorate resistance was not detected in any cultures without added chlorate. Chlorate resistant Salmonella were recovered at equivalent numbers regardless of treatment after 8h of aerobic or anaerobic culture with added chlorate; however, by 24h incubation chlorate sensitivity was completely restored to aerobic but not anaerobic cultures treated with shikimic acid or molybdate but not their combination. Results indicate that anaerobic adaptation of S. Typhimurium to sodium chlorate during pure culture is likely due to the selective propagation of low numbers of cells exhibiting spontaneous resistance to chlorate and this resistance is not reversible by

  6. Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

    Science.gov (United States)

    Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

    2011-01-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

  7. Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.

    Science.gov (United States)

    Vivancos, Pedro Diaz; Driscoll, Simon P; Bulman, Christopher A; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H

    2011-09-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.

  8. The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control

    NARCIS (Netherlands)

    Kemmerling, B.; Schwedt, A.; Rodriguez, P.; Mazzotta, S.; Frank, M.; Abu Qamar, S.; Mengiste, T.; Betsuyaku, S.; Parker, J.E.; Müssig, C.; Thomma, B.P.H.J.; Albrecht, C.; Vries, de S.C.; Hirt, H.; Nürnberger, T.

    2007-01-01

    Programmed cell death (PCD) is a common host response to microbial infection [1-3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled.

  9. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  10. RNAi-mediated silencing of MAP kinase signalling genes (Fmk1, Hog1, and Pbs2) in Fusarium oxysporum reduces pathogenesis on tomato plants.

    Science.gov (United States)

    Pareek, Manish; Rajam, Manchikatla Venkat

    2017-09-01

    Fusarium oxysporum is a soil-borne plant fungal pathogen, and causes colossal losses in several crop plants including tomato. Effective control measures include the use of harmful fungicides and resistant cultivars, but these methods have shown limited success. Conventional methods to validate fungal pathogenic genes are labour intensive. Therefore, an alternative strategy is required to efficiently characterize unknown pathogenic genes. RNA interference (RNAi) has emerged as a potential tool to functionally characterize novel fungal pathogenic genes and also to control fungal diseases. Here, we report an efficient method to produce stable RNAi transformants of F. oxysporum using Agrobacterium-mediated transformation (AMT). We have transformed F. oxysporum spores using RNAi constructs of Fmk1, Hog1, and Pbs2 MAP kinase signalling genes. Fmk1 RNAi fungal transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings. Hog1 and Pbs2 RNAi transformants showed altered conidial size, and reduced invasive growth and pathogenesis. These results showed that AMT using RNAi constructs is an effective approach for dissecting the role of genes involved in pathogenesis in F. oxysporum and this could be extended for other fungal systems. The obtained knowledge can be easily translated for developing fungal resistant crops by RNAi. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

    Science.gov (United States)

    Rodriguez, Alberto; Martínez, Juan A; Millard, Pierre; Gosset, Guillermo; Portais, Jean-Charles; Létisse, Fabien; Bolivar, Francisco

    2017-06-01

    Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. The volatile profiles of a rare apple (Malus domestica Borkh.) honey: shikimic acid-pathway derivatives, terpenes, and others.

    Science.gov (United States)

    Kuś, Piotr Marek; Jerković, Igor; Tuberoso, Carlo Ignazio Giovanni; Šarolić, Mladenka

    2013-09-01

    The volatile profiles of rare Malus domestica Borkh. honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44-45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid-pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H-indole-3-acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car-2-en-4-one (10.2%). CH2 Cl2 and pentane/Et2 O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9-3.9%), benzoic acid (2.0-11.2%), terpendiol I (0.3-7.4%), coumaran (0.0-2.8%), 2-phenylacetic acid (2.0-26.4%), methyl syringate (3.9-13.1%), vomifoliol (5.0-31.8%), and methyl 1H-indole-3-acetate (1.9-10.2%). Apple honey contained also benzyl alcohol, 2-phenylethanol, (E)-cinnamaldehyde, (E)-cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

  13. Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Roberts, J.K.M.; Aubert, S.; Gout, E.; Bligny, R.; Douce, R.

    1997-01-01

    Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport

  14. Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases.

    Science.gov (United States)

    Uchida, Naoyuki; Tasaka, Masao

    2013-12-01

    Plant vasculatures are complex tissues consisting of (pro)cambium, phloem, and xylem. The (pro)cambium serves as vascular stem cells that produce all vascular cells. The Arabidopsis ERECTA (ER) receptor kinase is known to regulate the architecture of inflorescence stems. It was recently reported that the er mutation enhances a vascular phenotype induced by a mutation of TDR/PXY, which plays a significant role in procambial proliferation, suggesting that ER participates in vascular development. However, detailed molecular mechanisms of the ER-dependent vascular regulation are largely unknown. Here, this work found that ER and its paralogue, ER-LIKE1, were redundantly involved in procambial development of inflorescence stems. Interestingly, their activity in the phloem was sufficient for vascular regulation. Furthermore, two endodermis-derived peptide hormones, EPFL4 and EPFL6, were redundantly involved in such regulation. It has been previously reported that EPFL4 and EPFL6 act as ligands of phloem-expressed ER for stem elongation. Therefore, these findings indicate that cell-cell communication between the endodermis and the phloem plays an important role in procambial development as well as stem elongation. Interestingly, similar EPFL-ER modules control two distinct developmental events by slightly changing their components: the EPFL4/6-ER module for stem elongation and the EPFL4/6-ER/ERL1 module for vascular development.

  15. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  16. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  17. A rice kinase-protein interaction map.

    Science.gov (United States)

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

    2009-03-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

  18. MAP kinase cascades in Arabidopsis innate immunity

    DEFF Research Database (Denmark)

    Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

    2012-01-01

    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 which trigger MAPK-dependent innate ...

  19. Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

    Science.gov (United States)

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

    2015-09-01

    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 showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

  20. 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.

  1. 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 Crenarcha...

  2. A CBL-Interacting Protein Kinase TaCIPK2 Confers Drought Tolerance in Transgenic Tobacco Plants through Regulating the Stomatal Movement.

    Science.gov (United States)

    Wang, Yan; Sun, Tao; Li, Tingting; Wang, Meng; Yang, Guangxiao; He, Guangyuan

    2016-01-01

    In plants, the CBL-CIPK signaling pathways play key roles in the response to abiotic stresses. However, functional studies of CIPKs in the important staple crop wheat are very rare. In this study, we identified a CIPK gene from wheat, designated TaCIPK2. Expression analysis results showed that TaCIPK2 could be up-regulated in wheat leaves by polyethylene glycol, abscisic acid and H2O2 treatments. Subcellular localization analyses revealed that TaCIPK2 was present in whole wheat epidermal cells. A yeast two-hybrid assay indicated that TaCIPK2 interacted with TaCBL1, 2, 3 and 4 in vitro. Transgenic tobacco plants over-expressing TaCIPK2 exhibited increased drought tolerance, indicated by a larger proportion of green cotyledons and higher survival rates under the osmotic and drought stress conditions compared with control plants. Additionally, physiological index analyses revealed that the transgenic tobacco plants had lower water loss rates and ion leakage, accumulated less malondialdehyde and H2O2, and had higher catalase and superoxide dismutase activities than the control plants. The transgenic plants also exhibited faster stomatal closure following exposure to osmotic stress conditions. The seed germination rates and stomatal aperture of TaCIPK2-overexpressing tobacco plants decreased after exogenous abscisic acid treatment was applied, implying that the transgenic tobacco plants were more sensitive to exogenous abscisic acid than the control plants. Our results indicate that TaCIPK2 plays a positive regulatory role in drought stress responses in transgenic tobacco plants.

  3. Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis

    DEFF Research Database (Denmark)

    Weinl, Christina; Marquardt, Sebastian; Kuijt, Suzanne J H

    2005-01-01

    numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act...... independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells...

  4. Enhanced expression of a calcium-dependent protein kinase

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  5. Avaliação do uso de glyphosate em soja geneticamente modificada e sua relação com o ácido chiquímico Evaluation of glyphosate application on transgenic soybean and its relationship with shikimic acid

    Directory of Open Access Journals (Sweden)

    D.A.S. Franco

    2012-09-01

    plantas de soja transgênica no campo quando tratadas de forma isolada com glyphosate. Os resultados também mostraram exsudação radicular do glyphosate por soja transgênica, com posterior absorção por soja convencional. Foram detectados resíduos de glyphosate e ácido aminometilfosfônico na solução nutritiva.Glyphosate [N-(phosphonomethyl glycine]-resistant crops (GRC are the transgenic crops most extensively grown worldwide, with soybean being the major GRC. It is important to evaluate the impact of glyphosate on transgenic soybean and its relationship with shikimic acid. A field experiment was conducted at Engenheiro Coelho-SP, Brazil, during the agricultural year 2007/2008 to evaluate the effect of glyphosate on the growth, development, and seed quality of GRC soybean variety BRS Valiosa RR. A randomized block design was used with four replications. Glyphosate was applied at 720 and 960 g a.e. ha-1 (acid equivalent and in sequence at the doses 720/720, 960/720, and 960/720/720 g a.e. ha-1 (acid equivalent. To evaluate transfer from GRC soybean to non GRC soybean cultivated in nutrient solution, a pot experiment was conducted at Instituto Biológico, SP, Brazil. Glyphosate was applied on the GRC soybean (M8045RR at 2,400 g a.e. ha-1. Both GRC soybean and non GRC soybean were sown in the same box with nutrient solution. At 0, 1, 3, 7, and 10 days after application, shikimic acid was measured by HPLC and the glyphosate and aminomethylphosphonic acid (AMPA levels in nutrient solution were determined by GC-MS. The results showed that yield, plant height, seed oil, and protein contents were not affected by glyphosate application. GRC soybean accumulated shikimic acid in the field. Glyphosate and AMPA were released through the roots of GRC soybean, and subsequently taken up by non-GRC soybean, exerting inhibitory effects on their shikimic pathway.

  6. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  7. Gene regulation by MAP kinase cascades

    DEFF Research Database (Denmark)

    Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

  8. Phenolics and Plant Allelopathy

    Directory of Open Access Journals (Sweden)

    De-An Jiang

    2010-12-01

    Full Text Available Phenolic compounds arise from the shikimic and acetic acid (polyketide metabolic pathways in plants. They are but one category of the many secondary metabolites implicated in plant allelopathy. Phenolic allelochemicals have been observed in both natural and managed ecosystems, where they cause a number of ecological and economic problems, such as declines in crop yield due to soil sickness, regeneration failure of natural forests, and replanting problems in orchards. Phenolic allelochemical structures and modes of action are diverse and may offer potential lead compounds for the development of future herbicides or pesticides. This article reviews allelopathic effects, analysis methods, and allelopathic mechanisms underlying the activity of plant phenolic compounds. Additionally, the currently debated topic in plant allelopathy of whether catechin and 8-hydroxyquinoline play an important role in Centaurea maculata and Centaurea diffusa invasion success is discussed. Overall, the main purpose of this review is to highlight the allelopacthic potential of phenolic compounds to provide us with methods to solve various ecology problems, especially in regard to the sustainable development of agriculture, forestry, nature resources and environment conservation.

  9. 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)....

  10. Silencing of the Mitogen-Activated Protein Kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants.

    Science.gov (United States)

    Onyilo, Francis; Tusiime, Geoffrey; Tripathi, Jaindra N; Chen, Li-Hung; Falk, Bryce; Stergiopoulos, Ioannis; Tushemereirwe, Wilberforce; Kubiriba, Jerome; Tripathi, Leena

    2018-01-01

    Pseudocercospora fijiensis , causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

  11. Silencing of the Mitogen-Activated Protein Kinases (MAPK Fus3 and Slt2 in Pseudocercospora fijiensis Reduces Growth and Virulence on Host Plants

    Directory of Open Access Journals (Sweden)

    Francis Onyilo

    2018-03-01

    Full Text Available Pseudocercospora fijiensis, causal agent of the black Sigatoka disease (BSD of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB. This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

  12. Wall-associated kinase-like polypeptide mediates nutritional status perception and response

    Science.gov (United States)

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

    The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

  13. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  14. Pyruvate kinase blood test

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003357.htm Pyruvate kinase blood test To use the sharing features on this page, ... energy when oxygen levels are low. How the Test is Performed A blood sample is needed. In the laboratory, white blood ...

  15. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  16. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  17. Enterococcus faecalis phosphomevalonate kinase

    Science.gov (United States)

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 μmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. PMID:15802646

  18. 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...

  19. Expression, purification and kinase activity analysis of maize ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Kinase activity is essential for a protein kinase to perform its biological function. In previous study we have cloned a novel plant SnRK2 subfamily gene from maize and named it as ZmSPK1. In this study the. cDNA of ZmSPK1 with dHA-His6 tag was amplified by PCR and was subcloned into the yeast.

  20. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    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.

  1. 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.

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

    KAUST Repository

    Fujii, Hiroaki; Verslues, Paul E.; Zhu, Jian-Kang

    2011-01-01

    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

  3. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Science.gov (United States)

    In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

  4. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants.

    Directory of Open Access Journals (Sweden)

    Gaoyi Cao

    Full Text Available A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

  5. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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.

  7. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.; Kwezi, Lusisizwe; Wheeler, Janet I.; Gehring, Christoph A

    2012-01-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  8. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  9. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    Science.gov (United States)

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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

  11. 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.

  12. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    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. PMID:24212825

  13. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available 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 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.

  14. ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead.

    Science.gov (United States)

    Huang, Tsai-Lien; Huang, Hao-Jen

    2008-04-01

    Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

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

    KAUST Repository

    Zhang, Xiujuan; Yang, Guanyu; Shi, Rui; Han, Xiaomin; Qi, Liwang; Wang, Ruigang; Xiong, Liming; Li, Guojing

    2013-01-01

    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

  16. 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....

  17. TOR signalling in plants.

    Science.gov (United States)

    Rexin, Daniel; Meyer, Christian; Robaglia, Christophe; Veit, Bruce

    2015-08-15

    Although the eukaryotic TOR (target of rapamycin) kinase signalling pathway has emerged as a key player for integrating nutrient-, energy- and stress-related cues with growth and metabolic outputs, relatively little is known of how this ancient regulatory mechanism has been adapted in higher plants. Drawing comparisons with the substantial knowledge base around TOR kinase signalling in fungal and animal systems, functional aspects of this pathway in plants are reviewed. Both conserved and divergent elements are discussed in relation to unique aspects associated with an autotrophic mode of nutrition and adaptive strategies for multicellular development exhibited by plants. © 2015 Authors; published by Portland Press Limited.

  18. Casein kinase 1-Like 3 is required for abscisic acid regulation of ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... root growth compared with ckl3 plants under different ABA concentration treatment. Also, compared with wild-type plants, the expressions of the ABA and abiotic stress-responsive ... CK1 isoforms show that the interaction between CK1 and ..... kinase I, in root development and plant hormone sensitivity.

  19. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  20. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  1. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe

    2018-01-22

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  2. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

    2018-01-01

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  3. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbling mode

    Science.gov (United States)

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP...

  4. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Science.gov (United States)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

  5. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    International Nuclear Information System (INIS)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2012-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  6. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  7. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  8. An Overview of Plant Phenolic Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Derong Lin

    2016-10-01

    Full Text Available In this paper, the biosynthesis process of phenolic compounds in plants is summarized, which includes the shikimate, pentose phosphate and phenylpropanoid pathways. Plant phenolic compounds can act as antioxidants, structural polymers (lignin, attractants (flavonoids and carotenoids, UV screens (flavonoids, signal compounds (salicylic acid and flavonoids and defense response chemicals (tannins and phytoalexins. From a human physiological standpoint, phenolic compounds are vital in defense responses, such as anti-aging, anti-inflammatory, antioxidant and anti-proliferative activities. Therefore, it is beneficial to eat such plant foods that have a high antioxidant compound content, which will cut down the incidence of certain chronic diseases, for instance diabetes, cancers and cardiovascular diseases, through the management of oxidative stress. Furthermore, berries and other fruits with low-amylase and high-glucosidase inhibitory activities could be regarded as candidate food items in the control of the early stages of hyperglycemia associated with type 2 diabetes.

  9. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    Science.gov (United States)

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. An Arabidopsis kinase cascade influences auxin-responsive cell expansion.

    Science.gov (United States)

    Enders, Tara A; Frick, Elizabeth M; Strader, Lucia C

    2017-10-01

    Mitogen-activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin-related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1-1 as a mutant that displays hypersensitivity in auxin-responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin-responsive cell expansion assays, suggesting that this MPK cascade affects auxin-influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho-like GTPases from Plants (ROP) small GTPase family. Similar to mpk1-1 and mkk3-1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin-responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin-responsive cell expansion are mediated through phosphorylation-dependent modulation of ROP activity. Our data suggest a MKK3 • MPK1 • RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  11. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    Science.gov (United States)

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  12. Sugar signals and the control of plant growth and development

    NARCIS (Netherlands)

    Lastdrager, Jeroen|info:eu-repo/dai/nl/357520076; Hanson, Johannes|info:eu-repo/dai/nl/304822299; Smeekens, Sjef|info:eu-repo/dai/nl/072489995

    2014-01-01

    Sugars have a central regulatory function in steering plant growth. This review focuses on information presented in the past 2 years on key players in sugar-mediated plant growth regulation, with emphasis on trehalose 6-phosphate, target of rapamycin kinase, and Snf1-related kinase 1 regulatory

  13. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  14. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  15. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

  16. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  17. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  18. Tackling Drought Stress: RECEPTOR-LIKE KINASES Present New Approaches

    Science.gov (United States)

    Marshall, Alex; Aalen, Reidunn B.; Audenaert, Dominique; Beeckman, Tom; Broadley, Martin R.; Butenko, Melinka A.; Caño-Delgado, Ana I.; de Vries, Sacco; Dresselhaus, Thomas; Felix, Georg; Graham, Neil S.; Foulkes, John; Granier, Christine; Greb, Thomas; Grossniklaus, Ueli; Hammond, John P.; Heidstra, Renze; Hodgman, Charlie; Hothorn, Michael; Inzé, Dirk; Østergaard, Lars; Russinova, Eugenia; Simon, Rüdiger; Skirycz, Aleksandra; Stahl, Yvonne; Zipfel, Cyril; De Smet, Ive

    2012-01-01

    Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops. PMID:22693282

  19. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  20. A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

    Science.gov (United States)

    Su, Shih-Heng; Krysan, Patrick J

    2016-12-01

    Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  1. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

    2007-01-01

    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  2. Signaling network of the Btk family kinases.

    Science.gov (United States)

    Qiu, Y; Kung, H J

    2000-11-20

    The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

  3. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  4. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD 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.

  5. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  6. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  7. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  8. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  9. S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; Liu, Zhen; Qiu, Wen-Li; Whitham, Steven A.; Qian, Wei-Jun

    2017-09-29

    It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse

  10. Alteration of plant physiology by glyphosate and its by-product aminomethylphosphonic acid: an overview.

    Science.gov (United States)

    Gomes, Marcelo P; Smedbol, Elise; Chalifour, Annie; Hénault-Ethier, Louise; Labrecque, Michel; Lepage, Laurent; Lucotte, Marc; Juneau, Philippe

    2014-09-01

    It is generally claimed that glyphosate kills undesired plants by affecting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, disturbing the shikimate pathway. However, the mechanisms leading to plant death may also be related to secondary or indirect effects of glyphosate on plant physiology. Moreover, some plants can metabolize glyphosate to aminomethylphosphonic acid (AMPA) or be exposed to AMPA from different environmental matrices. AMPA is a recognized phytotoxin, and its co-occurrence with glyphosate could modify the effects of glyphosate on plant physiology. The present review provides an overall picture of alterations of plant physiology caused by environmental exposure to glyphosate and its metabolite AMPA, and summarizes their effects on several physiological processes. It particularly focuses on photosynthesis, from photochemical events to C assimilation and translocation, as well as oxidative stress. The effects of glyphosate and AMPA on several plant physiological processes have been linked, with the aim of better understanding their phytotoxicity and glyphosate herbicidal effects. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  12. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-01-01

    is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

  13. Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

    Science.gov (United States)

    Montesano, M; Kõiv, V; Mäe, A; Palva, E T

    2001-11-01

    summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

  14. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

    Cann, Marissa L; McDonald, Ian M; East, Michael P; Johnson, Gary L; Graves, Lee M

    2017-11-01

    The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi

    International Nuclear Information System (INIS)

    Werner, I.; Bacher, A.; Eisenreich, W.

    1997-01-01

    The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this

  16. Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

    Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

  17. SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

    Science.gov (United States)

    Duan, Penggen; Rao, Yuchun; Zeng, Dali; Yang, Yaolong; Xu, Ran; Zhang, Baolan; Dong, Guojun; Qian, Qian; Li, Yunhai

    2014-02-01

    Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  18. Cytokinin metabolism of pathogenic fungus Leptosphaeria maculans involves isopentenyltransferase, adenosine kinase and cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Trdá, Lucie; Barešová, Monika; Šašek, Vladimír; Nováková, Miroslava; Zahajská, Lenka; Dobrev, Petre; Motyka, Václav; Burketová, Lenka

    2017-01-01

    Roč. 8, JUL 21 (2017), č. článku 1374. ISSN 1664-302X R&D Projects: GA ČR GA13-26798S; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Adenosine kinase * Cytokinin * Cytokinin oxidase/dehydrogenase * Isopentenyltransferase * Leptosphaeria maculans * Zeatin cis/trans isomerase Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection OBOR OECD: Microbiology Impact factor: 4.076, year: 2016

  19. Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

    KAUST Repository

    Almadanim, M. Cecí lia; Alexandre, Bruno M.; Rosa, Margarida T.G.; Sapeta, Helena; Leitã o, Antó nio E.; Ramalho, José C.; Lam, TuKiet T.; Negrã o, Só nia; Abreu, Isabel A.; Oliveira, M. Margarida

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here

  20. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase

  2. Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

    Science.gov (United States)

    Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

    2013-08-01

    Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  3. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  4. The Protein Kinase RSK Family - Roles in Prostate Cancer

    National Research Council Canada - National Science Library

    Lannigan, Deborah

    2006-01-01

    The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

  5. In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

    Science.gov (United States)

    Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

    2000-08-01

    The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

  6. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

    Science.gov (United States)

    Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

    2008-09-05

    The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

  7. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  8. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  9. A conserved cysteine motif is critical for rice ceramide kinase activity and function.

    Directory of Open Access Journals (Sweden)

    Fang-Cheng Bi

    Full Text Available Ceramide kinase (CERK is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare and investigate the effects of ceramides on rice cell viability.OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.

  10. 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...

  11. 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.

  12. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  13. How glyphosate affects plant disease development: it is more than enhanced susceptibility.

    Science.gov (United States)

    Hammerschmidt, Ray

    2018-05-01

    Glyphosate has been shown to affect the development of plant disease in several ways. Plants utilize phenolic and other shikimic acid pathway-derived compounds as part of their defense against pathogens, and glyphosate inhibits the biosynthesis of these compounds via its mode of action. Several studies have shown a correlation between enhanced disease and suppression of phenolic compound production after glyphosate. Glyphosate-resistant crop plants have also been studied for changes in resistance as a result of carrying the glyphosate resistance trait. The evidence indicates that neither the resistance trait nor application of glyphosate to glyphosate-resistant plants increases susceptibility to disease. The only exceptions to this are cases where glyphosate has been shown to reduce rust diseases on glyphosate-resistant crops, supporting a fungicidal role for this chemical. Finally, glyphosate treatment of weeds or volunteer crops can cause a temporary increase in soil-borne pathogens that may result in disease development if crops are planted too soon after glyphosate application. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. De novo biosynthesis of volatiles induced by insect herbivory in cotton plants

    International Nuclear Information System (INIS)

    Pare, P.W.; Tumlinson, J.H.

    1997-01-01

    In response to insect feeding on the leaves, cotton (Gossypium hirsutum L.) plants release elevated levels of volatiles, which can serve as a chemical signal that attracts natural enemies of the herbivore to the damaged plant. Pulse-labeling experiments with [13C]CO2 demonstrated that many of the volatiles released, including the acyclic terpenes (E,E)-alpha-farnesene, (E)-beta-farnesene, (E)-beta-ocimene, linalool,(E)-4,8-dimethyl-1,3,7-nonatriene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetrane, as well as the shikimate pathway product indole, are biosynthesized de novo following insect damage. However, other volatile constituents, including several cyclic terpenes, butyrates, and green leaf volatiles of the lipoxygenase pathway are released from storage or synthesized from stored intermediates. Analysis of volatiles from artificially damaged plants, with and without beet armyworm (Spodoptera exigua Hubner) oral secretions exogenously applied to the leaves, as well as volatiles from beet armyworm-damaged and -undamaged control plants, demonstrated that the application of caterpillar oral secretions increased both the production and release of several volatiles that are synthesized de novo in response to insect feeding. These results establish that the plant plays an active and dynamic role in mediating the interaction between herbivores and natural enemies of herbivores

  15. Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

    Science.gov (United States)

    Imran, Muhammad; Asad, Shaheen; Barboza, Andre Luiz; Galeano, Esteban; Carrer, Helaine; Mukhtar, Zahid

    2017-04-01

    Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4 - EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T 1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4 - EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

  16. The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis.

    Science.gov (United States)

    Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

    2015-11-01

    Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  17. Calcium-Dependent Protein Kinases in Phytohormone Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Wuwu Xu

    2017-11-01

    Full Text Available Calcium-dependent protein kinases (CPKs/CDPKs are Ca2+-sensors that decode Ca2+ signals into specific physiological responses. Research has reported that CDPKs constitute a large multigene family in various plant species, and play diverse roles in plant growth, development, and stress responses. Although numerous CDPKs have been exhaustively studied, and many of them have been found to be involved in plant hormone biosynthesis and response mechanisms, a comprehensive overview of the manner in which CDPKs participate in phytohormone signaling pathways, regulating nearly all aspects of plant growth, has not yet been undertaken. In this article, we reviewed the structure of CDPKs and the mechanism of their subcellular localization. Some CDPKs were elucidated to influence the intracellular localization of their substrates. Since little work has been done on the interaction between CDPKs and cytokinin signaling pathways, or on newly defined phytohormones such as brassinosteroids, strigolactones and salicylic acid, this paper mainly focused on discussing the integral associations between CDPKs and five plant hormones: auxins, gibberellins, ethylene, jasmonates, and abscisic acid. A perspective on future work is provided at the end.

  18. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    OpenAIRE

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun

    2011-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, wh...

  19. Resveratrol stimulates AMP kinase activity in neurons.

    Science.gov (United States)

    Dasgupta, Biplab; Milbrandt, Jeffrey

    2007-04-24

    Resveratrol is a polyphenol produced by plants that has multiple beneficial activities similar to those associated with caloric restriction (CR), such as increased life span and delay in the onset of diseases associated with aging. CR improves neuronal health, and the global beneficial effects of CR have been postulated to be mediated by the nervous system. One key enzyme thought to be activated during CR is the AMP-activated kinase (AMPK), a sensor of cellular energy levels. AMPK is activated by increases in the cellular AMP:ATP ratio, whereupon it functions to help preserve cellular energy. In this regard, the regulation of dietary food intake by hypothalamic neurons is mediated by AMPK. The suppression of nonessential energy expenditure by activated AMPK along with the CR mimetic and neuroprotective properties of resveratrol led us to hypothesize that neuronal activation of AMPK could be an important component of resveratrol activity. Here, we show that resveratrol activated AMPK in Neuro2a cells and primary neurons in vitro as well as in the brain. Resveratrol and the AMPK-activating compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) promoted robust neurite outgrowth in Neuro2a cells, which was blocked by genetic and pharmacologic inhibition of AMPK. Resveratrol also stimulated mitochondrial biogenesis in an AMPK-dependent manner. Resveratrol-stimulated AMPK activity in neurons depended on LKB1 activity but did not require the NAD-dependent protein deacetylase SIRT1 during this time frame. These findings suggest that neuronal activation of AMPK by resveratrol could affect neuronal energy homeostasis and contribute to the neuroprotective effects of resveratrol.

  20. Protein Kinase A in Cancer

    International Nuclear Information System (INIS)

    Caretta, Antonio; Mucignat-Caretta, Carla

    2011-01-01

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors

  1. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caretta, Antonio; Mucignat-Caretta, Carla, E-mail: carla.mucignat@unipd.it [Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova (Italy)

    2011-02-28

    In the past, many chromosomal and genetic alterations have been examined as possible causes of cancer. However, some tumors do not display a clear molecular and/or genetic signature. Therefore, other cellular processes may be involved in carcinogenesis. Genetic alterations of proteins involved in signal transduction have been extensively studied, for example oncogenes, while modifications in intracellular compartmentalization of these molecules, or changes in the expression of unmodified genes have received less attention. Yet, epigenetic modulation of second messenger systems can deeply modify cellular functioning and in the end may cause instability of many processes, including cell mitosis. It is important to understand the functional meaning of modifications in second messenger intracellular pathways and unravel the role of downstream proteins in the initiation and growth of tumors. Within this framework, the cAMP system has been examined. cAMP is a second messenger involved in regulation of a variety of cellular functions. It acts mainly through its binding to cAMP-activated protein kinases (PKA), that were suggested to participate in the onset and progression of various tumors. PKA may represent a biomarker for tumor detection, identification and staging, and may be a potential target for pharmacological treatment of tumors.

  2. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

    of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

  3. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens

  4. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  5. Protein Kinases in Human Breast Carcinoma

    National Research Council Canada - National Science Library

    Cane, William

    1998-01-01

    .... Rak is a novel nuclear tyrosine that our group has identified in breast cancer tissues and cell lines that has structural homology to the Src tyrosine kinase, with SH2 and SH3 domains at its amino terminus...

  6. 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

  7. Ror receptor tyrosine kinases: orphans no more

    OpenAIRE

    Green, Jennifer L.; Kuntz, Steven G.; Sternberg, Paul W.

    2008-01-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either act...

  8. Mediator kinase module and human tumorigenesis.

    Science.gov (United States)

    Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

    2015-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

  9. Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

    Science.gov (United States)

    Vemanna, Ramu S; Vennapusa, Amaranatha Reddy; Easwaran, Murugesh; Chandrashekar, Babitha K; Rao, Hanumantha; Ghanti, Kirankumar; Sudhakar, Chinta; Mysore, Kirankumar S; Makarla, Udayakumar

    2017-07-01

    In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Molecular dynamics simulations reveal the conformational dynamics of Arabidopsis thaliana BRI1 and BAK1 receptor-like kinases.

    Science.gov (United States)

    Moffett, Alexander S; Bender, Kyle W; Huber, Steven C; Shukla, Diwakar

    2017-07-28

    The structural motifs responsible for activation and regulation of eukaryotic protein kinases in animals have been studied extensively in recent years, and a coherent picture of their activation mechanisms has begun to emerge. In contrast, non-animal eukaryotic protein kinases are not as well understood from a structural perspective, representing a large knowledge gap. To this end, we investigated the conformational dynamics of two key Arabidopsis thaliana receptor-like kinases, brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1), through extensive molecular dynamics simulations of their fully phosphorylated kinase domains. Molecular dynamics simulations calculate the motion of each atom in a protein based on classical approximations of interatomic forces, giving researchers insight into protein function at unparalleled spatial and temporal resolutions. We found that in an otherwise "active" BAK1 the αC helix is highly disordered, a hallmark of deactivation, whereas the BRI1 αC helix is moderately disordered and displays swinging behavior similar to numerous animal kinases. An analysis of all known sequences in the A. thaliana kinome found that αC helix disorder may be a common feature of plant kinases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Fibronectin phosphorylation by ecto-protein kinase

    International Nuclear Information System (INIS)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

    1988-01-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

  12. The PIM kinases in hematological cancers.

    Science.gov (United States)

    Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

    2012-02-01

    The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

  13. Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment

    Directory of Open Access Journals (Sweden)

    Bruno Aquino

    2017-05-01

    Full Text Available Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK, have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK, the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1 is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

    Science.gov (United States)

    Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

    2015-01-01

    Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

  16. A role for barley calcium-dependent protein kinase CPK2a in the response to drought

    Directory of Open Access Journals (Sweden)

    Agata Cieśla

    2016-10-01

    Full Text Available Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L., one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

  17. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  18. Altered expression of Aurora kinases in Arabidopsis results in aneu- and polyploidization.

    Science.gov (United States)

    Demidov, Dmitri; Lermontova, Inna; Weiss, Oda; Fuchs, Joerg; Rutten, Twan; Kumke, Katrin; Sharbel, Timothy F; Van Damme, Daniel; De Storme, Nico; Geelen, Danny; Houben, Andreas

    2014-11-01

    Aurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase-promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, -2, and -3) of Arabidopsis we employed different combinations of T-DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP-competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1-TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  19. The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

    Directory of Open Access Journals (Sweden)

    Dong Sook Lee

    2017-10-01

    Full Text Available Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36 and overexpressing (CRK36OE plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity.

  20. A chemical genetic approach to engineer phototropin kinases for substrate labeling.

    Science.gov (United States)

    Schnabel, Jonathan; Hombach, Peter; Waksman, Thomas; Giuriani, Giovanni; Petersen, Jan; Christie, John M

    2018-04-13

    Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N 6 -benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N 6 -benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions. © 2018 Schnabel et al.

  1. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  2. Crystal structure of Cryptosporidium parvum pyruvate kinase.

    Directory of Open Access Journals (Sweden)

    William J Cook

    Full Text Available Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320 of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.

  3. A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  4. A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

    NARCIS (Netherlands)

    Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

    2015-01-01

    Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

  5. Role of adiponectin/phosphatidylinositol 3-kinase/protein kinase B ...

    African Journals Online (AJOL)

    The adiponectin/phosphatidylinositol 3-kinase/protein kinase B (ADP/PI3k/Akt) signal transduction pathway has an important role in promoting cell survival. This study was designed to determine if the ADP/PI3K/Akt signaling pathway has a role in the mechanism of ischemia–reperfusion injury in vivo. Sprague–Dawley rats ...

  6. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  7. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ting-Lei Gu

    Full Text Available Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23 of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

  8. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    Science.gov (United States)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  9. Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.

    Science.gov (United States)

    Armour, Sean M; Baur, Joseph A; Hsieh, Sherry N; Land-Bracha, Abigail; Thomas, Sheila M; Sinclair, David A

    2009-06-03

    Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

  10. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

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    Konrad Kubiński

    2017-02-01

    Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

  11. syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction

    Science.gov (United States)

    El-Hillal, O.; Kurosaki, T.; Yamamura, H.; Kinet, J.-P.; Scharenberg, A. M.

    1997-01-01

    Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well. PMID:9050880

  12. Mechanism of polyphosphate kinase from Propionibacterium shermanii

    International Nuclear Information System (INIS)

    Robinson, N.A.

    1986-01-01

    Polyphosphate kinase, which catalyzes the reaction shown below, is one of two enzymes which have been reported to catalyze the synthesis of polyphosphate. Purification performed by ammonium sulfate precipitation (0-40% fraction) was followed by chromatography. The enzyme represents 70% of the protein in the hydroxylapatite pool and is stable at this level of purity. The subunit molecular weight was determined by SDS polyacrylamide gel analysis, (83,000 +/- 3000), nondenaturing polyacrylamide gel electrophoresis, (80,000 and 86,000 daltons), gel filtration (Biogel A 0.5m column was 85,000 +/- 4000.) Polyphosphate kinase appears to be a monomeric enzyme of ∼83,000 daltons. Four assays were developed for polyphosphate kinase. Basic proteins such as polylysine stimulate the synthesis of polyphosphate, these proteins cause precipitation of polyphosphate kinase from relatively impure enzyme extracts: Synthesized polyphosphate interacts noncovalently with the basic protein-enzyme precipitate. Efficient synthesis of polyphosphate requires the addition of either phosphate or short chain polyphosphate. Synthesis did occur at 1/10 the rate when neither of these two compounds were included. Initiation, elongation, and termination events of polyphosphate synthesis were examined. Short chain polyphosphate acts as a primer, with [ 32 P] short-chain polyphosphate incorporation into long chain polyphosphate by the kinase

  13. Radioimmunoassay of bovine heart protein kinase

    International Nuclear Information System (INIS)

    Fleischer, N.; Rosen, O.M.; Reichlin, M.

    1976-01-01

    Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immunoreactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that cross reacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous

  14. The target landscape of clinical kinase drugs.

    Science.gov (United States)

    Klaeger, Susan; Heinzlmeir, Stephanie; Wilhelm, Mathias; Polzer, Harald; Vick, Binje; Koenig, Paul-Albert; Reinecke, Maria; Ruprecht, Benjamin; Petzoldt, Svenja; Meng, Chen; Zecha, Jana; Reiter, Katrin; Qiao, Huichao; Helm, Dominic; Koch, Heiner; Schoof, Melanie; Canevari, Giulia; Casale, Elena; Depaolini, Stefania Re; Feuchtinger, Annette; Wu, Zhixiang; Schmidt, Tobias; Rueckert, Lars; Becker, Wilhelm; Huenges, Jan; Garz, Anne-Kathrin; Gohlke, Bjoern-Oliver; Zolg, Daniel Paul; Kayser, Gian; Vooder, Tonu; Preissner, Robert; Hahne, Hannes; Tõnisson, Neeme; Kramer, Karl; Götze, Katharina; Bassermann, Florian; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Walch, Axel; Greif, Philipp A; Schneider, Sabine; Felder, Eduard Rudolf; Ruland, Juergen; Médard, Guillaume; Jeremias, Irmela; Spiekermann, Karsten; Kuster, Bernhard

    2017-12-01

    Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  15. Janus kinase inhibitors: jackpot or potluck?

    Directory of Open Access Journals (Sweden)

    Pavithran Keechilat

    2012-06-01

    Full Text Available The reports of a unique mutation in the Janus kinase-2 gene (JAK2 in polycythemia vera by several independent groups in 2005 quickly spurred the development of the Janus kinase inhibitors. In one of the great victories of translational research in recent times, the first smallmolecule Janus kinase inhibitor ruxolitinib entered a phase I trial in 2007. With the approval of ruxolitinib by the US Federal Drug Administration in November 2011 for high-risk and intermediate-2 risk myelofibrosis, a change in paradigm has occurred in the management of a subset of myeloproliferative neoplasms (MPN: primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Whereas the current evidence for ruxolitinib only covers high-risk and intermediate-2 risk myelofibrosis, inhibitors with greater potency are likely to offer better disease control and survival advantage in patients belonging to these categories, and possibly to the low-risk and intermediate-1 risk categories of MPN as well. But use of the Janus kinase inhibitors also probably has certain disadvantages, such as toxicity, resistance, withdrawal phenomenon, non-reversal of histology, and an implausible goal of disease clone eradication, some of which could offset the gains. In spite of this, Janus kinase inhibitors are here to stay, and for use in more than just myeloproliferative neoplasms.

  16. Protocols for the Design of Kinase-focused Compound Libraries.

    Science.gov (United States)

    Jacoby, Edgar; Wroblowski, Berthold; Buyck, Christophe; Neefs, Jean-Marc; Meyer, Christophe; Cummings, Maxwell D; van Vlijmen, Herman

    2018-05-01

    Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring

    Czech Academy of Sciences Publication Activity Database

    Andrs, M.; Kobarecny, J.; Jun, D.; Hodný, Zdeněk; Bartek, Jiří; Kuca, K.

    2015-01-01

    Roč. 58, č. 1 (2015), s. 41-71 ISSN 0022-2623 R&D Projects: GA MŠk(CZ) CZ.1.07/2.3.00/30.0044 Grant - others:University Hospital Hradec Kralove(CZ) 00179906; Faculty of Military Health Sciences, University of Defence(CZ) SV/FVZ201402 Institutional support: RVO:68378050 Keywords : DEPENDENT PROTEIN-KINASE * STRAND BREAK REPAIR * SELECTIVE PI3K-BETA INHIBITORS * TELANGIECTASIA MUTATED KINASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.589, year: 2015

  18. Receptor kinase-mediated control of primary active proton pumping at the plasma membrane

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Kristensen, Astrid; Cuin, Tracey A.

    2014-01-01

    Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro...... and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast...... heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2...

  19. Elucidating the roles of MAP kinases in the moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Stanimirovic, Sabrina

    changes of plant immunity required for the conquest of land by plants. I describe the role of MPKs (MPK3, MPK5, RAK1 & double knockout RAK1/RAK2) upon abiotic stress by characterizing the phenotypes and morphological changes there may be during stress treatments. I characterized the mutant phenotypes...... during treatment with phytohormones and osmotic and light stress. This thesis contains of a general introduction to plant immunity and the role of MPKs in signaling processes related to immunity, abiotic stress, and plant development in both vascular and non-vascular plants. The focus in this thesis......-Acetyltransferase-Kinase). This thesis and work on these MPK mutants gives the laboratory a great start on several future publications, since many of the mutant lines have interesting phenotypes with and without exposure to abiotic stresses...

  20. 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.

  1. The Role of PAS Kinase in PASsing the Glucose Signal

    Directory of Open Access Journals (Sweden)

    Julianne H. Grose

    2010-06-01

    Full Text Available PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration.

  2. Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley

    DEFF Research Database (Denmark)

    Rayapuram, Channabasavangowda; Jensen, Michael Krogh; Maiser, Fabian

    2012-01-01

    The receptor-like protein kinases (RLKs) constitute a large and diverse group of proteins controlling numerous plant physiological processes, including development, hormone perception and stress responses. The cysteine-rich RLKs (CRKs) represent a prominent subfamily of transmembrane-anchored RLKs...

  3. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  4. 2-Aminobenzimidazoles as potent Aurora kinase inhibitors.

    Science.gov (United States)

    Zhong, Min; Bui, Minna; Shen, Wang; Baskaran, Subramanian; Allen, Darin A; Elling, Robert A; Flanagan, W Michael; Fung, Amy D; Hanan, Emily J; Harris, Shannon O; Heumann, Stacey A; Hoch, Ute; Ivy, Sheryl N; Jacobs, Jeffrey W; Lam, Stuart; Lee, Heman; McDowell, Robert S; Oslob, Johan D; Purkey, Hans E; Romanowski, Michael J; Silverman, Jeffrey A; Tangonan, Bradley T; Taverna, Pietro; Yang, Wenjin; Yoburn, Josh C; Yu, Chul H; Zimmerman, Kristin M; O'Brien, Tom; Lew, Willard

    2009-09-01

    This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.

  5. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    Science.gov (United States)

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

  6. Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

    Science.gov (United States)

    Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

    2008-01-01

    The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

  7. Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.

    Science.gov (United States)

    Samuel, Marcus A; Mudgil, Yashwanti; Salt, Jennifer N; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R

    2008-08-01

    The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

  8. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  9. Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

    DEFF Research Database (Denmark)

    Niefind, K; Raaf, J; Issinger, Olaf-Georg

    2009-01-01

    the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP......Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

  10. Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

    Science.gov (United States)

    Barbosa, Inês C R; Shikata, Hiromasa; Zourelidou, Melina; Heilmann, Mareike; Heilmann, Ingo; Schwechheimer, Claus

    2016-12-15

    Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases. © 2016. Published by The Company of Biologists Ltd.

  11. Light-induced conformational changes of LOV1 (light oxygen voltage-sensing domain 1) and LOV2 relative to the kinase domain and regulation of kinase activity in Chlamydomonas phototropin.

    Science.gov (United States)

    Okajima, Koji; Aihara, Yusuke; Takayama, Yuki; Nakajima, Mihoko; Kashojiya, Sachiko; Hikima, Takaaki; Oroguchi, Tomotaka; Kobayashi, Amane; Sekiguchi, Yuki; Yamamoto, Masaki; Suzuki, Tomomi; Nagatani, Akira; Nakasako, Masayoshi; Tokutomi, Satoru

    2014-01-03

    Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

  12. Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

    Directory of Open Access Journals (Sweden)

    Amy eCurran

    2011-08-01

    Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

  13. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders. Several strategies .... family, the β-adrenergic receptor kinase (βARK), the ribosomal S6 ..... urinary bladder smooth muscle cells. While no ...

  14. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

    Brewster, Lizzy M.; Mairuhu, Gideon; Bindraban, Navin R.; Koopmans, Richard P.; Clark, Joseph F.; van Montfrans, Gert A.

    2006-01-01

    BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular

  15. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution...

  16. Deoxyribonucleoside kinases in mitochondrial DNA depletion.

    Science.gov (United States)

    Saada-Reisch, Ann

    2004-10-01

    Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

  17. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  18. Nonorthologous gene displacement of phosphomevalonate kinase

    NARCIS (Netherlands)

    Houten, S. M.; Waterham, H. R.

    2001-01-01

    Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes the phosphorylation of 5-phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis via the mevalonate pathway. So far, two nonorthologous genes encoding PMK have been described, the Saccharomyces cerevisiae ERG8

  19. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1992-01-01

    Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form...

  20. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

  1. Kinase-Centric Computational Drug Development

    NARCIS (Netherlands)

    Kooistra, Albert J.; Volkamer, Andrea

    2017-01-01

    Kinases are among the most studied drug targets in industry and academia, due to their involvement in a majority of cellular processes and, upon dysregulation, in a variety of diseases including cancer, inflammation, and autoimmune disorders. The high interest in this druggable protein family

  2. Kinases involved in Rec8 phosphorylation revealed

    Czech Academy of Sciences Publication Activity Database

    Anger, Martin

    2010-01-01

    Roč. 9, č. 14 (2010), s. 2708-2708 ISSN 1538-4101 Institutional research plan: CEZ:AV0Z50450515 Keywords : kinases * Rec8 * meisosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.999, year: 2010

  3. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

    International Nuclear Information System (INIS)

    Jensen, R.A.

    1983-01-01

    Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

  4. Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets.

    Science.gov (United States)

    Lai, Justine Y Q; Langston, Steven; Adams, Ruth; Beevers, Rebekah E; Boyce, Richard; Burckhardt, Svenja; Cobb, James; Ferguson, Yvonne; Figueroa, Eva; Grimster, Neil; Henry, Andrew H; Khan, Nawaz; Jenkins, Kerry; Jones, Mark W; Judkins, Robert; Major, Jeremy; Masood, Abid; Nally, James; Payne, Helen; Payne, Lloyd; Raphy, Gilles; Raynham, Tony; Reader, John; Reader, Valérie; Reid, Alison; Ruprah, Parminder; Shaw, Michael; Sore, Hannah; Stirling, Matthew; Talbot, Adam; Taylor, Jess; Thompson, Stephen; Wada, Hiroki; Walker, David

    2005-05-01

    This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. Copyright (c) 2004 Wiley Periodicals, Inc.

  5. Kinase detection with gallium nitride based high electron mobility transistors.

    Science.gov (United States)

    Makowski, Matthew S; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  6. p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

    204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...... MEK activity in a kinase independent manner, probably by serving as a scaffold to facilitate interaction of c-Raf....

  7. The Roles of Protein Kinases in Learning and Memory

    Science.gov (United States)

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  8. Specific RSK kinase inhibition by dibenzyl trisulfide and implication for therapeutic treatment of cancer.

    Science.gov (United States)

    Lowe, Henry I C; Facey, Caroline O B; Toyang, Ngeh J; Bryant, Joseph L

    2014-04-01

    The Jamaican "Guinea Hen Weed" (Petiveria alliacea L.) plant has been traditionally used in folklore medicine to treat a variety of diseases including cancer. In the present study we investigated on the therapeutic feasibility of dibenzyl trisulfide (DTS) (isolated from the Jamaican Guinea Hen Weed) as a potent small-molecule kinase inhibitor to treat cancer. We investigated the inhibitory effects of DTS against a large panel of kinases using a well-established competitive binding assay. Cell proliferation data were obtained using the WST-1 colorimetric assay. DTS inhibited the activity of the C-terminal kinase domain of RSK1 (80% compared to control) with a Kd of 1.3 μM. Anti-proliferative effects of DTS were observed in small lung, pancreatic, breast, and prostate cancer cells with IC50 values ranging from 0.34-0.84 μM. We have identified DTS as a highly selective and isoform-specific RSK1 kinase inhibitor with broad cancer therapeutic potential.

  9. Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study

    Science.gov (United States)

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2018-01-01

    Abstract Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5′-kinase fusion genes, combinatorial effects between 3′-KDR kinases and their 5′-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3′-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of ‘effective’ (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3′-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs’ clinical implications. PMID:28013235

  10. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

    OpenAIRE

    Halfter, Ursula; Ishitani, Manabu; Zhu, Jian-Kang

    2000-01-01

    The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na+ and K+ homeostasis and plant tolerance to high Na+ and low K+ environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis ...

  11. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

    OpenAIRE

    Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2011-01-01

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the cryst...

  12. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Science.gov (United States)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  13. OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

    Science.gov (United States)

    Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

    2016-02-01

    Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. © 2016 American Society of Plant Biologists. All Rights Reserved.

  14. Leucine-Rich repeat receptor kinases are sporadically distributed in eukaryotic genomes

    Directory of Open Access Journals (Sweden)

    Diévart Anne

    2011-12-01

    Full Text Available Abstract Background Plant leucine-rich repeat receptor-like kinases (LRR-RLKs are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs. Results We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle. Conclusions In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.

  15. Roles of Apicomplexan protein kinases at each life cycle stage.

    Science.gov (United States)

    Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

    2012-06-01

    Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals

    Science.gov (United States)

    Parthasarathy, Anutthaman; Cross, Penelope J.; Dobson, Renwick C. J.; Adams, Lily E.; Savka, Michael A.; Hudson, André O.

    2018-01-01

    Tyrosine, phenylalanine and tryptophan are the three aromatic amino acids (AAA) involved in protein synthesis. These amino acids and their metabolism are linked to the synthesis of a variety of secondary metabolites, a subset of which are involved in numerous anabolic pathways responsible for the synthesis of pigment compounds, plant hormones and biological polymers, to name a few. In addition, these metabolites derived from the AAA pathways mediate the transmission of nervous signals, quench reactive oxygen species in the brain, and are involved in the vast palette of animal coloration among others pathways. The AAA and metabolites derived from them also have integral roles in the health of both plants and animals. This review delineates the de novo biosynthesis of the AAA by microbes and plants, and the branching out of AAA metabolism into major secondary metabolic pathways in plants such as the phenylpropanoid pathway. Organisms that do not possess the enzymatic machinery for the de novo synthesis of AAA must obtain these primary metabolites from their diet. Therefore, the metabolism of AAA by the host animal and the resident microflora are important for the health of all animals. In addition, the AAA metabolite-mediated host-pathogen interactions in general, as well as potential beneficial and harmful AAA-derived compounds produced by gut bacteria are discussed. Apart from the AAA biosynthetic pathways in plants and microbes such as the shikimate pathway and the tryptophan pathway, this review also deals with AAA catabolism in plants, AAA degradation via the monoamine and kynurenine pathways in animals, and AAA catabolism via the 3-aryllactate and kynurenine pathways in animal-associated microbes. Emphasis will be placed on structural and functional aspects of several key AAA-related enzymes, such as shikimate synthase, chorismate mutase, anthranilate synthase, tryptophan synthase, tyrosine aminotransferase, dopachrome tautomerase, radical dehydratase, and type

  17. CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

    Science.gov (United States)

    Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

    2017-06-15

    Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  18. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling.

    Science.gov (United States)

    Nyati, Shyam; Schinske-Sebolt, Katrina; Pitchiaya, Sethuramasundaram; Chekhovskiy, Katerina; Chator, Areeb; Chaudhry, Nauman; Dosch, Joseph; Van Dort, Marcian E; Varambally, Sooryanarayana; Kumar-Sinha, Chandan; Nyati, Mukesh Kumar; Ray, Dipankar; Walter, Nils G; Yu, Hongtao; Ross, Brian Dale; Rehemtulla, Alnawaz

    2015-01-06

    Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion. Copyright © 2015, American Association for the Advancement of Science.

  19. Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

    Science.gov (United States)

    Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

    2008-01-01

    Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

  20. Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

    Science.gov (United States)

    Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

    2011-02-10

    Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

  1. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  2. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  3. Src family kinases in chronic kidney disease.

    Science.gov (United States)

    Wang, Jun; Zhuang, Shougang

    2017-09-01

    Src family kinases (SFKs) belong to nonreceptor protein tyrosine kinases and have been implicated in the regulation of numerous cellular processes, including cell proliferation, differentiation, migration and invasion, and angiogenesis. The role and mechanisms of SFKs in tumorgenesis have been extensively investigated, and some SFK inhibitors are currently under clinical trials for tumor treatment. Recent studies have also demonstrated the importance of SFKs in regulating the development of various fibrosis-related chronic diseases (e.g., idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, and systemic sclerosis). In this article, we summarize the roles of SFKs in various chronic kidney diseases, including glomerulonephritis, diabetic nephropathy, human immunodeficiency virus-associated nephropathy, autosomal dominant form of polycystic kidney disease, and obesity-associated kidney disease, and discuss the mechanisms involved. Copyright © 2017 the American Physiological Society.

  4. Tyrosine kinase signalling in breast cancer

    International Nuclear Information System (INIS)

    Hynes, Nancy E

    2000-01-01

    Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research

  5. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    International Nuclear Information System (INIS)

    Boura, Evzen; Nencka, Radim

    2015-01-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

  6. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  7. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

    Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

    2008-11-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

  8. Aurora kinase inhibitors: Progress towards the clinic

    Czech Academy of Sciences Publication Activity Database

    Kollareddy, M.; Zheleva, D.; Dzubak, P.; Brahmkshatriya, Pathik; Lepšík, Martin; Hajduch, M.

    2012-01-01

    Roč. 30, č. 6 (2012), s. 2411-2432 ISSN 0167-6997 Grant - others:GA ČR(CZ) GA301/08/1649; GA ČR(CZ) GD303/09/H048 Program:GA; GD Institutional research plan: CEZ:AV0Z40550506 Keywords : Aurora kinases * cancer * inhibitors Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2012

  9. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Jørgen; Seidelin, Jakob Benedict

    2011-01-01

    The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...... 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....

  10. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Nencka, Radim

    2015-01-01

    Roč. 337, č. 2 (2015), s. 136-145 ISSN 0014-4827 R&D Projects: GA ČR GJ15-21030Y; GA MŠk LO1302; GA ČR GA15-09310S EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : phosphatidylinositol 4-kinase * inhibitor * crystal structure * virus Subject RIV: CC - Organic Chemistry Impact factor: 3.378, year: 2015

  11. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  12. Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens

    Science.gov (United States)

    Harkenrider, Mitch; Sharma, Rita; De Vleesschauwer, David; Tsao, Li; Zhang, Xuting; Chern, Mawsheng; Canlas, Patrick; Zuo, Shimin; Ronald, Pamela C.

    2016-01-01

    Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus. PMID:26795719

  13. The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity.

    Science.gov (United States)

    Li, Guowei; Boudsocq, Marie; Hem, Sonia; Vialaret, Jérôme; Rossignol, Michel; Maurel, Christophe; Santoni, Véronique

    2015-07-01

    The hydraulic conductivity of plant roots (Lp(r)) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp(r) of knockout Arabidopsis plants for four Ca(2+)-dependent protein kinases. cpk7 plants showed a 30% increase in Lp(r) because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp(r) of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins. © 2014 John Wiley & Sons Ltd.

  14. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

    Science.gov (United States)

    Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

    2000-09-12

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

  15. CIKS, a connection to IκB kinase and stress-activated protein kinase

    Science.gov (United States)

    Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

    2000-01-01

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

  16. SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    Burg, van den H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  17. SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    van den Burg, H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  18. 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....

  19. Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases.

    Science.gov (United States)

    Ho, Chin-Min Kimmy; Paciorek, Tomasz; Abrash, Emily; Bergmann, Dominique C

    2016-08-22

    Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The First Pentacyclic Triterpenoid Gypsogenin Derivative Exhibiting Anti-ABL1 Kinase and Anti-chronic Myelogenous Leukemia Activities.

    Science.gov (United States)

    Ciftci, Halil Ibrahim; Ozturk, Safiye Emirdag; Ali, Taha F S; Radwan, Mohamed O; Tateishi, Hiroshi; Koga, Ryoko; Ocak, Zeynep; Can, Mustafa; Otsuka, Masami; Fujita, Mikako

    2018-04-01

    The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC 50 value of 9.3 µM. In contrast, the IC 50 against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC 50 =8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

  1. RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

    Science.gov (United States)

    Chen, Jiwei; Zhang, Qian; Wang, Qigang; Feng, Ming; Li, Yang; Meng, Yonglu; Zhang, Yi; Liu, Guoqin; Ma, Zhimin; Wu, Hongzhi; Gao, Junping; Ma, Nan

    2017-02-23

    Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration. We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter. Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.

  2. The Pim kinases: new targets for drug development.

    Science.gov (United States)

    Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis

    2011-12-01

    The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.

  3. A Comparative Study of the Aneugenic and Polyploidy-inducing Effects of Fisetin and Two Model Aurora Kinase Inhibitors

    Science.gov (United States)

    Gollapudi, P.; Hasegawa, L.S.; Eastmond, D.A.

    2014-01-01

    Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981

  4. PAK4 crystal structures suggest unusual kinase conformational movements.

    Science.gov (United States)

    Zhang, Eric Y; Ha, Byung Hak; Boggon, Titus J

    2018-02-01

    In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077). We find an unusual motion between the N-lobe and C-lobe of PAK4 that manifests as a partial unwinding of helix αC. Principal component analysis of the crystal structures rationalizes these movements into three major states, and analysis of the kinase hydrophobic spines indicates concerted movements that create an accessible back pocket cavity. The conformational changes that we observe for PAK4 differ from previous descriptions of kinase motions, and although we observe these differences in crystal structures there is the possibility that the movements observed may suggest a diversity of kinase conformational changes associated with regulation. Protein kinases are key signaling proteins, and are important drug targets, therefore understanding their regulation is important for both basic research and clinical points of view. In this study, we observe unusual conformational 'hinging' for protein kinases. Hinging, the opening and closing of the kinase sub-domains to allow nucleotide binding and release, is critical for proper kinase regulation and for targeted drug discovery. We determine new crystal structures of PAK4, an important Rho-effector kinase, and conduct analyses of these and previously determined structures. We find that PAK4 crystal structures can be classified into specific conformational groups, and that these groups are associated with previously unobserved hinging motions and an unusual conformation for the kinase hydrophobic core. Our findings therefore indicate that there may be a diversity of kinase hinging motions, and that these may

  5. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

    Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

  6. Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator

    Science.gov (United States)

    Akten, Bikem; Tangredi, Michelle M.; Jauch, Eike; Roberts, Mary A.; Ng, Fanny; Raabe, Thomas; Jackson, F. Rob

    2009-01-01

    There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the MAPK pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the Casein Kinase 2 regulatory subunit (CK2β), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism. PMID:19144847

  7. Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

    OpenAIRE

    Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

    2000-01-01

    A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

  8. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

    , but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

  9. Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana BRI1-associated kinase 1 (BAK1) cytoplasmic domain

    International Nuclear Information System (INIS)

    Gao, Jian; Ma, Yuanyuan; Sun, Yuna; Zhao, Huadong; Hong, Dapeng; Yan, Liming; Lou, Zhiyong

    2012-01-01

    The cytoplasmic domain of BRI1-associated kinase 1 from A. thaliana has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution. BRI1-associated kinase 1 (BAK1) is a member of the plant receptor-like kinase (RLK) superfamily. BAK1 has been shown to initiate brassinosteroid (BR) signalling and innate immune responses in plants by forming receptor complexes with both brassinosteroid-insensitive 1 (BRI1) and flagellin-sensing 2 (FLS2). To gain a better understanding of the structural details and the mechanism of action of the BAK1 kinase domain, recombinant BAK1 cytoplasmic domain has been expressed, purified and crystallized at 291 K using PEG 3350 as a precipitant. A 2.6 Å resolution data set was collected from a single flash-cooled crystal at 100 K. This crystal belonged to space group C2, with unit-cell parameters a = 70.3, b = 75.6, c = 71.9 Å, β = 93.1°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient was 2.6 Å 3 Da −1

  10. Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

    International Nuclear Information System (INIS)

    Mariano, Andrea C.; Andrade, Maxuel O.; Santos, Anesia A.; Carolino, Sonia M.B.; Oliveira, Marli L.; Baracat-Pereira, Maria Cristina; Brommonshenkel, Sergio H.; Fontes, Elizabeth P.B.

    2004-01-01

    Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed

  11. The secret life of kinases: functions beyond catalysis.

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  12. SH2 domains: modulators of nonreceptor tyrosine kinase activity

    OpenAIRE

    Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

    2009-01-01

    The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed ...

  13. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

    Johansson, K.; Ramaswamy, S.; Ljungcrantz, C.

    2001-01-01

    Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine ki......; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases....

  14. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    Energy Technology Data Exchange (ETDEWEB)

    Knecht, Wolfgang [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Mikkelsen, Nils Egil [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Clausen, Anders Ranegaard [Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden); Willer, Mette [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Gojkovic, Zoran [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Piskur, Jure, E-mail: Jure.Piskur@cob.lu.se [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden)

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  15. Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

    DEFF Research Database (Denmark)

    Naik, M U; Benedikz, Eirikur; Hernandez, I

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region...

  16. Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2.

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

    Full Text Available Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32P [counts per minute (CPM] for each peptide substrate was determined by the radiolabel assay using [γ-(32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135 were phosphorylated by other enzymes (PKA, PKCα, and ERK1, R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

  17. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    Science.gov (United States)

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  18. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

  19. Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition

    DEFF Research Database (Denmark)

    Soundararajan, M.; Roos, A.K.; Savitsky, P.

    2013-01-01

    Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK sub...

  20. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    International Nuclear Information System (INIS)

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-01-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification

  1. Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

    Directory of Open Access Journals (Sweden)

    Inger Lindin

    2014-03-01

    Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

  2. How protein kinases co-ordinate mitosis in animal cells.

    Science.gov (United States)

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  3. SH2 domains: modulators of nonreceptor tyrosine kinase activity.

    Science.gov (United States)

    Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

    2009-12-01

    The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

  4. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Directory of Open Access Journals (Sweden)

    Saadat U Aleem

    Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  5. Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption

    DEFF Research Database (Denmark)

    Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo

    2012-01-01

    Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation of replic......Activation of oncogenes or inhibition of WEE1 kinase deregulates Cyclin-dependent kinase (CDK) activity and leads to replication stress, however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibiting WEE1 kinase rapidly increases initiation...... of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted...

  6. A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

    DEFF Research Database (Denmark)

    Götz, C; Koenig, M G; Issinger, O G

    1995-01-01

    by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

  7. SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

    Science.gov (United States)

    Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

    1997-10-31

    Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

  8. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    OpenAIRE

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in tr...

  9. Radioimmunoassay of creatine kinase BB isoenzyme

    Energy Technology Data Exchange (ETDEWEB)

    Jianguo, Geng [Shanghai Medical Univ. (China). Zhongshan Hospital; and others

    1988-11-01

    A radioimmunoassay of creatine kinase BB isoenzyme (CK-BB) was developed by using CK-BB purified from human brain. The CK-BB antiserum was raised by immunizing rabbite and {sup 125}I-CK-BB iodinated with Bolton-Hunter reagent. The affinity constant was 3.0 x 10{sup 9} mol/L. No cross reactions with creatine kinase MM isoenzyme and neuron-specific enolase were found. The measuring range was 3.5 x 10{sup -8} {approx} 1.2 x 10{sup -5} mmol/L, the average recovery rate 97.5%, with the inter and intrassay CV 3.1% and 12%, respectively. The average serum CK-BB concentration in 83 normal persons was 1.5 x 10{sup -7} +- 8.1 x 10{sup -8} mmol/L, quite different from the values of acute myocardial infarction (5.2 x 10{sup -6} +- 1.2 x 10{sup -4} mmol/L, n = 28) and cerebral vascular accident (8.4 x 10{sup -4} +- 5.0 x 10{sup -4} mmol/L, n = 10).

  10. Targeting the Pim kinases in multiple myeloma.

    LENUS (Irish Health Repository)

    Keane, N A

    2015-07-17

    Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine\\/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and\\/or with novel drugs targeting other survival pathways in MM.

  11. Radioimmunoassay of creatine kinase BB isoenzyme

    International Nuclear Information System (INIS)

    Geng Jianguo

    1988-01-01

    A radioimmunoassay of creatine kinase BB isoenzyme (CK-BB) was developed by using CK-BB purified from human brain. The CK-BB antiserum was raised by immunizing rabbite and 125 I-CK-BB iodinated with Bolton-Hunter reagent. The affinity constant was 3.0 x 10 9 mol/L. No cross reactions with creatine kinase MM isoenzyme and neuron-specific enolase were found. The measuring range was 3.5 x 10 -8 ∼ 1.2 x 10 -5 mmol/L, the average recovery rate 97.5%, with the inter and intrassay CV 3.1% and 12%, respectively. The average serum CK-BB concentration in 83 normal persons was 1.5 x 10 -7 +- 8.1 x 10 -8 mmol/L, quite different from the values of acute myocardial infarction (5.2 x 10 -6 +- 1.2 x 10 -4 mmol/L, n = 28) and cerebral vascular accident (8.4 x 10 -4 +- 5.0 x 10 -4 mmol/L, n = 10)

  12. Activation of oocyte phosphatidylinositol kinase by polyamines

    International Nuclear Information System (INIS)

    Allende, J.E.; Carrasco, D.; Allende, C.C.

    1987-01-01

    Membrane bound phosphatidylinositol is phosphorylated by a specific membrane enzyme to form phosphatidylinositol 4 phosphate (PIP) which in turn is again phosphorylated to generate phosphatidylinositol 4,5 biphosphate (PIPP). The regulation of phosphatidylinositol phosphorylation and hydrolysis is relevant to the possible role of inositol phosphates as second messengers of hormone action. The membranes of Xenopus laevis oocytes contain a phosphatidylinositol kinase that can generate radioactive PIP after incubation with [ 32 ATP]. The radioactive product is extracted with methanol-chloroform and isolated by thin layer chromatography. The oocyte enzyme has an app Km for ATP of 80 μM and cannot use GTP as a phosphate donor. The formation of PIP is greatly stimulated by the addition of synthetic peptides containing clusters of polylysine at concentrations 0.5 mM. A similar effect is observed with a lysine rich peptide that corresponds to the 14 amino acids of the carboxyl terminus of the Kirstein ras 2 protein and also by polyornithine. Polyarginine and histone H 1 have much lower effects. Peptides containing polylysine clusters have also been found to affect the activity of other key membrane enzymes such as protein kinases and adenylate cyclase

  13. ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis

    OpenAIRE

    Rodrigues, A.; Adamo, M.; Crozet, P.; Margalha, L.; Confraria, A.; Martinho, C.; Elias, A.; Rabissi, A.; Lumbreras, V.; Gonzalez-Guzman, M.; Antoni, R.; Rodriguez, P. L.; Baena-Gonzalez, E.

    2013-01-01

    Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs),...

  14. Simultaneous Expression of PDH45 with EPSPS Gene Improves Salinity and Herbicide Tolerance in Transgenic Tobacco Plants.

    Science.gov (United States)

    Garg, Bharti; Gill, Sarvajeet S; Biswas, Dipul K; Sahoo, Ranjan K; Kunchge, Nandkumar S; Tuteja, Renu; Tuteja, Narendra

    2017-01-01

    To cope with the problem of salinity- and weed-induced crop losses, a multi-stress tolerant trait is need of the hour but a combinatorial view of such traits is not yet explored. The overexpression of PDH45 (pea DNA helicase 45) and EPSPS (5-enoylpruvyl shikimate-3-phosphate synthase) genes have been reported to impart salinity and herbicide tolerance. Further, the understanding of mechanism and pathways utilized by PDH45 and EPSPS for salinity and herbicide tolerance will help to improve the crops of economical importance. In the present study, we have performed a comparative analysis of salinity and herbicide tolerance to check the biochemical parameters and antioxidant status of tobacco transgenic plants. Collectively, the results showed that PDH45 overexpressing transgenic lines display efficient tolerance to salinity stress, while PDH45+EPSPS transgenics showed tolerance to both the salinity and herbicide as compared to the control [wild type (WT) and vector control (VC)] plants. The activities of the components of enzymatic antioxidant machinery were observed to be higher in the transgenic plants indicating the presence of an efficient antioxidant defense system which helps to cope with the stress-induced oxidative-damages. Photosynthetic parameters also showed significant increase in PDH45 and PDH45+EPSPS overexpressing transgenic plants in comparison to WT, VC and EPSPS transgenic plants under salinity stress. Furthermore, PDH45 and PDH45+EPSPS synergistically modulate the jasmonic acid and salicylic acid mediated signaling pathways for combating salinity stress. The findings of our study suggest that pyramiding of the PDH45 gene with EPSPS gene renders host plants tolerant to salinity and herbicide by enhancing the antioxidant machinery thus photosynthesis.

  15. A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.

    Science.gov (United States)

    Thamm, Antje M; Li, Gengnan; Taja-Moreno, Marlene; Gerdes, Svetlana Y; de Crécy-Lagard, Valérie; Bruner, Steven D; Hanson, Andrew D

    2017-07-20

    The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover. ©2017 The Author(s).

  16. Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity.

    Science.gov (United States)

    Harper, Shannon M; Christie, John M; Gardner, Kevin H

    2004-12-28

    Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

  17. Interaction of Medicago truncatula Lysin Motif Receptor-Like Kinases, NFP and LYK3, Produced in Nicotiana benthamiana Induces Defence-Like Responses

    NARCIS (Netherlands)

    Pietraszewska-Bogiel, A.; Lefebvre, B.; Koini, A.M.; Klaus-Heisen, D.; Takken, F.L.W.; Geurts, R.; Cullimore, J.V.; Gadella, Th.W.J.

    2013-01-01

    Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP

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

    Energy Technology Data Exchange (ETDEWEB)

    Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr

    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. - Highlights: • Multiple regions on the Arabidopsis Raptor protein were found to be involved in substrate binding. • N-terminal end of the Arabidopsis ribosomal S6 kinase 1 (AtS6K1) was responsible for interacting with AtRaptor1. • The Raptor-interacting fragment of AtS6K1 could be utilized as an effective inhibitor of plant TOR signaling.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • Multiple regions on the Arabidopsis Raptor protein were found to be involved in substrate binding. • N-terminal end of the Arabidopsis ribosomal S6 kinase 1 (AtS6K1) was responsible for interacting with AtRaptor1. • The Raptor-interacting fragment of AtS6K1 could be utilized as an effective inhibitor of plant TOR signaling.

  20. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (Purdue); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-{angstrom} resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases.

  1. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Energy Technology Data Exchange (ETDEWEB)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  2. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    International Nuclear Information System (INIS)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-01-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

  3. Creatine kinase isozyme expression in embryonic chicken heart

    NARCIS (Netherlands)

    Lamers, W. H.; Geerts, W. J.; Moorman, A. F.; Dottin, R. P.

    1989-01-01

    The distribution pattern of creatine kinase (EC 2.7.3.2) isozymes in developing chicken heart was studied by immunohistochemistry. Creatine kinase M, which is absent from adult heart, is transiently expressed between 4 and 11 days of incubation. During that period, numerous muscular cells in the

  4. Role of Bruton's tyrosine kinase in B cells and malignancies

    NARCIS (Netherlands)

    Pal Singh, S. (Simar); F. Dammeijer (Floris); R.W. Hendriks (Rudi)

    2018-01-01

    textabstractBruton's tyrosine kinase (BTK) is a non-receptor kinase that plays a crucial role in oncogenic signaling that is critical for proliferation and survival of leukemic cells in many B cell malignancies. BTK was initially shown to be defective in the primary immunodeficiency X-linked

  5. Oral protein kinase c β inhibition using ruboxistaurin

    DEFF Research Database (Denmark)

    Aiello, Lloyd Paul; Vignati, Louis; Sheetz, Matthew J

    2011-01-01

    To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C β Inhibitor-Diabetic Retinopathy Study and Protein Kinase C β Inhibitor-Diabetic Retinopathy Study 2 ruboxi...

  6. Molecular cloning and characterization of a novel human kinase ...

    Indian Academy of Sciences (India)

    throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the ...

  7. Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

    NARCIS (Netherlands)

    Tavanti, E.; Sero, V.; Vella, S.; Fanelli, M.; Michelacci, F.; Landuzzi, L.; Magagnoli, G.; Versteeg, R.; Picci, P.; Hattinger, C. M.; Serra, M.

    2013-01-01

    Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell

  8. ATR kinase regulates its attenuation via PPM1D phosphatase ...

    Indian Academy of Sciences (India)

    In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogationleads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition ofATR kinase activity specifically during replication stress recovery results in persistent ...

  9. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  10. Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity

    Directory of Open Access Journals (Sweden)

    Vijayata Singh

    2017-08-01

    Full Text Available The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1 functions as a co-receptor with several receptor kinases including the brassinosteroid (BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1, which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2 and EF-TU RECEPTOR (EFR, respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F in the bak1-4 null mutant. Neither BAK1 (Y610F transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive

  11. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...... that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...

  12. Tyrosine kinases, drugs, and Shigella flexneri dissemination.

    Science.gov (United States)

    Dragoi, Ana-Maria; Agaisse, Hervé

    2014-01-01

    Shigella flexneri is an enteropathogenic bacterium responsible for approximately 100 million cases of severe dysentery each year. S. flexneri colonization of the human colonic epithelium is supported by direct spread from cell to cell, which relies on actin-based motility. We have recently uncovered that, in intestinal epithelial cells, S. flexneri actin-based motility is regulated by the Bruton's tyrosine kinase (Btk). Consequently, treatment with Ibrutinib, a specific Btk inhibitor currently used in the treatment of B-cell malignancies, effectively impaired S. flexneri spread from cell to cell. Thus, therapeutic intervention capitalizing on drugs interfering with host factors supporting the infection process may represent an effective alternative to treatments with antimicrobial compounds.

  13. 2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

    Science.gov (United States)

    Dow, Robert L; Ammirati, Mark; Bagley, Scott W; Bhattacharya, Samit K; Buckbinder, Leonard; Cortes, Christian; El-Kattan, Ayman F; Ford, Kristen; Freeman, Gary B; Guimarães, Cristiano R W; Liu, Shenping; Niosi, Mark; Skoura, Athanasia; Tess, David

    2018-04-12

    Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

  14. Protein kinase substrate identification on functional protein arrays

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    2008-02-01

    Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

  15. Src protein-tyrosine kinase structure and regulation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2004-01-01

    Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

  16. Structural studies of Schistosoma mansoni adenylate kinases

    International Nuclear Information System (INIS)

    Marques, I.A.; Pereira, H.M.; Garrat, R.C.

    2012-01-01

    Full text: Parasitic diseases are a major cause of death in developing countries, however receive little or no attention from pharmaceutical companies for the development of novel therapies. In this respect, the Center for Structural Molecular Biology (CBME) of the Institute of Physics of Sao Carlos (IFSC / USP) has developed expertise in all stages of the development of active compounds against target enzymes from parasitic diseases. The present work focuses on the adenylate kinase enzymes (ADK's) from Schistosoma mansoni. These enzymes are widely distributed and catalyze the reaction of phosphoryl exchange between nucleotides in the reaction 2ADP to ATP + AMP, which is critical for the cells life cycle. Due to the particular property of the reaction catalyzed, the ADK's are recognized as reporters of the cells energetic state, translating small changes in the balance between ATP and ADP into a large change in concentration of AMP. The genome of S. mansoni was recently sequenced by the Sanger Center in England. On performing searches for genes encoding adenylate kinases we found two such genes. The corresponding gene products were named ADK1 (197 residues) and ADK2 (239 residues), and the two sequences share only 28 percent identity. Both have been cloned into the pET-28a(+)vector, expressed in E. coli and purified. Preliminary tests of activity have been performed only for ADK1 showing it to be catalytically active. Crystallization trials were performed for both proteins and thus far, crystals of ADK1 have been obtained which diffract to 2.05 at the LNLS beamline MX2 and the structure solved by molecular replacement. Understanding, at the atomic level, the function of these enzymes may help in the development of specific inhibitors and may provide tools for developing diagnostic tests for schistosomiasis. (author)

  17. Hybrid and rogue kinases encoded in the genomes of model eukaryotes.

    Directory of Open Access Journals (Sweden)

    Ramaswamy Rakshambikai

    Full Text Available The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes-S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens-and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.

  18. Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1.

    Science.gov (United States)

    Oide, Mao; Okajima, Koji; Kashojiya, Sachiko; Takayama, Yuki; Oroguchi, Tomotaka; Hikima, Takaaki; Yamamoto, Masaki; Nakasako, Masayoshi

    2016-09-16

    Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    Science.gov (United States)

    2017-01-01

    v e V ia b il it y Figure 8. PC3-LN4 cells in normoxia or hypoxia were treated with Pim inhibitors. Left panel shows a Western blot and the...3728-36, PMID 25241892 4. Warfel, NA, Kraft, AS. Pim kinase (and Akt) biology and signaling in tumors. Pharmacol Ther. 2015 Jul; 151: 41 - 9. doi: 10.1016...Associated Fibroblast Biology in Prostate Cancer These studies will accelerate and significantly advance the rational development of targeted agents

  20. Creatine kinase and creatine kinase subunit-B in coronary sinus blood in pacing-induced angina pectoris

    DEFF Research Database (Denmark)

    Bagger, J P; Ingerslev, J; Heinsvig, E M

    1982-01-01

    In nine out of 10 patients with angiographic documented coronary artery disease, pacing-induced angina pectoris provoked myocardial production of lactate, whereas no significant release of either creatine kinase or creatine kinase subunit-B to coronary sinus and peripheral venous blood could...

  1. Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

    Science.gov (United States)

    Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

    2015-12-01

    Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

    Science.gov (United States)

    Polte, T R; Hanks, S K

    1995-11-07

    The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

  3. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  4. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  5. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    International Nuclear Information System (INIS)

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-01-01

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

  6. Genome-wide Identification and Expression Analysis of Calcium-dependent Protein Kinase and Its Closely Related Kinase Genes in Capsicum annuum

    Directory of Open Access Journals (Sweden)

    hanyang ecai

    2015-09-01

    Full Text Available As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs play important roles in regulating the downstream components of calcium signaling, which are ubiquitously involved in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a comprehensive analysis of genes encoding pepper CDPKs and CDPK-related protein kinases (CRKs was performed, and 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CaCPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the Capsicum annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and eight CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.

  7. STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) Interacts with Protein Kinase SnRK11[OPEN

    Science.gov (United States)

    Nietzsche, Madlen; Guerra, Tiziana; Fernie, Alisdair R.

    2018-01-01

    Sucrose nonfermenting related kinase1 (SnRK1) is a conserved energy sensor kinase that regulates cellular adaptation to energy deficit in plants. Activation of SnRK1 leads to the down-regulation of ATP-consuming biosynthetic processes and the stimulation of energy-generating catabolic reactions by transcriptional reprogramming and posttranslational modifications. Although considerable progress has been made during the last years in understanding the SnRK1 signaling pathway, many of its components remain unidentified. Here, we show that the catalytic α-subunits KIN10 and KIN11 of the Arabidopsis (Arabidopsis thaliana) SnRK1 complex interact with the STOREKEEPER RELATED1/G-Element Binding Protein (STKR1) inside the plant cell nucleus. Overexpression of STKR1 in transgenic Arabidopsis plants led to reduced growth, a delay in flowering, and strongly attenuated senescence. Metabolite profiling revealed that the transgenic lines exhausted their carbohydrates during the dark period to a greater extent than the wild type and accumulated a range of amino acids. At the global transcriptome level, genes affected by STKR1 overexpression were broadly associated with systemic acquired resistance, and transgenic plants showed enhanced resistance toward a virulent strain of the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis Noco2. We discuss a possible connection of STKR1 function, SnRK1 signaling, and plant immunity. PMID:29192025

  8. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

  9. Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

    Science.gov (United States)

    Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

    2012-01-01

    To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

  10. Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

    Science.gov (United States)

    Dalton, George D; Dewey, William L

    2006-02-01

    Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

  11. 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.

  12. Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

    Science.gov (United States)

    Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-01-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

  13. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

    Science.gov (United States)

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-03-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

  14. Cycloartane-3,24,25-triol inhibits MRCKα kinase and demonstrates promising anti prostate cancer activity in vitro

    Directory of Open Access Journals (Sweden)

    Lowe Henry I C

    2012-11-01

    Full Text Available Abstract Background Given the high occurrence of prostate cancer worldwide and one of the major sources of the discovery of new lead molecules being medicinal plants, this research undertook to investigate the possible anti-cancer activity of two natural cycloartanes; cycloartane-3,24,25-diol (extracted in our lab from Tillandsia recurvata and cycloartane-3,24,25-triol (purchased. The inhibition of MRCKα kinase has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Methods Kinase inhibition was investigated using competition binding (to the ATP sites assays which have been previously established and authenticated and cell proliferation was measured using the WST-1 assay. Results Cycloartane-3,24,25-triol demonstrated strong selectivity towards the MRCKα kinase with a Kd50 of 0.26 μM from a total of 451 kinases investigated. Cycloartane-3,24,25-triol reduced the viability of PC-3 and DU145 cell lines with IC50 values of 2.226 ± 0.28 μM and 1.67 ± 0.18 μM respectively. Conclusions These results will prove useful in drug discovery as Cycloartane-3,24,25-triol has shown potential for development as an anti-cancer agent against prostate cancer.

  15. Cycloartane-3,24,25-triol inhibits MRCKα kinase and demonstrates promising anti prostate cancer activity in vitro

    Science.gov (United States)

    2012-01-01

    Background Given the high occurrence of prostate cancer worldwide and one of the major sources of the discovery of new lead molecules being medicinal plants, this research undertook to investigate the possible anti-cancer activity of two natural cycloartanes; cycloartane-3,24,25-diol (extracted in our lab from Tillandsia recurvata) and cycloartane-3,24,25-triol (purchased). The inhibition of MRCKα kinase has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Methods Kinase inhibition was investigated using competition binding (to the ATP sites) assays which have been previously established and authenticated and cell proliferation was measured using the WST-1 assay. Results Cycloartane-3,24,25-triol demonstrated strong selectivity towards the MRCKα kinase with a Kd50 of 0.26 μM from a total of 451 kinases investigated. Cycloartane-3,24,25-triol reduced the viability of PC-3 and DU145 cell lines with IC50 values of 2.226 ± 0.28 μM and 1.67 ± 0.18 μM respectively. Conclusions These results will prove useful in drug discovery as Cycloartane-3,24,25-triol has shown potential for development as an anti-cancer agent against prostate cancer. PMID:23151005

  16. Cycloartane-3,24,25-triol inhibits MRCKα kinase and demonstrates promising anti prostate cancer activity in vitro.

    Science.gov (United States)

    Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

    2012-11-14

    Given the high occurrence of prostate cancer worldwide and one of the major sources of the discovery of new lead molecules being medicinal plants, this research undertook to investigate the possible anti-cancer activity of two natural cycloartanes; cycloartane-3,24,25-diol (extracted in our lab from Tillandsia recurvata) and cycloartane-3,24,25-triol (purchased). The inhibition of MRCKα kinase has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays which have been previously established and authenticated and cell proliferation was measured using the WST-1 assay. Cycloartane-3,24,25-triol demonstrated strong selectivity towards the MRCKα kinase with a Kd50 of 0.26 μM from a total of 451 kinases investigated. Cycloartane-3,24,25-triol reduced the viability of PC-3 and DU145 cell lines with IC50 values of 2.226 ± 0.28 μM and 1.67 ± 0.18 μM respectively. These results will prove useful in drug discovery as Cycloartane-3,24,25-triol has shown potential for development as an anti-cancer agent against prostate cancer.

  17. MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability.

    Science.gov (United States)

    Zhao, Chunzhao; Wang, Pengcheng; Si, Tong; Hsu, Chuan-Chih; Wang, Lu; Zayed, Omar; Yu, Zheping; Zhu, Yingfang; Dong, Juan; Tao, W Andy; Zhu, Jian-Kang

    2017-12-04

    Mitogen-activated protein kinase cascades are important signaling modules that convert environmental stimuli into cellular responses. We show that MPK3, MPK4, and MPK6 are rapidly activated after cold treatment. The mpk3 and mpk6 mutants display increased expression of CBF genes and enhanced freezing tolerance, whereas constitutive activation of the MKK4/5-MPK3/6 cascade in plants causes reduced expression of CBF genes and hypersensitivity to freezing, suggesting that the MKK4/5-MPK3/6 cascade negatively regulates the cold response. MPK3 and MPK6 can phosphorylate ICE1, a basic-helix-loop-helix transcription factor that regulates the expression of CBF genes, and the phosphorylation promotes the degradation of ICE1. Interestingly, the MEKK1-MKK2-MPK4 pathway constitutively suppresses MPK3 and MPK6 activities and has a positive role in the cold response. Furthermore, the MAPKKK YDA and two calcium/calmodulin-regulated receptor-like kinases, CRLK1 and CRLK2, negatively modulate the cold activation of MPK3/6. Our results uncover important roles of MAPK cascades in the regulation of plant cold response. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

    Science.gov (United States)

    Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

    2017-06-29

    Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

  19. Constitutive Activity of the Arabidopsis MAP Kinase 3 Confers Resistance to Pseudomonas syringae and Drives Robust Immune Responses

    KAUST Repository

    Lang, Julien

    2017-08-02

    Mitogen Activated Protein Kinases (MAPKs) are known to be important mediators of plant responses to biotic and abiotic stresses. In a recent report, we enlarged the understanding of the Arabidopsis thaliana MPK3 functions showing that the expression of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation of several defense genes. Consistently with these data, we present here results demonstrating that, compared to wild type controls, CA-MPK3 plants are more resistant to the hemibiotrophic pathogen Pseudomonas syringae DC3000. Based on our previous work, we also discuss the mechanisms of robust plant immunity controlled by sustained MPK3 activity, focusing especially on the roles of disease resistance proteins.

  20. RhoA/Rho-Kinase in the Cardiovascular System.

    Science.gov (United States)

    Shimokawa, Hiroaki; Sunamura, Shinichiro; Satoh, Kimio

    2016-01-22

    Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system. © 2016 American Heart Association, Inc.

  1. Kinase inhibition by the Jamaican ball moss, Tillandsia recurvata L.

    Science.gov (United States)

    Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

    2012-10-01

    This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd(50)s were below 20 μg/ml. Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.

  2. Structure of the intact ATM/Tel1 kinase

    Science.gov (United States)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  3. Medicinal Plants.

    Science.gov (United States)

    Phillipson, J. David

    1997-01-01

    Highlights the demand for medicinal plants as pharmaceuticals and the demand for health care treatments worldwide and the issues that arise from this. Discusses new drugs from plants, anticancer drugs, antiviral drugs, antimalarial drugs, herbal remedies, quality, safety, efficacy, and conservation of plants. Contains 30 references. (JRH)

  4. Plant Physiology: FERONIA Defends the Cell Walls against Corrosion.

    Science.gov (United States)

    Verger, Stéphane; Hamant, Olivier

    2018-03-05

    A new study uncovers the role of wall sensing and remodeling in the plant response to salt stress, identifying the FERONIA receptor kinase as a key player in that process, likely through direct sensing of cell wall pectins. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. The 'retro-design' concept for novel kinase inhibitors.

    Science.gov (United States)

    Müller, Gerhard; Sennhenn, Peter C; Woodcock, Timothy; Neumann, Lars

    2010-07-01

    Protein kinases are among the most attractive therapeutic targets for a broad range of diseases. This feature review highlights and classifies the main design principles employed to generate active and selective kinase inhibitors. In particular, emphasis is focused on a fragment-based lead-generation approach, which constitutes a novel design method for developing type II kinase inhibitors with distinct binding kinetic attributes. This 'retro-design' strategy relies on a customized fragment library, and contrasts the traditional approach used in the design of type II inhibitors.

  6. Second-generation inhibitors of Bruton tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Jingjing Wu

    2016-09-01

    Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

  7. Nucleolin (C23), a physiological substrate for casein kinase II

    DEFF Research Database (Denmark)

    Schneider, H R; Issinger, O G

    1988-01-01

    Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin...... phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated...... by purified casein kinase II with about two moles phosphate per one mole of nucleolin....

  8. Structural Studies of Archaealthermophilic Adenylate Kinase; TOPICAL

    International Nuclear Information System (INIS)

    Konisky, J.

    2002-01-01

    Through this DOE-sponsored program Konisky has studied the evolution and molecular biology of microbes that live in extreme environments. The emphasis of this work has been the determination of the structural features of thermophilic enzymes that allow them to function optimally at near 100 C. The laboratory has focused on a comparative study of adenylate kinase (ADK), an enzyme that functions to interconvert adenine nucleotides. Because of the close phylogenetic relatedness of members of the Methanococci, differences in the structure of their ADKs will be dominated by structural features that reflect contributions to their optimal temperature for activity, rather than differences due to phylogenetic divergence. We have cloned, sequenced and modeled the secondary structure for several methanococcal ADKs. Using molecular modeling threading approaches that are based on the solved structure for the porcine ADK, we have also proposed a general low resolution three dimensional structure for each of the methanococcal enzymes. These analyses have allowed us to propose structural features that confer hyperthermoactivity to those enzymes functioning in the hyperthermophilic members of the Methanococci. Using protein engineering methodologies, we have tested our hypotheses by examining the effects of selective structural changes on thermoactivity. Despite possessing between 68-81% sequence identity, the methanococcal AKs had significantly different stability against thermal denaturation, with melting points ranging from 69-103 C. The construction of several chimerical AKs by linking regions of the MVO and MJA AKs demonstrated the importance of cooperative interactions between amino- and carboxyl-terminal regions in influencing thermostability. Addition of MJA terminal fragments to the MVO AK increased thermal stability approximately 20 C while maintaining 88% of the mesophilic sequence. Further analysis using structural models suggested that hydrophobic interactions are

  9. Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

    Science.gov (United States)

    Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

    2009-06-15

    Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

  10. Plant walkdown

    International Nuclear Information System (INIS)

    Kostov, M.

    2000-01-01

    This report covers the following: preparatory steps for performing plant walk-down; the objective of the first plant walk-down; plant walk-down procedures; earthquake screening evaluation; walk-down documentation; second plant walk-down. The following objectives concerning the plant walk-down(s) were achieved. The plant system configuration is verified in order to proceed with event tree and fault tree analyses. Systems interactions, other types of dependencies or plant unique features are identified. he safety related components that are judged to generically possess high capacities (i.e., larger than the earthquake review level) have been verified to contain no weaknesses. Further analyses needed to establish the capacities of remaining safety-related components are identified and necessary field data are obtained. Information on components is obtained to assist in HCLPF (fragility) evaluation and peer review of the seismic margin study

  11. Aquatic plants

    DEFF Research Database (Denmark)

    Madsen, T. V.; Sand-Jensen, K.

    2006-01-01

    Aquatic fl owering plants form a relatively young plant group on an evolutionary timescale. The group has developed over the past 80 million years from terrestrial fl owering plants that re-colonised the aquatic environment after 60-100 million years on land. The exchange of species between terre...... terrestrial and aquatic environments continues today and is very intensive along stream banks. In this chapter we describe the physical and chemical barriers to the exchange of plants between land and water.......Aquatic fl owering plants form a relatively young plant group on an evolutionary timescale. The group has developed over the past 80 million years from terrestrial fl owering plants that re-colonised the aquatic environment after 60-100 million years on land. The exchange of species between...

  12. Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

    Science.gov (United States)

    López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

    2018-03-01

    The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

  13. Potassium sensing histidine kinase in Bacillus subtilis.

    Science.gov (United States)

    López, Daniel; Gontang, Erin A; Kolter, Roberto

    2010-01-01

    The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. 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...... 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...

  15. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    Science.gov (United States)

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  16. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

    Science.gov (United States)

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  17. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  18. Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

    Science.gov (United States)

    Jones, Mathew J K; Jallepalli, Prasad V

    2016-01-01

    During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

  19. Localization of two mammalian cyclin dependent kinases during mammalian meiosis

    NARCIS (Netherlands)

    Ashley, T.; Walpita, D.; de rooij, D. G.

    2001-01-01

    Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids)

  20. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. ... have shown that the stress associated with cold temperature ..... vation by cyclic-AMP-dependent protein kinase, studied using.

  1. Profiling bacterial kinase activity using a genetic circuit

    DEFF Research Database (Denmark)

    van der Helm, Eric; Bech, Rasmus; Lehning, Christina Eva

    Phosphorylation is a post-translational modification that regulates the activity of several key proteins in bacteria and eukaryotes. Accordingly, a variety of tools has been developed to measure kinase activity. To couple phosphorylation to an in vivo fluorescent readout we used the Bacillus...... subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously...... reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool....

  2. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Jaouani Mouna

    2015-09-26

    Sep 26, 2015 ... to several mutations at the Pyruvate Kinase gene (PKLR) located on chromosome .... Tunisians (Fig. 2) [21]. The screening of whole PKLR gene revealed the presence of ..... newborns: the pitfalls of diagnosis. J Pediatr 2007 ...

  3. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  4. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family. Jaouani Mouna, Hamdi Nadia, Chaouch Leila, Kalai Miniar, Mellouli Fethi, Darragi Imen, Boudriga Imen, Chaouachi Dorra, Bejaoui Mohamed, Abbes Salem ...

  5. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  6. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2001-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  7. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2002-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  8. 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. © 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist © 2015 New Phytologist Trust.

  9. Janus Associated Kinases Inhibitors in the Pharmacological Thera

    Directory of Open Access Journals (Sweden)

    Daniela Santos1

    2017-01-01

    Full Text Available Janus associated kinases inhibitors are a new strategy for the treatment of different clinical conditions like immunologic, inflammatory and oncology disorders. The aim of this study was to perform a review of all Janus associated kinases inhibitors available in national and international pharmaceutical market, their therapeutic indications and adverse effects, and the potential indications for investigation of those already available in the pharmaceutical market. It was also performed a review of the main new Janus associated kinases inhibitors that are still in clinical research. A literature review was conducted by consulting the summary of product characteristics of Janus associated kinases inhibitors available in the pharmaceutical market and a research in the bibliographic database PubMed using the terms «JAK inhibitors», «Janus associated kinases inhibitors» and «Janus kinases inhibitors». Ninety-five publications were included in the present review, published from January 2014 to January 2015. Drug databases of the European Medicines Agency and United States Food and Drug Administration were also consulted to search for Janus associated kinases inhibitors authorized in clinical practice. Currently, ruxolitinib and tofacitinib are available in the pharmaceutical market and oclatinib is approved as a veterinary medicinal product. Both drugs approved for human use have major adverse effects at hematological and immunological levels, which enhance the importance of the pharmacist’s role in the monitoring of patients involved in these treatments. However, several molecules are in pre-clinical and clinical studies trying to prove its potential in the treatment of several immunologic, inflammatory and oncology disorders. Thus, it is still necessary to deepen the knowledge in this area in order to overcome the risks of therapy with these agents. These risks weighed against the benefits of its clinical use have compromised the progress of

  10. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  11. Structural analysis of the Csk homologous kinase CHK

    International Nuclear Information System (INIS)

    Mulhern, T.; Chong, Y.-P.; Cheng, H.-C.

    2003-01-01

    Full text: CHK (Csk homologous kinase) is an intracellular protein tyrosine kinase, which is highly expressed in the haematopoietic system and the brain. The in vivo role of CHK is to specifically phosphorylate and deactivate the Src family of protein tyrosine kinases. The members of the Src family: Src, Blk, Fyn, Fgr, Hck, Lck, Lyn, Yes and Yrk are major players in numerous cell signalling pathways and exquisitely tuned control of Src family activity is fundamental to many processes in normal cells (reviewed in Lowell and Soriano, 1996). For example, the Src family kinase Fyn is highly expressed in the brain and its activity is vital for memory and learning. In the haematopoietic system, the Src family kinase Hck controls cytoskeletal reorganization, cell motility and immunologic activation. While the Csk family enzymes are closely related to the Src proteins (∼37% identity), the x-ray crystal structures of Src (Xu et al., 1997) and Csk (Ogawa et al., 2002) do display several important differences. Unlike Src, the Csk the SH2 and SH3 domains do not bind intramolecular ligands and they adopt a strikingly different disposition to that observed in Src. Another interesting feature is that the linkers between the SH3 and SH2 domains and between the SH2 and kinase domains, are in intimate contact with the N-lobe of kinase and both appear to play important roles in regulation of the kinase activity. However, the structural and functional basis of how this can be altered is still unclear. We describe the results of biochemical analyses of CHK mediated deactivation of Hck, which suggest that in addition to direct tail-phosphorylation, protein-protein interactions are important. We also describe heteronuclear NMR studies of the structure and ligand binding properties of the CHK SH2 and SH3 domains with a particular emphasis on the transmission of regulatory signals from the ligand binding sites to the interdomain linkers

  12. High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38 alpha

    NARCIS (Netherlands)

    Simard, J.R.; Grutter, C.; Pawar, V.; Aust, B.; Wolf, A.; Rabiller, M.; Wulfert, S.; Robubi, A.; Kluter, S.; Ottmann, C.; Rauh, D.

    2009-01-01

    Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases

  13. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    Science.gov (United States)

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  14. Identification of the protein kinase C phosphorylation site in neuromodulin

    International Nuclear Information System (INIS)

    Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

    1990-01-01

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

  15. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    Science.gov (United States)

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  16. Crystal structure of human protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

  17. Putative tyrosine kinases expressed in K-562 human leukemia cells

    International Nuclear Information System (INIS)

    Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

    1990-01-01

    Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

  18. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    M Kasim Diril

    2016-09-01

    Full Text Available The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

  19. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

    Science.gov (United States)

    Diril, M Kasim; Bisteau, Xavier; Kitagawa, Mayumi; Caldez, Matias J; Wee, Sheena; Gunaratne, Jayantha; Lee, Sang Hyun; Kaldis, Philipp

    2016-09-01

    The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

  20. SH2-dependent autophosphorylation within the Tec family kinase Itk.

    Science.gov (United States)

    Joseph, Raji E; Severin, Andrew; Min, Lie; Fulton, D Bruce; Andreotti, Amy H

    2009-08-07

    The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the betaD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.

  1. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.

    Science.gov (United States)

    Kim, Dennis H; Feinbaum, Rhonda; Alloing, Geneviève; Emerson, Fred E; Garsin, Danielle A; Inoue, Hideki; Tanaka-Hino, Miho; Hisamoto, Naoki; Matsumoto, Kunihiro; Tan, Man-Wah; Ausubel, Frederick M

    2002-07-26

    A genetic screen for Caenorhabditis elegans mutants with enhanced susceptibility to killing by Pseudomonas aeruginosa led to the identification of two genes required for pathogen resistance: sek-1, which encodes a mitogen-activated protein (MAP) kinase kinase, and nsy-1, which encodes a MAP kinase kinase kinase. RNA interference assays and biochemical analysis established that a p38 ortholog, pmk-1, functions as the downstream MAP kinase required for pathogen defense. These data suggest that this MAP kinase signaling cassette represents an ancient feature of innate immune responses in evolutionarily diverse species.

  2. Blue light-excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis.

    Science.gov (United States)

    Oide, Mao; Okajima, Koji; Nakagami, Hirofumi; Kato, Takayuki; Sekiguchi, Yuki; Oroguchi, Tomotaka; Hikima, Takaaki; Yamamoto, Masaki; Nakasako, Masayoshi

    2018-01-19

    Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage-sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from Arabidopsis to study their molecular properties in the dark and under BL irradiation. Phosphorylation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. 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.

  4. Electronic plants

    Science.gov (United States)

    Stavrinidou, Eleni; Gabrielsson, Roger; Gomez, Eliot; Crispin, Xavier; Nilsson, Ove; Simon, Daniel T.; Berggren, Magnus

    2015-01-01

    The roots, stems, leaves, and vascular circuitry of higher plants are responsible for conveying the chemical signals that regulate growth and functions. From a certain perspective, these features are analogous to the contacts, interconnections, devices, and wires of discrete and integrated electronic circuits. Although many attempts have been made to augment plant function with electroactive materials, plants’ “circuitry” has never been directly merged with electronics. We report analog and digital organic electronic circuits and devices manufactured in living plants. The four key components of a circuit have been achieved using the xylem, leaves, veins, and signals of the plant as the template and integral part of the circuit elements and functions. With integrated and distributed electronics in plants, one can envisage a range of applications including precision recording and regulation of physiology, energy harvesting from photosynthesis, and alternatives to genetic modification for plant optimization. PMID:26702448

  5. Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A; Kuiper, Raoul V; Curbo, Sophie; Karlsson, Anna

    2013-02-15

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK(+/-) transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK(+/-)TK2(-/-) mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK(+/-)TK2(-/-) mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

  6. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  7. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  8. Prodrugs of herpes simplex thymidine kinase inhibitors.

    Science.gov (United States)

    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  9. Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    David E Garcia

    Full Text Available The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2 from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3 and purified on a Ni²⁺ column. The KM of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.

  10. Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

    OpenAIRE

    Thauerer, Bettina; zur Nedden, Stephanie; Baier-Bitterlich, Gabriele

    2010-01-01

    Exposure of pheochromocytoma (PC12) cells to hypoxia (1% O2) favors differentiation at the expense of cell viability. Additional incubation with nerve growth factor (NGF) and guanosine, a purine nucleoside with neurotrophin characteristics, rescued cell viability and further enhanced the extension of neurites. In parallel, an increase in the activity of protein kinase C-related kinase (PRK1), which is known to be involved in regulation of the actin cytoskeleton, was observed in hypoxic cells....

  11. Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

    Science.gov (United States)

    Stavenger, Robert A; Cui, Haifeng; Dowdell, Sarah E; Franz, Robert G; Gaitanopoulos, Dimitri E; Goodman, Krista B; Hilfiker, Mark A; Ivy, Robert L; Leber, Jack D; Marino, Joseph P; Oh, Hye-Ja; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Zhang, Daohua; Zhao, Yongdong; Jolivette, Larry J; Head, Martha S; Semus, Simon F; Elkins, Patricia A; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Doe, Christopher P; Bentley, Ross; Chen, Zunxuan X; Hu, Erding; Lee, Dennis

    2007-01-11

    The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

  12. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  13. Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

    OpenAIRE

    Martos, C; Plana, M; Guasch, M D; Itarte, E

    1985-01-01

    Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partia...

  14. Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

    OpenAIRE

    Elbing, Karin; McCartney, Rhonda R.; Schmidt, Martin C.

    2006-01-01

    Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerpr...

  15. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Science.gov (United States)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  16. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  17. The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

    Science.gov (United States)

    Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

    2017-09-22

    The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

    Science.gov (United States)

    Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

    2017-08-18

    Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  20. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    OpenAIRE

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulat...

  1. A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

    Science.gov (United States)

    Yuan, Hushan; Pupo, Monica T.; Blois, Joe; Smith, Adam; Weissleder, Ralph; Clardy, Jon; Josephson, Lee

    2009-01-01

    The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min vs Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC50 of 44 nM, compared to Wm’s IC50 of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. PMID:19523825

  2. Plant embryogenesis

    NARCIS (Netherlands)

    Vries, de Sacco C.; Weijers, Dolf

    2017-01-01

    Land plants are called ‘embryophytes’ and thus, their collective name is defined by their ability to form embryos. Indeed, embryogenesis is a widespread phenomenon in plants, and much of our diet is composed of embryos (just think of grains, beans or nuts; Figure 1). However, in addition to embryos

  3. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  4. Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

    Science.gov (United States)

    Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya; Liu, Yin; James, Michael; Herrero, Juan; Shaw, Stephen

    2005-03-18

    To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

  5. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  6. Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Varley, C L; Royds, J A; Brown, B L; Dobson, P R

    2001-01-01

    We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

  7. 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.

  8. Phylogenetic analysis of the Neks reveals early diversification of ciliary-cell cycle kinases.

    Directory of Open Access Journals (Sweden)

    Jeremy D K Parker

    2007-10-01

    Full Text Available NIMA-related kinases (Neks have been studied in diverse eukaryotes, including the fungus Aspergillus and the ciliate Tetrahymena. In the former, a single Nek plays an essential role in cell cycle regulation; in the latter, which has more than 30 Neks in its genome, multiple Neks regulate ciliary length. Mammalian genomes encode an intermediate number of Neks, several of which are reported to play roles in cell cycle regulation and/or localize to centrosomes. Previously, we reported that organisms with cilia typically have more Neks than organisms without cilia, but were unable to establish the evolutionary history of the gene family.We have performed a large-scale analysis of the Nek family using Bayesian techniques, including tests of alternate topologies. We find that the Nek family had already expanded in the last common ancestor of eukaryotes, a ciliated cell which likely expressed at least five Neks. We suggest that Neks played an important role in the common ancestor in regulating cilia, centrioles, and centrosomes with respect to mitotic entry, and that this role continues today in organisms with cilia. Organisms that lack cilia generally show a reduction in the number of Nek clades represented, sometimes associated with lineage specific expansion of a single clade, as has occurred in the plants.This is the first rigorous phylogenetic analysis of a kinase family across a broad array of phyla. Our findings provide a coherent framework for the study of Neks and their roles in coordinating cilia and cell cycle progression.

  9. Nitrate Activation of Cytosolic Protein Kinases Diverts Photosynthetic Carbon from Sucrose to Amino Acid Biosynthesis

    Science.gov (United States)

    Champigny, Marie-Louise; Foyer, Christine

    1992-01-01

    The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis. PMID:16653003

  10. Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan; Reddy, Kanamata; Kurinov, Igor; Carper, Andria; Langfald, Alyssa; Oi, Naomi; Kim, Myoung Ok; Zhu, Feng; Sosa, Carlos P.; Zhou, Keyuan; Bode, Ann M.; Dong, Zigang (Cornell); (Guangdong); (UMM)

    2012-06-27

    Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

  11. Effects of obesity on protein kinase C, brain creatine kinase, transcription, and autophagy in cochlea.

    Science.gov (United States)

    Hwang, Juen-Haur

    2017-06-01

    Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-β (PKC-β), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-β, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.

  12. Kinase inhibitors: a new class of antirheumatic drugs

    Directory of Open Access Journals (Sweden)

    Kyttaris VC

    2012-09-01

    Full Text Available Vasileios C KyttarisDivision of Rheumatology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAAbstract: The outlook for patients with rheumatoid arthritis has improved significantly over the last three decades with the use of disease-modifying antirheumatic drugs. However, despite the use of methotrexate, cytokine inhibitors, and molecules targeting T and B cells, a percentage of patients do not respond or lose their response over time. The autoimmune process in rheumatoid arthritis depends on activation of immune cells, which utilize intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. In the past decade, small molecules targeting several kinases, such as p38 MAPK, Syk, and JAK have been developed. Several p38 MAPK inhibitors proved ineffective in treating rheumatoid arthritis. The Syk inhibitor, fostamatinib, proved superior to placebo in Phase II trials and is currently under Phase III investigation. Tofacitinib, a JAK1/3 inhibitor, was shown to be efficacious in two Phase III trials, while VX-509, a JAK3 inhibitor, showed promising results in a Phase II trial. Fostamatinib and tofacitinib were associated with increased rates of infection, elevation of liver enzymes, and neutropenia. Moreover, fostamatinib caused elevations of blood pressure and diarrhea, while tofacitinib was associated with an increase in creatinine and elevation of lipid levels.Keywords: rheumatoid arthritis, kinase inhibitors, mitogen-activated phosphokinase p38, spleen tyrosine kinase, Janus kinases

  13. Cloning and expression of human deoxycytidine kinase cDNA

    International Nuclear Information System (INIS)

    Chottiner, E.G.; Shewach, D.S.; Datta, N.S.; Ashcraft, E.; Gribbin, D.; Ginsburg, D.; Fox, I.H.; Mitchell, B.S.

    1991-01-01

    Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, the authors have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine

  14. Fragment-based approaches to the discovery of kinase inhibitors.

    Science.gov (United States)

    Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

    2014-01-01

    Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

  15. The long and the short of SAD-1 kinase.

    Science.gov (United States)

    Kim, Joanne S M; Hung, Wesley; Zhen, Mei

    2010-05-01

    The Ser/Thr SAD kinases are evolutionarily conserved, critical regulators of neural development. Exciting findings in recent years have significantly advanced our understanding of the mechanism through which SAD kinases regulate neural development. Mammalian SAD-A and SAD-B, activated by a master kinase LKB1, regulate microtubule dynamics and polarize neurons. In C. elegans, the sad-1 gene encodes two isoforms, namely the long and the short, which exhibit overlapping and yet distinct functions in neuronal polarity and synaptic organization. Surprisingly, our most recent findings in C. elegans revealed a SAD-1-independent LKB1 activity in neuronal polarity. We also found that the long SAD-1 isoform directly interacts with a STRADalpha pseudokinase, STRD-1, to regulate neuronal polarity and synaptic organization. We elaborate here a working model of SAD-1 in which the two isoforms dimer/oligomerize to form a functional complex, and STRD-1 clusters and localizes the SAD-1 complex to synapses. While the mechanistic difference between the vertebrate and invertebrate SAD kinases may be puzzling, a recent discovery of the functionally distinct SAD-B isoforms predicts that the difference likely arises from our incomplete understanding of the SAD kinase mechanism and may eventually be reconciled as the revelation continues.

  16. Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

    Science.gov (United States)

    Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2014-01-01

    WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

  17. Functions of Aurora kinase C in meiosis and cancer

    Directory of Open Access Journals (Sweden)

    Suzanne M. Quartuccio

    2015-08-01

    Full Text Available The mammalian genome encodes three Aurora kinase protein family members: A, B, and C. While Aurora kinase A (AURKA and B (AURKB are found in cells throughout the body, significant protein levels of Aurora kinase C (AURKC are limited to cells that undergo meiosis (sperm and oocyte. Despite its discovery nearly 15 years ago, we know little about the function of AURKC compared to that of the other 2 Aurora kinases. This lack of understanding can be attributed to the high sequence homology between AURKB and AURKC preventing the use of standard approaches to understand non-overlapping and meiosis I (MI-specific functions of the two kinases. Recent evidence has revealed distinct functions of AURKC in meiosis and may aid in our understanding of why chromosome segregation during MI often goes awry in oocytes. Many cancers aberrantly express AURKC, but because we do not fully understand AURKC function in its normal cellular context, it is difficult to predict the biological significance of this expression on the disease. Here, we consolidate and update what is known about AURKC signaling in meiotic cells to better understand why it has oncogenic potential.

  18. Purification and characterization of a thylakoid protein kinase

    International Nuclear Information System (INIS)

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs

  19. A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

    Science.gov (United States)

    Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

    2017-10-13

    Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

  20. Functional and Structural Characterization of a Receptor-Like Kinase Involved in Germination and Cell Expansion in Arabidopsis

    Science.gov (United States)

    Wu, Zhen; Liang, Shan; Song, Wen; Lin, Guangzhong; Wang, Weiguang; Zhang, Heqiao; Han, Zhifu; Chai, Jijie

    2017-01-01

    Leucine-rich repeat receptor-like kinases (LRR-RLKs) are widespread in different plant species and play important roles in growth and development. Germination inhibition is vital for the completion of seed maturation and cell expansion is a fundamental cellular process driving plant growth. Here, we report genetic and structural characterizations of a functionally uncharacterized LRR-RLK, named GRACE (Germination Repression and Cell Expansion receptor-like kinase). Overexpression of GRACE in Arabidopsis exhibited delayed germination, enlarged cotyledons, rosette leaves and stubbier petioles. Conversely, these phenotypes were reversed in the T-DNA insertion knock-down mutant grace-1 plants. A crystal structure of the extracellular domain of GRACE (GRACE-LRR) determined at the resolution of 3.0 Å revealed that GRACE-LRR assumed a right-handed super-helical structure with an island domain (ID). Structural comparison showed that structure of the ID in GRACE-LRR is strikingly different from those observed in other LRR-RLKs. This structural observation implies that GRACE might perceive a new ligand for signaling. Collectively, our data support roles of GRACE in repressing seed germination and promoting cell expansion of Arabidopsis, presumably by perception of unknown ligand(s). PMID:29213277

  1. Two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Willson, V J.C.; Jones, H M; Thompson, R J [Cambridge Univ. (UK). Clinical School

    1981-06-18

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and /sup 125/I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive.

  2. A two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    International Nuclear Information System (INIS)

    Willson, V.J.C.; Jones, H.M.; Thompson, R.J.

    1981-01-01

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and 125 I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive. (Auth.)

  3. Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants

    Directory of Open Access Journals (Sweden)

    Pankaj Dhonukshe

    2012-01-01

    Full Text Available Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration.

  4. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  5. GsLRPK, a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja, is a positive regulator to cold stress tolerance.

    Science.gov (United States)

    Yang, Liang; Wu, Kangcheng; Gao, Peng; Liu, Xiaojuan; Li, Guangpu; Wu, Zujian

    2014-02-01

    Plant LRR-RLKs serve as protein interaction platforms, and as regulatory modules of protein activation. Here, we report the isolation of a novel plant-specific LRR-RLK from Glycine soja (termed GsLRPK) by differential screening. GsLRPK expression was cold-inducible and shows Ser/Thr protein kinase activity. Subcellular localization studies using GFP fusion protein indicated that GsLRPK is localized in the plasma membrane. Real-time PCR analysis indicated that temperature, salt, drought, and ABA treatment can alter GsLRPK gene transcription in G. soja. However, just protein induced by cold stress not by salinity and ABA treatment in tobacco was found to possess kinase activity. Furthermore, we found that overexpression of GsLRPK in yeast and Arabidopsis can enhance resistance to cold stress and increase the expression of a number of cold responsive gene markers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. Expression and Purification of PI3 Kinase {alpha} and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

    DEFF Research Database (Denmark)

    Boldyreff, Brigitte; Rasmussen, Tine L; Jensen, Hans H

    2008-01-01

    Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal....... In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. (Journal of Biomolecular Screening XXXX:xx-xx)....

  7. Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

    Science.gov (United States)

    Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

    2016-10-02

    Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

  8. Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo.

    Science.gov (United States)

    Han, Xiaomin; Li, Guojing; Zhang, Shuqun

    2017-01-01

    Ethylene, a gaseous phytohormone, has profound effects on plant growth, development, and adaptation to the environment. Ethylene-regulated processes begin with the induction of ethylene biosynthesis. There are two key steps in ethylene biosynthesis. The first is the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl-Methionine (SAM), a common precursor in many metabolic pathways, which is catalyzed by ACC synthase (ACS). The second is the oxidative cleavage of ACC to form ethylene under the action of ACC oxidase (ACO). ACC biosynthesis is the committing and generally the rate-limiting step in ethylene biosynthesis. As a result, characterizing the cellular ACS activity and understanding its regulation are important. In this chapter, we detail the methods used to measure, (1) the enzymatic activity of both recombinant and native ACS proteins, and (2) the phosphorylation of ACS protein by mitogen-activated protein kinases (MAPKs) in vivo and in vitro.

  9. Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis

    KAUST Repository

    Chardin, Camille

    2017-04-14

    Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

  10. Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis

    KAUST Repository

    Chardin, Camille; Schenk, Sebastian T.; Hirt, Heribert; Colcombet, Jean; Krapp, Anne

    2017-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

  11. 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.

  12. Complete inhibition of creatine kinase in isolated perfused rat hearts

    International Nuclear Information System (INIS)

    Fossel, E.T.; Hoefeler, H.

    1987-01-01

    Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts are able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure. 31 P-NMR of the heart was carried out

  13. Kinase fusions are frequent in Spitz tumors and spitzoid melanomas

    Science.gov (United States)

    Esteve-Puig, Rosaura; Botton, Thomas; Yeh, Iwei; Lipson, Doron; Otto, Geoff; Brennan, Kristina; Murali, Rajmohan; Garrido, Maria; Miller, Vincent A.; Ross, Jeffrey S; Berger, Michael F.; Sparatta, Alyssa; Palmedo, Gabriele; Cerroni, Lorenzo; Busam, Klaus J.; Kutzner, Heinz; Cronin, Maureen T; Stephens, Philip J; Bastian, Boris C.

    2014-01-01

    Spitzoid neoplasms are a group of melanocytic tumors with distinctive histopathologic features. They include benign tumors (Spitz nevi), malignant tumors (spitzoid melanomas), and tumors with borderline histopathologic features and uncertain clinical outcome (atypical Spitz tumors). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbor kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%), and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signaling pathways, are tumorigenic, and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz nevi, 56% of atypical Spitz tumors, and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signaling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms, and may serve as therapeutic targets for metastatic spitzoid melanomas. PMID:24445538

  14. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

    Science.gov (United States)

    Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

    2017-01-01

    ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  15. Novel Bruton's tyrosine kinase inhibitors currently in development

    Directory of Open Access Journals (Sweden)

    D'Cruz OJ

    2013-03-01

    Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

  16. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    that phosphorylates all the natural deoxyribonucleosides and like insects, C. elegans only contains a single deoxyribonucleoside kinase-like gene. In contrast to the insects, however, the protein encoded by the elegans gene is 46 % identical to human TK1 (HuTK1) and have no homology to the insect kinase. Like HuTK1...... the C. elegans kinase (CeTK1) has thymidine as the preferred substrate, but it also displays activity with deoxyguanosine, though with high Km. A number of point mutations have been introduced in the active site of both the human and elegans TK's in order to change the substrate specificity away from...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site....

  17. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    International Nuclear Information System (INIS)

    Sprowles, Amy; Robinson, Dan; Wu Yimi; Kung, H.-J.; Wisdom, Ron

    2005-01-01

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli

  18. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

    Directory of Open Access Journals (Sweden)

    Caroline Lund Dahlberg

    Full Text Available Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  19. Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

    Science.gov (United States)

    Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

    2006-06-01

    This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  20. A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB

    OpenAIRE

    López-Berges, Manuel S.; Rispail, Nicolas; Prados-Rosales, Rafael C.; Pietro, Antonio D.

    2010-01-01

    During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine s...

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

    Science.gov (United States)

    Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

    2015-01-01

    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.

  2. A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

    Science.gov (United States)

    Nanjareddy, Kalpana; Blanco, Lourdes; Arthikala, Manoj-Kumar; Alvarado-Affantranger, Xóchitl; Quinto, Carmen; Sánchez, Federico; Lara, Miguel

    2016-11-01

    The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. Molecular cloning and in silico analysis of three somatic embryogenesis receptor kinase mRNA from date palm

    Directory of Open Access Journals (Sweden)

    Rekik Imen

    2013-01-01

    Full Text Available We report here the isolation and characterizations of three somatic embryogenesis receptor kinase (PhSERK genes from palm date by a rapid amplification of cDNA ends (RACE approach. PhSERKs belong to a small family of receptor kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. Sequence analysis of these genes revealed the sequence size of 11051 pb (PhSERK1, 7981 pb (PhSERK2 and 10510 pb (PhSERK3. The open reading frames of PhSERK1, PhSERK2 and PhSERK3 are 1914 pb, 1797 pb and 1719 pb respectively. PhSERKs belongs to the LRR-type cell surface RLKs, which possess a number of characteristic domains. These include an extracellular domain (EX containing a variable number of LRR units, signal pepetide (SP immediately followed by a single transmembrane domain (TM and an intracellular kinase domain. The phylogenetic tree shows that the protein PhSERK1, PhSERK2 and PhSERK3 clustered within monocots SERKs proteins groups. We also predicted the secondary and tertiary with ligand binding sites structure of the protein PhSERKs.

  4. 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.

  5. 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.

  6. Plant Macrofossils

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Records of past vegetation and environmental change derived from plant remains large enough to be seen without a microscope (macrofossils), such as leaves, needles,...

  7. T Plant

    Data.gov (United States)

    Federal Laboratory Consortium — Arguably the second most historic building at Hanford is the T Plant.This facility is historic in that it's the oldest remaining nuclear facility in the country that...

  8. Lunar Plants

    Data.gov (United States)

    National Aeronautics and Space Administration — We present an open design for a first plant growth module on the Moon (LPX). The primary science goal of lunar habitat is to investigate germination and initial...

  9. Alien plants

    Science.gov (United States)

    2018-04-01

    No-one’s ever travelled to an extrasolar planet, or even observed one that we’re sure harbours life. But if plants do exist on such alien worlds, we can have fun speculating what form they might take.

  10. Semiconductor technology in protein kinase research and drug discovery: sensing a revolution.

    Science.gov (United States)

    Bhalla, Nikhil; Di Lorenzo, Mirella; Estrela, Pedro; Pula, Giordano

    2017-02-01

    Since the discovery of protein kinase activity in 1954, close to 600 kinases have been discovered that have crucial roles in cell physiology. In several pathological conditions, aberrant protein kinase activity leads to abnormal cell and tissue physiology. Therefore, protein kinase inhibitors are investigated as potential treatments for several diseases, including dementia, diabetes, cancer and autoimmune and cardiovascular disease. Modern semiconductor technology has recently been applied to accelerate the discovery of novel protein kinase inhibitors that could become the standard-of-care drugs of tomorrow. Here, we describe current techniques and novel applications of semiconductor technologies in protein kinase inhibitor drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Human adenylate kinases – classification, structure, physiological and pathological importance

    Directory of Open Access Journals (Sweden)

    Magdalena Wujak

    2015-01-01

    Full Text Available Adenylate kinase (AK, EC 2.7.4.3 is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy β – and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.

  12. Characterization of pathogenic germline mutations in human Protein Kinases

    Directory of Open Access Journals (Sweden)

    Orengo Christine A

    2011-07-01

    Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

  13. Plant simulator

    International Nuclear Information System (INIS)

    Fukumitsu, Hiroyuki

    1998-01-01

    A simulator of a reactor plant of the present invention comprises a plurality of distributed computers, an indication processing section and an operation section. The simulation calculation functions of various kinds of plant models in the plant are shared by the plurality of computers. The indication processing section controls collection of data of the plant simulated by the computers and instructions of an operator. The operation section is operated by the operator and the results of operation are transmitted to the indication processing section, to conduct operation trainings and display the results of the simulation. Each of the computers and the indication processing portion are connected with each other by a network having a memory for common use. Data such as the results of calculation of plant models and various kinds of parameters of the plant required commonly to the calculators and the indication processing section are stored in the common memory, and adapted to be used by way of the network. (N.H.)

  14. Development of schizogenous intercellular spaces in plants

    Directory of Open Access Journals (Sweden)

    Kimitsune eIshizaki

    2015-07-01

    Full Text Available Gas exchange is essential for multicellular organisms. In contrast to the circulatory systems of animals, land plants have tissues with intercellular spaces (ICSs, called aerenchyma, that are critical for efficient gas exchange. Plants form ICSs by two different mechanisms: schizogeny, where localized cell separation creates spaces; and lysogeny, where cells die to create intercellular spaces. In schizogenous ICS formation, specific molecular mechanisms regulate the sites of cell separation and coordinate extensive reorganization of cell walls. Emerging evidence suggests the involvement of extracellular signaling, mediated by peptide ligands and leucine-rich repeat receptor-like kinases, in the regulation of cell wall remodeling during cell separation. Recent work on the liverwort Marchantia polymorpha has demonstrated a critical role for a plasma membrane-associated plant U-box E3 ubiquitin ligase in ICS formation. In this review, I discuss the mechanism of schizogenous ICS formation, focusing on the potential role of extracellular signaling in the regulation of cell separation.

  15. Structural Changes of Creatine Kinase upon Substrate Binding

    OpenAIRE

    Forstner, Michael; Kriechbaum, Manfred; Laggner, Peter; Wallimann, Theo

    1998-01-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzym...

  16. A structural informatics approach to mine kinase knowledge bases.

    Science.gov (United States)

    Brooijmans, Natasja; Mobilio, Dominick; Walker, Gary; Nilakantan, Ramaswamy; Denny, Rajiah A; Feyfant, Eric; Diller, David; Bikker, Jack; Humblet, Christine

    2010-03-01

    In this paper, we describe a combination of structural informatics approaches developed to mine data extracted from existing structure knowledge bases (Protein Data Bank and the GVK database) with a focus on kinase ATP-binding site data. In contrast to existing systems that retrieve and analyze protein structures, our techniques are centered on a database of ligand-bound geometries in relation to residues lining the binding site and transparent access to ligand-based SAR data. We illustrate the systems in the context of the Abelson kinase and related inhibitor structures. 2009 Elsevier Ltd. All rights reserved.

  17. Expression of casein kinase 2 during mouse embryogenesis

    DEFF Research Database (Denmark)

    Mestres, P; Boldyreff, B; Ebensperger, C

    1994-01-01

    This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohisto......This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level...

  18. Proteinase K processing of rabbit muscle creatine kinase

    DEFF Research Database (Denmark)

    Leydier, C; Andersen, Jens S.; Couthon, F

    1997-01-01

    Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However...

  19. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  20. The Azaindole Framework in the Design of Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Jean-Yves Mérour

    2014-11-01

    Full Text Available This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.