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Sample records for surface adenylate kinase

  1. Adenylate kinase locus 1 polymorphism and feto-placental development.

    Science.gov (United States)

    Fulvia, Gloria-Bottini; Antonio, Pietroiusti; Anna, Neri; Patrizia, Saccucci; Ada, Amante; Egidio, Bottini; Andrea, Magrini

    2011-12-01

    Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK₁) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK₁2-1 phenotype than in those with high activity AK₁1 phenotype. The difference between AK₁ and AK₁2-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK₁ phenotype. These results suggest that the difference in enzymatic activity between AK₁ phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    Directory of Open Access Journals (Sweden)

    Andre Terzic

    2009-04-01

    Full Text Available Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7 are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.

  3. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  4. Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions.

    NARCIS (Netherlands)

    Bruin, W.C.C. de; Oerlemans, F.T.J.J.; Wieringa, B.

    2004-01-01

    Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand

  5. The arsonomethyl analogue of adenosine 5'-phosphate. An uncoupler of adenylate kinase.

    Science.gov (United States)

    Adams, S R; Sparkes, M J; Dixon, H B

    1984-01-01

    Adenosine was converted into the arsonomethyl analogue of AMP. The reactions used provide a general route for converting an alcohol, R-CH2-OH, into the arsonomethyl analogue, R-CH2-CH2-AsO3H2, of its phosphate, R-CH2-O-PO3H2. The analogue of AMP proves to be a substrate for rabbit adenylate kinase, which shows a limiting velocity with it of 1/17 that with AMP, a Michaelis constant raised 70-fold to about 10 mM, and hence a specificity constant lowered about 1200-fold. The product of transfer of a phospho group from ATP to the analogue is, like all anhydrides of arsonic acids, unstable to hydrolysis, and so breaks down to yield orthophosphate and regenerate the analogue. Hence adenylate kinase is converted into an ATPase by the presence of the analogue. PMID:6089748

  6. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    Science.gov (United States)

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  7. Mechanism of adenylate kinase: Site-directed mutagenesis versus x-ray and NMR

    International Nuclear Information System (INIS)

    Tsai, Mingdaw; Yan, Honggao

    1991-01-01

    Controversy is an integral part of scientific research and is often a precursor to the truth. However, this lesson has been learned in a very hard way in the case of the structure-function relationship of adenylate kinase (AK), which catalyzes the interconversion between MgATP+AMP and MgADP+ADP. While this small kinase has been considered a model kinase and the enzyme-substrate interaction of AK was among the first investigated by X-ray crystallography and NMR the substrate binding sites deduced from the early studies by these two powerful techniques (termed the X-ray model and the NMR model, respectively) were dramatically different. Ironically, both models have had substantial impact on researchers in related fields. The problems have finally been dealt with since 1987 by the interplay between site-directed mutagenesis, X-ray, and NMR. The purpose of this review is not only to summarize the current knowledge in the structure-function relationship of adenylate kinase but also to accurately document and critically analyze historical developments in the hope that history will not be repeated

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

    Directory of Open Access Journals (Sweden)

    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

  9. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    Science.gov (United States)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

  10. Role of water in the enzymatic catalysis: study of ATP + AMP → 2ADP conversion by adenylate kinase.

    Science.gov (United States)

    Adkar, Bharat V; Jana, Biman; Bagchi, Biman

    2011-04-28

    The catalytic conversion ATP + AMP → 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

  11. Recombinant adenylate kinase 3 from liver fluke Clonorchis sinensis for histochemical analysis and serodiagnosis of clonorchiasis.

    Science.gov (United States)

    Kwon, Soon Bin; Kim, Paul; Woo, Hae Sun; Kim, Tae Yun; Kim, Ju Yeong; Lee, Hye Min; Jang, Yun Soo; Kim, Eun-Min; Yong, Tai-Soon; Seong, Baik Lin

    2018-03-27

    Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

  12. Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions.

    Science.gov (United States)

    de Bruin, Wieke; Oerlemans, Frank; Wieringa, Bé

    2004-07-01

    Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand management of proliferating cells, AK1 and AK1beta were expressed in mouse neuroblastoma N2a cells and in human colon carcinoma SW480 cells. Glucose deprivation, galactose feeding, and metabolic inhibitor tests revealed a differential energy dependency for these two cell lines. N2a cells showed a faster proliferation rate and strongest coupling to mitochondrial activity, SW480 proliferation was more dependent on glycolysis. Despite these differences, ectopic expression of AK1 or AK1beta did not affect their growth characteristics under normal conditions. Also, no differential effects were seen under metabolic stress upon treatment with mitochondrial and glycolytic inhibitors in in vitro culture or in solid tumors grown in vivo. Although many intimate connections have been revealed between cell death and metabolism, our results suggest that AK1- or AK1beta-mediated high-energy phosphoryl transfer is not a modulating factor in the survival of tumor cells during episodes of metabolic crisis.

  13. Deciphering hierarchical features in the energy landscape of adenylate kinase folding/unfolding

    Science.gov (United States)

    Taylor, J. Nicholas; Pirchi, Menahem; Haran, Gilad; Komatsuzaki, Tamiki

    2018-03-01

    Hierarchical features of the energy landscape of the folding/unfolding behavior of adenylate kinase, including its dependence on denaturant concentration, are elucidated in terms of single-molecule fluorescence resonance energy transfer (smFRET) measurements in which the proteins are encapsulated in a lipid vesicle. The core in constructing the energy landscape from single-molecule time-series across different denaturant concentrations is the application of rate-distortion theory (RDT), which naturally considers the effects of measurement noise and sampling error, in combination with change-point detection and the quantification of the FRET efficiency-dependent photobleaching behavior. Energy landscapes are constructed as a function of observation time scale, revealing multiple partially folded conformations at small time scales that are situated in a superbasin. As the time scale increases, these denatured states merge into a single basin, demonstrating the coarse-graining of the energy landscape as observation time increases. Because the photobleaching time scale is dependent on the conformational state of the protein, possible nonequilibrium features are discussed, and a statistical test for violation of the detailed balance condition is developed based on the state sequences arising from the RDT framework.

  14. 31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

    International Nuclear Information System (INIS)

    Kim, Hyun Won

    2011-01-01

    Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

  15. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-01-01

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 μM isoproterenol and 50 μM GTP-γ-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 μM GTP-γ-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of β-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes

  16. Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

    OpenAIRE

    Konisky, J; Michels, P C; Clark, D S

    1995-01-01

    The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

  17. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase.

    Science.gov (United States)

    Jana, Biman; Adkar, Bharat V; Biswas, Rajib; Bagchi, Biman

    2011-01-21

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  18. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    Science.gov (United States)

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  19. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    International Nuclear Information System (INIS)

    Hass, Mathias A. S.; Liu, Wei-Min; Agafonov, Roman V.; Otten, Renee; Phung, Lien A.; Schilder, Jesika T.; Kern, Dorothee; Ubbink, Marcellus

    2015-01-01

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements

  20. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  1. Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

    Science.gov (United States)

    Pabst, A M; Walter, C; Grassmann, L; Weyhrauch, M; Brüllmann, D D; Ziebart, T; Scheller, H; Lehmann, K M

    2014-05-01

    The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each materials at all points of measurement (between -36 % and -71 %; p each ceramic materials could be investigated. This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

  2. A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

    Energy Technology Data Exchange (ETDEWEB)

    Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di; Trincao, J.; Pickering, I.J.; George, G.N.; Calvete, J.J.; Brondino, C.; Pereira, A.S.; Lampreia, J.; Tavares, P.; Moura, J.J.G.; Moura, I.

    2009-05-18

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  3. A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774.

    Science.gov (United States)

    Gavel, Olga Yu; Bursakov, Sergey A; Di Rocco, Giulia; Trincão, José; Pickering, Ingrid J; George, Graham N; Calvete, Juan J; Shnyrov, Valery L; Brondino, Carlos D; Pereira, Alice S; Lampreia, Jorge; Tavares, Pedro; Moura, José J G; Moura, Isabel

    2008-01-01

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  4. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  5. Adenyl cyclase.

    Science.gov (United States)

    Coppe, D; Steer, M L

    1978-02-01

    Certain hormones regulate the activity of their target cells by stimulating adenyl cyclase, which is an enzyme located within the target cell's plasma membrane. Adenyl cyclase catalyzes the formation of cyclic AMP, which is released into the cell and modulates cell functions. In this communication the characteristics of adenyl cyclase are reviewed. The coupling between hormone receptors and this enzyme is discussed as is the ability of agents such as hormones, ATP, magnesium, calcium, guanine nucleotides and prostaglandins to alter cyclase activity. Several diseases that result from derangements of the adenyl cyclase system are known and the molecular bases for these diseases are discussed in this review.

  6. Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability.

    Science.gov (United States)

    Davlieva, Milya; Shamoo, Yousif

    2010-02-01

    The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes. (c) 2009 Wiley-Liss, Inc.

  7. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Kladova, A. V.; Gavel, O. Yu.; Mukhopaadhyay, A.; Boer, D. R.; Teixeira, S.; Shnyrov, V. L.; Moura, I.; Moura, J. J. G.; Romão, M. J.; Trincão, J.; Bursakov, S. A.

    2009-01-01

    Adenylate kinase (AK) from D. gigas was purified and crystallized in three different metal-bound forms: Zn 2+ –AK, Co 2+ –AK and Fe 2+ –AK. Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn 2+ –AK and Fe 2+ –AK crystallized in space group I222 with similar unit-cell parameters, whereas Co 2+ –AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn 2+ –AK and Fe 2+ –AK forms and a dimer was present for the Co 2+ –AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes

  8. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  9. Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

    Science.gov (United States)

    Luo, A; Tian, Y; Jin, S

    2000-01-01

    The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

  10. Alterations in adenylate kinase activity in human PBMCs after in vitro exposure to electromagnetic field: comparison between extremely low frequency electromagnetic field (ELF) and therapeutic application of a musically modulated electromagnetic field (TAMMEF).

    Science.gov (United States)

    Albanese, Antonietta; Battisti, Emilio; Vannoni, Daniela; Aceto, Emilia; Galassi, Gianmichele; Giglioni, Stefania; Tommassini, Valentina; Giordano, Nicola

    2009-01-01

    This study investigated the effects of electromagnetic fields on enzymes involved in purine metabolism in human peripheral blood mononuclear cells in vitro. Cells were obtained from 20 volunteers. We tested both low-energy, extremely low frequency (ELF; 100-Hz) electromagnetic fields and the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEFs); the latter is characterized by variable frequencies, intensities, and wave shapes. Adenylate kinase activity was increased after ELF field exposure but decreased slightly after TAMMEF exposure. Neither of the two electromagnetic field affected the activities of the purine metabolism enzymes ecto-5'-nucleotidase, adenosine deaminase, and adenosine kinase. We concluded that ELF fields may influence cellular electrical charge stability; stimulation of adenylate kinase activity could restore the cell to a state of equilibrium. In contrast, TAMMEF fields may be useful for maintaining and regulating the cellular electrical charge.

  11. Down-regulation of Cell Surface Cyclic AMP Receptors and Desensitization of Cyclic AMP-stimulated Adenylate Cyclase by Cyclic AMP in Dictyostelium discoideum. Kinetics and Concentration Dependence

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylate cyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of

  12. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  13. On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

    Science.gov (United States)

    Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

    2004-08-01

    The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

  14. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  15. Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

    Science.gov (United States)

    Etxebarria, Aitor; González-Bullón, David; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2013-01-01

    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface. PMID:24058533

  16. Impairment of F1F0-ATPase, adenine nucleotide translocator and adenylate kinase causes mitochondrial energy deficit in human skin fibroblasts with chromosome 21 trisomy.

    Science.gov (United States)

    Valenti, Daniela; Tullo, Apollonia; Caratozzolo, Mariano F; Merafina, Riccardo S; Scartezzini, Paolo; Marra, Ersilia; Vacca, Rosa A

    2010-10-15

    A central role for mitochondrial dysfunction has been proposed in the pathogenesis of DS (Down's syndrome), a multifactorial disorder caused by trisomy of human chromosome 21. To explore whether and how abnormalities in mitochondrial energy metabolism are involved in DS pathogenesis, we investigated the catalytic properties, gene expression and protein levels of certain proteins involved in mitochondrial ATP synthesis, such as F1F0-ATPase, ANT (adenine nucleotide translocator) and AK (adenylate kinase), in DS-HSF (human skin fibroblasts with trisomic karyotype), comparing them with euploid fibroblasts. In DS-HSF, we found a strong impairment of mitochondrial ATP synthesis due to a reduction in the catalytic efficiency of each of the investigated proteins. This impairment occurred in spite of unchanged gene expression and an increase in ANT and AK protein content, whereas the amount of ATPase subunits was selectively reduced. Interestingly, exposure of DS-HSF to dibutyryl-cAMP, a permanent derivative of cAMP, stimulated ANT, AK and ATPase activities, whereas H89, a specific PKA (protein kinase A) inhibitor, suppressed this cAMPdependent activation, indicating an involvement of the cAMP/PKA-mediated signalling pathway in the ATPase, ANT and AK deficit. Consistently, DS-HSF showed decreased basal levels of cAMP and reduced PKA activity. Despite the impairment of mitochondrial energy apparatus, no changes in cellular energy status, but increased basal levels of L-lactate, were found in DS-HSF, which partially offset for the mitochondrial energy deficit by increasing glycolysis and mitochondrial mass.These results provide new insight into the molecular basis for mitochondrial dysfunction in DS and might provide a molecular explanation for some clinical features of the syndrome.

  17. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All

  18. A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress.

    Science.gov (United States)

    Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A

    2017-09-01

    Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.

  19. The central regulation of plant physiology by adenylates.

    Science.gov (United States)

    Geigenberger, Peter; Riewe, David; Fernie, Alisdair R

    2010-02-01

    There have been many recent developments concerning the metabolic, transport and signalling functions of adenylates in plants, suggesting new roles for these compounds as central regulators of plant physiology. For example, altering the expression levels of enzymes involved in the equilibration, salvaging, synthesis and transport of adenylates leads to perturbations in storage, growth and stress responses, implying a role for adenylates as important signals. Furthermore, sensing of the internal energy status involves SNF1-related kinases, which control the expression and phosphorylation of key metabolic enzymes. ATP also acts as an apoplastic signalling molecule to control cell growth and pathogen responses. These new results could shed light on the emerging question of whether energy homeostasis in plant cells differs from mechanisms found in microbes and mammals. Copyright 2009 Elsevier Ltd. All rights reserved.

  20. Regulation of brain adenylate cyclase by calmodulin

    International Nuclear Information System (INIS)

    Harrison, J.K.

    1988-01-01

    This thesis examined the interaction between the Ca 2+ -binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-[ 125 I]-CaM-diazopyruvamide ( 125 I-CAM-DAP) behaved like native CaM with respect to Ca 2+ -enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca 2+ -dependent stimulation of adenylate cyclase. 125 I-CaM-DAP cross-linked to CaM-binding proteins in a Ca 2+ -dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125 I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

  1. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie

    2004-01-01

    The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface...... as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell...

  2. Lymphocyte adenylate cyclase activity in immunosuppressed patients

    NARCIS (Netherlands)

    Michel, M. C.; Brodde, O. E.

    1989-01-01

    In order to determine whether alterations of adenylate cyclase are involved in the immunosuppressive effect of glucocorticoid/cyclosporin treatment we measured basal, prostaglandin E1-, and forskolin-stimulated cAMP production in lymphocyte membranes from kidney transplant patients undergoing

  3. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas

    2012-01-01

    The potassium channel Kv7.1 is expressed in the heart, where it contributes to the repolarization of the cardiac action potential. Additionally, Kv7.1 is expressed in epithelial tissues playing a role in salt and water transport. We recently demonstrated that surface-expressed Kv7.1 is internalized...... in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...

  4. The hormonal regulation of adenylate cyclase.

    Science.gov (United States)

    Feder, D; Im, M J; Pfeuffer, T; Hekman, M; Helmreich, E J; Levitzki, A

    1986-01-01

    The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.

  5. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

    Science.gov (United States)

    Martín, César; Etxaniz, Asier; Uribe, Kepa B; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M; Aréchaga, Juan; Ostolaza, Helena

    2015-09-08

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

  6. Pituitary adenylate cyclase activating polypeptide and migraine

    DEFF Research Database (Denmark)

    Zagami, Alessandro S; Edvinsson, Lars; Goadsby, Peter J

    2014-01-01

    Pituitary adenylate cyclase activating peptide (PACAP) is found in human trigeminocervical complex and can trigger migraine. PACAP levels were measured using a sensitive radioimmunoassay. Stimulation of the superior sagittal sinus (SSS) in cat elevated PACAP levels in cranial blood. Patients...... with moderate or severe migraine headache had elevated PACAP in the external jugular vein during headache (n = 15), that was reduced 1 h after treatment with sumatriptan 6 mg (n = 11), and further reduced interictally (n = 9). The data suggest PACAP, or its receptors, are a promising target for migraine...

  7. Receptors, adenylate cyclase, depression, and lithium.

    Science.gov (United States)

    Belmaker, R H

    1981-04-01

    Although numerous studies have suggested that depression may be associated with a reduction in synaptic noradrenaline in the brain, direct beta-adrenergic receptor agonists have not been tested in the treatment of depression until recently. Moreover, newer theories of antidepressant action suggest that a reduction in beta-adrenergic receptor sensitivity is a better correlate of antidepressant treatment than noradrenaline turnover changes. It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta-2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. Plasma cyclic AMP before treatment with salbutamol rose 26% in response to salbutamol 0.25 mg iv. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to iv salbutamol declined over 60%. The beta-adrenergic adenylate cyclase remained subsensitive 4 days after cessation of salbutamol therapy. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. The plasma cyclic GMP response to subcutaneous epinephrine, suggested as a model for presynaptic alpha-noradrenergic mechanisms, is also partially inhibited by Li therapy. Since cyclic AMP and cyclic GMP may be viewed as balancing substances, their interaction may provide a mechanism for Li's dual clinical effects in mania

  8. Adenylate cyclases involvement in pathogenicity, a minireview.

    Science.gov (United States)

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  9. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Directory of Open Access Journals (Sweden)

    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  10. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Science.gov (United States)

    Martín, César; Uribe, Kepa B; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2011-02-23

    Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  11. Adenylate Cyclase-the More Membrane Associated, the Less Radiosensitive

    OpenAIRE

    ATTILA, TAKATS; VU HOA, BINH; KATALIN, OFFENMULLER; KATALIN, BODO; LORAND, BERTOK; GYORGY J., KOTELES; Frederic Joliot-Curie National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology

    1993-01-01

    A dose dependent but not parallel decreases were observed both in SH content and catalytic activity of "free" catalytic subunit after irradiation (0-3200 Gy), while SR groups of membrane-associated adenylate cyclase were insensitive (under 3200 Gy). An initial "radioactivation" of membrane-associated enzyme was found under 800 Gy, then an inhibition above 1600 Gy. The SH alkylating agent, N-ethylmaleimide resulted in a complete inactivation, both of membrane associated form of adenylate cycla...

  12. Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

    Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

  13. Receptor-like kinases as surface regulators for RAC/ROP-mediated pollen tube growth and interaction with the pistil

    Science.gov (United States)

    Zou, Yanjiao; Aggarwal, Mini; Zheng, Wen-Guang; Wu, Hen-Ming; Cheung, Alice Y.

    2011-01-01

    Background RAC/ROPs are RHO-type GTPases and are known to play diverse signalling roles in plants. Cytoplasmic RAC/ROPs are recruited to the cell membrane and activated in response to extracellular signals perceived and mediated by cell surface-located signalling assemblies, transducing the signals to regulate cellular processes. More than any other cell types in plants, pollen tubes depend on continuous interactions with an extracellular environment produced by their surrounding tissues as they grow within the female organ pistil to deliver sperm to the female gametophyte for fertilization. Scope We review studies on pollen tube growth that provide compelling evidence indicating that RAC/ROPs are crucial for regulating the cellular processes that underlie the polarized cell growth process. Efforts to identify cell surface regulators that mediate extracellular signals also point to RAC/ROPs being the molecular switches targeted by growth-regulating female factors for modulation to mediate pollination and fertilization. We discuss a large volume of work spanning more than two decades on a family of pollen-specific receptor kinases and some recent studies on members of the FERONIA family of receptor-like kinases (RLKs). Significance The research described shows the crucial roles that two RLK families play in transducing signals from growth regulatory factors to the RAC/ROP switch at the pollen tube apex to mediate and target pollen tube growth to the female gametophyte and signal its disintegration to achieve fertilization once inside the female chamber. PMID:22476487

  14. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    Science.gov (United States)

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  15. Surface Electromyography Assessments of the Vastus medialis and Rectus femoris Muscles and Creatine Kinase after Eccentric Contraction Following Glutamine Supplementation.

    Science.gov (United States)

    Rahmani-Nia, Farhad; Farzaneh, Esmail; Damirchi, Arsalan; Majlan, Ali Shamsi; Tadibi, Vahid

    2014-03-01

    L-glutamine is the most abundant amino acid found in human muscle and plays an important role in protein synthesis and can reduce the levels of inflammation biomarkers and creatine kinase (CK) after training sessions. Delayed onset muscle soreness (DOMS) develops after intense exercise and is associated with an inflammatory response. The purpose of this study was to investigate the effect of glutamine supplementation on surface electromyography activity of the vastus medialis muscle (VMM) and rectus femoris muscle (RFM) and levels of creatine kinase after an eccentric contraction. SEVENTEEN HEALTHY MEN (AGE: 22.35±2.27yr; body mass: 69.91± 9.78kg; height: 177.08±4.32cm) were randomly assigned to experimental (n=9) and control groups (n=8) in a double-blind manner. In both groups, subjects were given L-glutamine supplementation (0.1g.kg(-1)) or placebo three times a week for 4 weeks. Median frequency (MDF) and mean power frequency (MPF) for VMM and RFM muscles and also CK measurements were performed before, 24h and 48 h after a resistance training session. The resistance training included 6 sets of eccentric leg extensions to exhaustion with 75% of 1RM. There was no significant difference between groups for MDF or MPF in VMM and RFM. The difference of CK level between the groups was also not significant. The results of this study indicate that glutamine supplementation has no positive effect on muscle injury markers after a resistance training session.

  16. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    OpenAIRE

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explai...

  17. The effect of adenyl compounds on the rat heart.

    OpenAIRE

    Burnstock, G.; Meghji, P.

    1983-01-01

    1 The effects of adenyl compounds were examined on the rat atrium and ventricle. 2 Adenosine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-triphosphate (ATP) and beta, gamma-methylene ATP (APPCP) produced negative inotropic effects on the rat atrium. These inhibitory effects were antagonized by 8-phenyltheophylline (8-PT), a P1-purinoceptor antagonist, and potentiated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, but were not affected...

  18. Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines

    Czech Academy of Sciences Publication Activity Database

    Šebo, Peter; Osička, Radim; Mašín, Jiří

    2014-01-01

    Roč. 13, č. 10 (2014), s. 1215-1227 ISSN 1476-0584 R&D Projects: GA ČR GA13-14547S; GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * antigen delivery * Bordetella pertussis Subject RIV: EE - Microbiology, Virology Impact factor: 4.210, year: 2014

  19. Renoprotection by pituitary adenylate cyclase-activating polypeptide in multiple myeloma and other kidney diseases.

    Science.gov (United States)

    Li, Min; Maderdrut, Jerome L; Lertora, Juan J L; Arimura, Akira; Batuman, Vecihi

    2008-01-10

    Renal involvement in patients with multiple myeloma complicates their treatment and shortens their life-span. The main renal lesion is a tubulointerstitial transformation with fibrosis, frequently associated with cast formation in the distal nephron that results from co-precipitation of pathological immunoglobulin light chains with Tamm-Horsfall proteins. The human renal proximal tubular reabsorption of excessive light chains by endocytosis causes cellular protein overload and activates the transcription factor nuclear factor kappa B (NFkappaB). The activation of NFkappaB promotes the synthesis of inflammatory cytokines and activates signaling pathways, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2, Jun kinase, and p38 MAPK, thus promoting interstitial inflammation and fibrosis. We tested the concept that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/vasoactive intestinal peptide family, could prevent the development of cast nephropathies. PACAP38 inhibited myeloma light chain-induced proinflammatory cytokine expression with greater potency than dexamethasone, and attenuated the resulting cell damage in the renal proximal tubule epithelial cells. The results indicated that its effects are mediated through inhibition of phosphorylation of p38 MAPK and nuclear translocation of the p50 subunit of NFkappaB via both the PAC(1) and VPAC(1) receptors. PACAP was also shown to be efficacious in other common in vivo animal models for kidney hypertrophies, including streptozotocin-induced diabetic nephropathy and gentamicin-induced nephrotoxicity. Thus, our studies suggest that PACAP38 could be used as a cytoprotective agent that would be effective in the treatment of renal tubule injury in multiple myeloma and other chronic kidney diseases.

  20. Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

    Science.gov (United States)

    Boarder, M R; Plevin, R; Marriott, D B

    1988-10-25

    The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

  1. Does treatment with beta-adrenoceptor antagonists in vivo alter human adenylate cyclase responsiveness in vitro?

    NARCIS (Netherlands)

    Michel, M. C.; Klüppel, M.; Philipp, T.; Brodde, O. E.

    1991-01-01

    1. Treatment with beta-adrenoceptor antagonists in vivo can alter adenylate cyclase responsiveness in the human heart. We have determined the effects of treatment with four different beta-adrenoceptor antagonists in vivo on the responsiveness of lymphocyte and platelet adenylate cyclase in vitro in

  2. Interactions of fluoride and guanine nucleotides with thyroid adenylate cyclase.

    Science.gov (United States)

    Goldhammer, A; Wolff, J

    1982-02-18

    The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.

  3. Third Acivity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin

    Czech Academy of Sciences Publication Activity Database

    Fišer, Radovan; Mašín, Jiří; Basler, Marek; Krůšek, Jan; Špuláková, V.; Konopásek, Ivo; Šebo, Peter

    2007-01-01

    Roč. 282, č. 5 (2007), s. 2808-2820 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA AV ČR IAA5020406 Grant - others:XE(XE) LSHB-CT-2003-503582; Univerzita Karlova(CZ) 146/2005/B-BIO Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50110509 Source of funding: R - rámcový projekt EK ; V - iné verejné zdroje Keywords : bordetella * adenylate cyclase toxin * enzymatic aktivity Subject RIV: EE - Microbiology, Virology Impact factor: 5.581, year: 2007

  4. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    Science.gov (United States)

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  5. The Escherichia coli adenylate cyclase complex. Stimulation by potassium and phosphate.

    Science.gov (United States)

    Liberman, E; Reddy, P; Gazdar, C; Peterkofsky, A

    1985-04-10

    In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.

  6. Food restriction modulates β-adrenergic-sensitive adenylate cyclase in rat liver during aging

    International Nuclear Information System (INIS)

    Katz, M.S.

    1988-01-01

    Adenylate cyclase activities were studied in rat liver during postmaturational aging of male Fischer 344 rats fed ad libitum or restricted to 60% of the ad libitum intake. Catecholamine-stimulated adenylate cyclase activity increased by 200-300% between 6 and 24-27 mo of age in ad libitum-fed rats, whereas in food-restricted rats catecholamine response increased by only 58-84% between 6 and 30 mo. In ad libitum-fed rats, glucagon-stimulated enzyme activity also increased by 40% between 6 and 12 mo and in restricted rats a similar age-related increase was delayed until 18 mo. β-Adrenergic receptor density increased by 50% between 6 and 24 mo in livers from ad libitum-fed but not food-restricted rats and showed a highly significant correlation with maximal isoproterenol-stimulated adenylate cyclase activity over the postmaturational life span. Age-related increases in unstimulated (basal) adenylate cyclase activity and nonreceptor-mediated enzyme activation were retarded by food restriction. The results demonstrate that food restriction diminishes a marked age-related increase in β-adrenergic-sensitive adenylate cyclase activity of rat liver. Alterations of adrenergic-responsive adenylate cyclase with age and the modulatory effects of food restriction appear to be mediated by changes in both receptor and nonreceptor components of adenylate cyclase

  7. Gated dynamic 31P MRS shows reduced contractile phosphocreatine breakdown in mice deficient in cytosolic creatine kinase and adenylate kinase.

    NARCIS (Netherlands)

    Kan, H.E.; Veltien, A.A.; Arnts, H.F.G.; Nabuurs, C.I.H.C.; Luijten, B.M.A.; Haan, A. de; Wieringa, B.; Heerschap, A.

    2009-01-01

    We developed a new dedicated measurement protocol for dynamic (31)P MRS analysis in contracting calf muscles of the mouse, using minimally invasive assessment of the contractile force combined with the acquisition of spectroscopic data gated to muscle contraction and determination of phosphocreatine

  8. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    Energy Technology Data Exchange (ETDEWEB)

    Tumbale, Percy [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Williams, Jessica S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Schellenberg, Matthew J. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Kunkel, Thomas A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. of Molecular Genetics; Williams, R. Scott [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. Molecular Genetics

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  9. Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity

    Science.gov (United States)

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2014-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases1,2. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for deadenylation repair is unclear. Here we examine the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. PMID:24362567

  10. Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site.

    Science.gov (United States)

    Morris, S A; Bilezikian, J P

    1983-02-01

    The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.

  11. Structure of the turkey erythrocyte adenylate cyclase system.

    Science.gov (United States)

    Nielsen, T B; Lad, P M; Preston, M S; Kempner, E; Schlegel, W; Rodbell, M

    1981-02-01

    Target analysis of the turkey erythrocyte adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system showed that the molecular weight of the ground state enzyme increases from 92,000 with MnATP as substrate and no stimulatory ligands to 226,000 when activated by fluoride ion or by 5'-guanyl imidodiphosphate (p[NH]ppG) subsequent to clearance of previously bound GDP. The identical increment in size (130,000) suggests that the same regulatory unit is involved in the activation by both effectors. When assayed with isoproterenol and p[NH]ppG, the enzyme system displayed a further increment in size of 90,000 daltons. Based on binding of the antagonist 125I-labeled hydroxybenzylpindolol, the beta-adrenergic receptor is about 90,000 daltons or the same as that seen for activation of the enzyme by isoproterenol through the beta-adrenergic-receptor. Because single targets were seen for the ground state enzyme system under all conditions, it would appear that the various regulatory and catalytic components are structurally linked prior to activation by hormone, guanine nucleotides, and fluoride ion. Furthermore, based on reported subunit sizes of the nucleotide regulatory and receptor components are composed of multiple subunits, either homologous or heterologous in structure.

  12. Effects of PTH and Ca2+ on renal adenyl cyclase

    International Nuclear Information System (INIS)

    Nielsen, S.T.; Neuman, W.F.

    1978-01-01

    The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca 2+ . Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 x 10 -8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex

  13. Restricted Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Pediatric B-Lineage Acute Lymphoblastic Leukemia Suggests Targetability with Therapeutic Monoclonal Antibodies

    Science.gov (United States)

    Dave, Hema; Anver, Miriam R.; Butcher, Donna O.; Brown, Patrick; Khan, Javed; Wayne, Alan S.; Baskar, Sivasubramanian; Rader, Christoph

    2012-01-01

    Background Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. Methodology and Principal Findings Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. Conclusions and Significance Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies. PMID:23285131

  14. Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Hema Dave

    Full Text Available Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL, short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies.Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues.Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.

  15. Determination of the turn-off reaction for the hormone-activated adenylate cyclase.

    Science.gov (United States)

    Cassel, D; Eckstein, F; Lowe, M; Selinger, Z

    1979-10-10

    Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase.

  16. Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

    Science.gov (United States)

    Douiri, Salma; Bahdoudi, Seyma; Hamdi, Yosra; Cubì, Roger; Basille, Magali; Fournier, Alain; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Vaudry, David; Masmoudi-Kouki, Olfa

    2016-06-01

    Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the

  17. Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

    Science.gov (United States)

    Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-04-01

    Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  18. Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

    DEFF Research Database (Denmark)

    Krintel, Christian; Mörgelin, Matthias; Logan, Derek T

    2009-01-01

    Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well...... as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study......, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have...

  19. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  20. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Science.gov (United States)

    Bhargava, Amol; Cotton, James A; Dixon, Brent R; Gedamu, Lashitew; Yates, Robin M; Buret, Andre G

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  1. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    International Nuclear Information System (INIS)

    Niles, L.P.; Hashemi, F.

    1990-01-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [ 125 I]iodomelatonin, was examined using an incubation temperature (30 degree C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [ 125 I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus

  2. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    Science.gov (United States)

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

  3. Adenylate cyclase regulates elongation of mammalian primary cilia

    International Nuclear Information System (INIS)

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-01-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3β by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  4. Enhanced adenylate cyclase activity of turkey erythrocytes following treatment with beta-adrenergic receptor antagonists.

    Science.gov (United States)

    Peters, J R; Nambi, P; Sibley, D R; Lefkowitz, R J

    1984-12-15

    The turkey erythrocyte contains a beta 1-adrenergic receptor-linked adenylate cyclase system. We have examined the effects of pretreatment with receptor antagonists on adenylate cyclase activity and the individual components in the pathway of enzyme activation in this system. Isoproterenol-stimulated adenylate cyclase activity is increased by 30% (P less than 0.01) over control in membranes derived from cells preincubated with the antagonist propranolol. The effect is stereospecific and dose-related with a EC50 of 100 nM for the (-) isomer. The time course of effect is rapid being complete by 90 min. Non-receptor mediated stimulation of adenylate cyclase activity by manganese ion, forskolin and NaF is similarly enhanced following propranolol pretreatment. Sensitization of adenylate cyclase activity also occurs following pretreatment with a number of antagonists but is not seen after preincubation with pindolol or practolol. Quantitation of beta-adrenergic receptor (R) density using [125I]cyanopindolol indicates no difference between membranes derived from control and antagonist pretreated cells. Coupling of R with the guanine nucleotide regulatory protein (N) as assessed by high affinity agonist binding is unchanged following pretreatment. The efficacy of 5'-guanylylimidodiphosphate Gpp(NH)p in producing a shift of agonist binding curves associated with destabilization of high affinity H-R-N complexes, is also the same (EC50 = 0.2 microM) in membranes from control and antagonist treated cells. The isoproterenol stimulated rate of release of [3H]GDP from membranes preloaded with [3H]GTP as an index of formation of an active form of the N protein is similarly unaffected by antagonist preincubation. We conclude that the mechanism of the observed sensitization of turkey erythrocyte adenylate cyclase by beta-adrenergic antagonists is receptor mediated and likely involves facilitation of N interaction with the catalytic subunit of the enzyme.

  5. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    Science.gov (United States)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  6. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

    2016-01-01

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM 1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM 1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM 1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [ 125 I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β 2 -adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM 1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  7. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  8. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Osička, Radim; Bumba, Ladislav; Šebo, Peter

    2015-01-01

    Roč. 73, č. 8 (2015) ISSN 2049-632X R&D Projects: GA ČR GAP302/12/0460; GA ČR GA15-09157S; GA ČR(CZ) GA15-11851S Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * membrane penetration * pore-formation Subject RIV: EE - Microbiology, Virology Impact factor: 2.483, year: 2015

  9. Kinases and Cancer.

    Science.gov (United States)

    Cicenas, Jonas; Zalyte, Egle; Bairoch, Amos; Gaudet, Pascale

    2018-03-01

    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 [...].

  10. 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 [...

  11. Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases

    Czech Academy of Sciences Publication Activity Database

    Břehová, Petra; Šmídková, Markéta; Skácel, Jan; Dračínský, Martin; Mertlíková-Kaiserová, Helena; Velasquez, M. P. S.; Watts, V. J.; Janeba, Zlatko

    2016-01-01

    Roč. 11, č. 22 (2016), s. 2534-2546 ISSN 1860-7179 R&D Projects: GA MV VG20102015046; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : adenylate cyclase toxin * acyclic nucleoside phosphonates * anthranilic acid Subject RIV: CC - Organic Chemistry Impact factor: 3.225, year: 2016

  12. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    International Nuclear Information System (INIS)

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E.

    1987-01-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the β-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the β-adrenergic pathway, adenylate cyclase activity and β-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. β-Adrenergic receptors were identified in BAT using [ 125 I]iodocyanopindolol. Binding sites had the characteristics of mixed β 1 - and β 2 -type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in β-adrenergic receptor density due to a loss of the β 1 -adrenergic subtype. This BAT β-adrenergic receptor downregulation was tissue specific, since myocardial β-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of β-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability

  13. Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site.

    Science.gov (United States)

    Skurat, A V; Yurkova, M S; Baranova, L A; Gulyaev, N N; Bulargina, T V; Severin, E S

    1985-03-01

    6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.

  14. Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

    DEFF Research Database (Denmark)

    Hautmann, Matthias; Friis, Ulla G; Desch, Michael

    2007-01-01

    Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because...

  15. Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

    DEFF Research Database (Denmark)

    Andersen, Martin Nybo; Hefting, Louise Leth; Steffensen, Annette Buur

    2015-01-01

    The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the IKs current, which is enhanced by PKA mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.......1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not co-assemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin Darby Canine Kidney cells. While PKA inhibition...... reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition lead to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role...

  16. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...... reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12...

  17. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

    International Nuclear Information System (INIS)

    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-01-01

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists

  18. Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

    Science.gov (United States)

    He, Shuai; Kyaw, Yi Mon Ei; Tan, Eddie Khay Ming; Bekale, Laurent; Kang, Malvin Wei Cherng; Kim, Susana Soo-Yeon; Tan, Ivan; Lam, Kong-Peng; Kah, James Chen Yong

    2018-04-26

    The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)-kemptide (Kem) bioconjugate (AuNS-BSA-Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm -1 , whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS-BSA-Kem as a SERS probe for cancer screening based on PKA activity.

  19. AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

    Science.gov (United States)

    Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia; Caplan, Michael J

    2010-12-01

    The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

  20. Mutations in the hydrophobic surface of an amphipathic groove of 14-3-3zeta disrupt its interaction with Raf-1 kinase.

    Science.gov (United States)

    Wang, H; Zhang, L; Liddington, R; Fu, H

    1998-06-26

    14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.

  1. 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

  2. Functional exchange of components between light-activated photoreceptor phosphodiesterase and hormone-activated adenylate cyclase systems

    Energy Technology Data Exchange (ETDEWEB)

    Bitensky, M.W.; Wheeler, M.A.; Rasenick, M.M.; Yamazaki, A.; Stein, P.J.; Halliday, K.R.; Wheeler, G.L.

    1982-06-01

    Previous studies have noted profound similarities between the regulation of light-activated (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) in a variety of tissues. We report ere the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was found to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-(..beta..,..gamma..)imidotriphosphate (p(NH)ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p(NH)ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn/sup 2 +/-activated adenylate cyclase activites and this inhibition was reversed by photoreceptor GBPp(NH)ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.

  3. Vanadate stimulates adenylate cyclase via the guanine nucleotide regulatory protein by a mechanism differing from that of fluoride.

    Science.gov (United States)

    Krawietz, W; Downs, R W; Spiegel, A M; Aurbach, G D

    1982-03-01

    Vanadate stimulates adenylate cyclase activity in turkey erythrocyte membranes. The maximal stimulation is 7-fold over basal at 3 mM vanadate; higher concentrations are inhibitory. A suboptimal concentration of fluoride (1 mM) together with vanadate (3 mM) activates adenylate cyclase in a non-additive manner; cyclase activation by optimal fluoride (10 mM) is inhibited by vanadate (3 mM). There is no stimulation by vanadate of adenylate cyclase activity (measured either with Mg2+ or Mn2+) in CYC- S49 lymphoma cell membranes. Vanadate (3 mM) shows no effect on binding of Beta-adrenergic agonists or antagonists to the [3H] (-)-dihydroalprenolol binding site in turkey erythrocyte membranes. These results suggest that the effect of vanadate on Adenylate cyclase is mediated through the nucleotide regulatory protein and may act by a mechanism similar to fluoride. However, in cholera toxic-treated membranes as well as in GDP-beta-S plus isoproterenol-treated membranes, fluoride-stimulated adenylate cyclase activity is significantly reduced, but vanadate stimulation is not. Our results suggest that although the actions of vanadate and fluoride in adenylate cyclase may each involve the nucleotide regulatory unit, the exact mechanisms of activation by the two anions differ.

  4. Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

    Directory of Open Access Journals (Sweden)

    Rouhiainen Leo

    2008-09-01

    Full Text Available Abstract Background Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy. mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer. Results A comparative analysis of the adenylation domains from the first module of McyB (McyB1 and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants. Conclusion We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may

  5. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    International Nuclear Information System (INIS)

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-01-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands 3 H-N-methylscopolamine ( 3 H-NMS), 3 H-quinuclidinyl benzilate ( 3 H-QNB), and 3 H-pirenzepine. 3 H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, 3 H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by 3 H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for 3 H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure

  6. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    Energy Technology Data Exchange (ETDEWEB)

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  7. Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Wald, Tomáš; Petry-Podgorska, Inga; Fišer, Radovan; Matoušek, Tomáš; Dědina, Jiří; Osička, Radim; Šebo, Peter; Mašín, Jiří

    2014-01-01

    Roč. 450, APR 2014 (2014), s. 57-62 ISSN 0003-2697 R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR GA13-14547S; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 ; RVO:68081715 Keywords : Potassium * Adenylate cyclase toxin * RTX Subject RIV: CE - Biochemistry Impact factor: 2.219, year: 2014

  8. Interaction of Bordetella adenylate cyclase toxin with complement receptor 3 involves multivalent glycan binding

    Czech Academy of Sciences Publication Activity Database

    Hasan, Shakir; Osičková, Adriana; Bumba, Ladislav; Novák, Petr; Šebo, Peter; Osička, Radim

    2015-01-01

    Roč. 589, č. 3 (2015), s. 374-379 ISSN 0014-5793 R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR(CZ) GA15-09157S; GA ČR(CZ) GA15-11851S Institutional support: RVO:61388971 Keywords : Adenylate cyclase toxin * CD11b/CD18 * Complement receptor type 3 Subject RIV: CE - Biochemistry Impact factor: 3.519, year: 2015

  9. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    Czech Academy of Sciences Publication Activity Database

    Osička, Radim; Osičková, Adriana; Hasan, Shakir; Bumba, Ladislav; Černý, Jiří; Šebo, Peter

    2015-01-01

    Roč. 4, DEC 9 (2015) ISSN 2050-084X R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR(CZ) GA15-11851S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 ; RVO:86652036 Keywords : E. coli * adenylate cyclase toxin * biochemistry Subject RIV: CE - Biochemistry Impact factor: 8.282, year: 2015

  10. Different strictuctural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Konopásek, I.; Svobodová, J.; Šebo, Peter

    2004-01-01

    Roč. 1660, - (2004), s. 144-154 ISSN 0005-2736 R&D Projects: GA AV ČR IPP1050128; GA AV ČR IAA5020907 Grant - others:GA Howard Hughes Medical Institut(US) 55000334; GA(XE) QLK2-CT-1999-00556 Institutional research plan: CEZ:AV0Z5020903 Keywords : bordetella pertussis * adenylate cyclase toxin * membrane interaction Subject RIV: EE - Microbiology, Virology Impact factor: 3.441, year: 2004

  11. Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling

    Czech Academy of Sciences Publication Activity Database

    Kamanová, Jana; Kofroňová, Olga; Mašín, Jiří; Genth, H.; Vojtová, Jana; Linhartová, Irena; Benada, Oldřich; Just, I.; Šebo, Peter

    2008-01-01

    Roč. 181, č. 8 (2008), s. 5587-5597 ISSN 0022-1767 R&D Projects: GA MŠk 1M0506; GA MŠk 2B06161; GA ČR GA310/08/0447 Grant - others:XE(XE) LSHB-CT-2003-503582 Institutional research plan: CEZ:AV0Z50200510 Keywords : bordetella * adenylate cyclase toxin * rhoa Subject RIV: EC - Immunology Impact factor: 6.000, year: 2008

  12. Age-associated alterations in hepatic β-adrenergic receptor/adenylate cyclase complex

    International Nuclear Information System (INIS)

    Graham, S.M.; Herring, P.A.; Arinze, I.J.

    1987-01-01

    The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 22-mo-old rats. The Mn 2+ -dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and ∼ 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the β-adrenergic receptor, as measured by the binding of [ 125 I]-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase in the proportion of β-receptors in the high-affinity state. These observations suggest that β-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of β-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase

  13. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

    OpenAIRE

    Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

    2010-01-01

    Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a...

  14. 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.

  15. Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

    Science.gov (United States)

    Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

    2006-11-24

    The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

  16. cAMP-dependent protein kinase inhibits α7 nicotinic receptor activity in layer 1 cortical interneurons through activation of D1/D5 dopamine receptors.

    Science.gov (United States)

    Komal, Pragya; Estakhr, Jasem; Kamran, Melad; Renda, Anthony; Nashmi, Raad

    2015-08-15

    Protein kinases can modify the function of many proteins including ion channels. However, the role of protein kinase A in modifying nicotinic receptors in the CNS has never been investigated. We showed through whole-cell recordings of layer 1 prefrontal cortical interneurons that α7 nicotinic responses are negatively modulated by protein kinase A. Furthermore, we show that stimulation of dopamine receptors can similarly attenuate α7 nicotinic responses through the activation of protein kinase A. These results suggest how the interaction of the cholinergic and dopaminergic systems may influence neuronal excitability in the brain. Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4β2. Serine 365 in the M3-M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase-PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer 1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine

  17. Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid.

    Directory of Open Access Journals (Sweden)

    Simon Vobruba

    Full Text Available Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R-4-propyl-proline (PPL. Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.

  18. Comparison of the in vivo and in vitro activities of adenylate cyclase from Mycobacterium tuberculosis H37Ra(NCTC 7417)

    International Nuclear Information System (INIS)

    Padh, Harish; Venkitsubramanian, T.A.

    1979-01-01

    The incorporation of [ 14 C] adenine into the adenosine 3', 5'-monophosphate (cyclic AMP) fraction by whole cells of Mycobacterium tuberculosis was taken as a measure of the in vivo activity of adenylate cyclase. The in vivo activity of adenylate cyclase was significantly inhibited by glucose, thus suggesting that the low level of cyclic AMP in the presence of glucose is due to the inhibited synthesis of cyclic AMP. In vitro activity of adenylate cyclase had optimum pH of 8.5 and Km of 1.33 mM for ATP. Glucose and other sugars did not show significant inhibition of in vitro activity. The results suggest that the adenylate cyclase activity becomes less sensitive to glucose when the bacterial cells are disrupted, an analogy with eukaryotic adenylate cyclase which loses sensitivity to hormones when the cells are disrupted. (auth.)

  19. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

    Czech Academy of Sciences Publication Activity Database

    Novák, Jakub; Černý, Ondřej; Osičková, Adriana; Linhartová, Irena; Mašín, Jiří; Bumba, Ladislav; Šebo, Peter; Osička, Radim

    2017-01-01

    Roč. 9, č. 10 (2017), s. 1-28, č. článku 300. E-ISSN 2072-6651 R&D Projects: GA ČR GA15-09157S; GA ČR(CZ) GA16-05919S; GA MŠk(CZ) LM2015064; GA MZd(CZ) NV16-28126A Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * Bordetella * cAMP Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.030, year: 2016

  20. Alteration in adenylate cyclase response to aminergic stimulation following neonatal x-irradiation

    International Nuclear Information System (INIS)

    Chronister, R.B.; Palmer, G.C.; Gerbrandt, L.

    1980-01-01

    X-irradiation of the rat neonatal hippocampus produces severe alterations in the architectonic features of the mature hippocampus. The most prominent alteration is a marked depletion of the granule cells of the dentate gyrus, with a subsequent realignment of CA 4 cells. The present data also show that norepinephrine (NE), dopamine and histamine stimulation of adenylate cyclase activity is severely attenuated in the hippocampi of irradiated animals. This failure suggests that the NE fibers of irradiated subjects, although normal in content of NE, are not functional in some of their NE-effector actions

  1. Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

    Czech Academy of Sciences Publication Activity Database

    Basler, Marek; Knapp, O.; Mašín, Jiří; Fišer, R.; Maier, E.; Benz, R.; Šebo, Peter; Osička, Radim

    2007-01-01

    Roč. 282, č. 17 (2007), s. 12419-12429 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA AV ČR IAA5020406 Grant - others:XE(XE) European Union 6th FP contract LSHB-CT-2003-503582 THERAVAC Institutional research plan: CEZ:AV0Z50200510 Source of funding: R - rámcový projekt EK Keywords : bordetella * adenylate cyclase toxin * ac membrane translocation Subject RIV: EE - Microbiology, Virology Impact factor: 5.581, year: 2007

  2. Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis*

    Science.gov (United States)

    Boll, Björn; Taubitz, Tatjana; Heide, Lutz

    2011-01-01

    MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes. PMID:21890635

  3. The role of transcriptional regulation in maintaining the availability of mycobacterial adenylate cyclases

    Directory of Open Access Journals (Sweden)

    Sarah J. Casey

    2014-03-01

    Full Text Available Mycobacterium species have a complex cAMP regulatory network indicated by the high number of adenylate cyclases annotated in their genomes. However the need for a high level of redundancy in adenylate cyclase genes remains unknown. We have used semiquantitiative RT-PCR to examine the expression of eight Mycobacterium smegmatis cyclases with orthologs in the human pathogen Mycobacterium tuberculosis, where cAMP has recently been shown to be important for virulence. All eight cyclases were transcribed in all environments tested, and only four demonstrated environmental-mediated changes in transcription. M. smegmatis genes MSMEG_0545 and MSMEG_4279 were upregulated during starvation conditions while MSMEG_0545 and MSMEG_4924 were downregulated in H2O2 and MSMEG_3780 was downregulated in low pH and starvation. Promoter fusion constructs containing M. tuberculosis H37Rv promoters showed consistent regulation compared to their M. smegmatis orthologs. Overall our findings indicate that while low levels of transcriptional regulation occur, regulation at the mRNA level does not play a major role in controlling cellular cyclase availability in a given environment.

  4. Effects of sevoflurane on adenylate cyclase and phosphodiesterases activity in brain of rats

    International Nuclear Information System (INIS)

    Feng Changdong; Yang Jianping; Dai Tijun

    2009-01-01

    Objective: To investigate the effects of sevoflurane on c adenylate cyclase (AC) and phosphodiesterases (PDE) activity in the cerebrocortex, hippocampus and brain stem of rats, and to examine the role of cAMP in sevoflurane anesthesia. Methods: Fourty SD rats were delaminately designed and allocated randomly to 5 groups inhaling 1.5% sevoflurane i.e., no recovery (recovery group, n=8) and one hour after righting reflexrecovery (aware group, n=8). The brain tissues were rapidly dissected into cerebrocortex and hippocampus and brain stem.Then the adenylate cyclase and phosphodiesterases activity were assessed. Results: So far as the activity of AC is concerned, compared with the control group, the activity of AC in the cerebrocortex, hippocampus and brain stem brain stem of induction group and anesthesia group, the cerebrocortex, and hippocampus in the recovery group were significantly increased; compared with those in the anesthesia group, the activity of AC in the cerebrocortex, hippocampus and brain stem of aware group were significantly decreased (P<0.05); For the activity of PDE, compared with the control group, the activity of PDE in the cerebrocortex, hippocampus and brain stem in the induction group and anesthesia group was significantly decreased, compared with that in anesthesia group, the activity of PDE in the cerebrocortex, hippocampus and brain stem of recovery group and aware group was significantly increased (P<0.05). Conclusion: cAMP may play an important role in sevoflurane anesthesia. (authors)

  5. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

  6. Effect of drugs on lipid methylation, receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

    NARCIS (Netherlands)

    Van Waarde, Aren; Van Haastert, P.J.M.

    1986-01-01

    Intercellular communication in Dictyostelium discoldeum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on

  7. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  8. Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP.

    Science.gov (United States)

    Cassel, D; Selinger, Z

    1978-09-01

    The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.

  9. Amidate Prodrugs of 9-[2-(Phosphonomethoxy)Ethyl]Adenine as Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Šmídková, Markéta; Dvořáková, Alexandra; Tloušťová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Roč. 58, č. 2 (2014), s. 664-671 ISSN 0066-4804 R&D Projects: GA MV VG20102015046 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : Bordetella pertussis * adenylate cyclase toxin * ACT * inhibitors * PMEA * amidate prodrugs Subject RIV: CC - Organic Chemistry Impact factor: 4.476, year: 2014

  10. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    Energy Technology Data Exchange (ETDEWEB)

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. (Duke Univ. Medical Center, Durham, NC (USA))

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  11. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    International Nuclear Information System (INIS)

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J.

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a β-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of β-adrenergic receptors: in fact, [ 125 I]pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level

  12. Distribution and protective function of pituitary adenylate cyclase-activating polypeptide (PACAP in the retina

    Directory of Open Access Journals (Sweden)

    Tomoya eNakamachi

    2012-11-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP, which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells.

  13. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

    DEFF Research Database (Denmark)

    Sasaki, S; Watanabe, J; Ohtaki, H

    2017-01-01

    BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required...... and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression....... Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin...

  14. Selective splitting of 3'-adenylated dinucleoside polyphosphates by specific enzymes degrading dinucleoside polyphosphates.

    Science.gov (United States)

    Guranowski, Andrzej; Sillero, Antonio; Günther Sillero, María Antonia

    2003-01-01

    Several 3'-[(32)P]adenylated dinucleoside polyphosphates (Np(n)N'p*As) were synthesized by the use of poly(A) polymerase (Sillero MAG et al., 2001, Eur J Biochem.; 268: 3605-11) and three of them, ApppA[(32)P]A or ApppAp*A, AppppAp*A and GppppGp*A, were tested as potential substrates of different dinucleoside polyphosphate degrading enzymes. Human (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17) acted almost randomly on both AppppAp*A, yielding approximately equal amounts of pppA + pAp*A and pA + pppAp*A, and GppppGp*, yielding pppG + pGp*A and pG + pppGp*A. Narrow-leafed lupin (Lupinus angustifolius) tetraphosphatase acted preferentially on the dinucleotide unmodified end of both AppppAp*A (yielding 90% of pppA + pAp*A and 10 % of pA + pppAp*A) and GppppGp*A (yielding 89% pppG + pGp*A and 11% of pG + pppGp*A). (Symmetrical) dinucleoside tetraphosphatase (EC 3.6.1.41) from Escherichia coli hydrolyzed AppppAp*A and GppppGp*A producing equal amounts of ppA + ppAp*A and ppG + ppGp*A, respectively, and, to a lesser extent, ApppAp*A producing pA + ppAp*A. Two dinucleoside triphosphatases (EC 3.6.1.29) (the human Fhit protein and the enzyme from yellow lupin (Lupinus luteus)) and dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53) from Saccharomyces cerevisiae did not degrade the three 3'-adenylated dinucleoside polyphosphates tested.

  15. Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase

    International Nuclear Information System (INIS)

    Verkman, A.S.; Skorecki, K.L.; Ausiello, D.A.

    1986-01-01

    Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone

  16. Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation.

    Science.gov (United States)

    Magnone, Mirko; Basile, Giovanna; Bruzzese, Debora; Guida, Lucrezia; Signorello, Maria Grazia; Chothi, Madhu Parakkottil; Bruzzone, Santina; Millo, Enrico; Qi, Ai-Dong; Nicholas, Robert A; Kassack, Matthias U; Leoncini, Giuliana; Zocchi, Elena

    2008-09-05

    Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 microm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

  17. Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

    Science.gov (United States)

    Nathanson, James A.; Greengard, Paul

    1974-01-01

    An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

  18. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    Directory of Open Access Journals (Sweden)

    Chen Yun-Ru

    2012-09-01

    Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

  19. Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

    Science.gov (United States)

    Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

    2018-05-02

    Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

  20. 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)....

  1. Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin

    Czech Academy of Sciences Publication Activity Database

    Staneková, Z.; Adkins, Irena; Kosová, Martina; Janulíková, J.; Šebo, Peter; Varečková, E.

    2013-01-01

    Roč. 97, č. 1 (2013), s. 24-35 ISSN 0166-3542 R&D Projects: GA ČR GA310/08/0447; GA ČR GP310/09/P582 Institutional support: RVO:61388971 Keywords : Bordetella adenylate cyclase toxoid * Influenza A infection * Cross-protection Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.434, year: 2013

  2. Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

    Czech Academy of Sciences Publication Activity Database

    Knapp, O.; Maier, E.; Mašín, Jiří; Šebo, Peter; Benz, R.

    2008-01-01

    Roč. 1778, č. 1 (2008), s. 260-269 ISSN 0005-2736 R&D Projects: GA AV ČR(CZ) IAA5020406 Grant - others:XE(XE) QLK2-CT-1999-00556 Institutional research plan: CEZ:AV0Z50200510 Keywords : adenylate cyclase toxin * act * voltage Subject RIV: EE - Microbiology, Virology Impact factor: 4.180, year: 2008

  3. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the circulation after sumatriptan

    DEFF Research Database (Denmark)

    Hansen, Jakob Møller; Fahrenkrug, Jan; Petersen, Jesper Troensegaard

    2013-01-01

    The origin of migraine pain is still elusive, but increasingly researchers focus on the neuropeptides in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. The parasympathetic neurotransmitters, pituitary adenylate cyclase activating...... peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) may be released from parasympathetic fibres and activate sensory nerve fibres during migraine attacks. Triptans are effective and well tolerated in acute migraine management but the exact mechanism of action is still debated. Triptans might...

  4. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    Energy Technology Data Exchange (ETDEWEB)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  5. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB.20

    International Nuclear Information System (INIS)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT 1C receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing [ 3 H]serotonin, [ 3 H]lysergic acid diethylamide or [ 3 H]dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor

  6. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    International Nuclear Information System (INIS)

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-01-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the [ 3 H]-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the [ 3 H]DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the β-adrenergic receptor in rat heart

  7. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

  8. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  9. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Antal

    Full Text Available Adenylate cyclase type III (AC3 is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells. In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

  10. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    International Nuclear Information System (INIS)

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-01-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine

  11. Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

    Science.gov (United States)

    Johns, Christian W; Finley, Natosha L

    2017-11-30

    Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α -helical content to CaMWt, and only modest differences in α -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N -terminal CaM and CyaA-ACD.

  12. Adenyl cyclase activator forskolin protects against Huntington's disease-like neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Sidharth Mehan

    2017-01-01

    Full Text Available Long term suppression of succinate dehydrogenase by selective inhibitor 3-nitropropionic acid has been used in rodents to model Huntington's disease where mitochondrial dysfunction and oxidative damages are primary pathological hallmarks for neuronal damage. Improvements in learning and memory abilities, recovery of energy levels, and reduction of excitotoxicity damage can be achieved through activation of Adenyl cyclase enzyme by a specific phytochemical forskolin. In this study, intraperitoneal administration of 10 mg/kg 3-nitropropionic acid for 15 days in rats notably reduced body weight, worsened motor cocordination (grip strength, beam crossing task, locomotor activity, resulted in learning and memory deficits, greatly increased acetylcholinesterase, lactate dehydrogenase, nitrite, and malondialdehyde levels, obviously decreased adenosine triphosphate, succinate dehydrogenase, superoxide dismutase, catalase, and reduced glutathione levels in the striatum, cortex and hippocampus. Intragastric administration of forskolin at 10, 20, 30 mg/kg dose-dependently reversed these behavioral, biochemical and pathological changes caused by 3-nitropropionic acid. These results suggest that forskolin exhibits neuroprotective effects on 3-nitropropionic acid-induced Huntington's disease-like neurodegeneration.

  13. Adenyl cyclases and cAMP in plant signaling - Past and present

    KAUST Repository

    Gehring, Christoph A.

    2010-06-25

    In lower eukaryotes and animals 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins. 2010 Gehring; licensee BioMed Central Ltd.

  14. [Effect of Qishen Yiqi droplet on adenylate contents in myocardium of cor pulmonale rats].

    Science.gov (United States)

    Geng, Shi-Han; Shan, Qiu-Yue; Xu, Meng-Xi; Zhang, Yi-Dan; Song, Li-Li; Hou, Ning; Zhang, Wen; Zhang, Mi-Xia; Zhuang, Peng-Wei; Zhang, Yan-Jun

    2017-01-01

    Cor pulmonale rat models were induced by a single intraperitoneal injection of monocrotaline(MCT), and the sham group received a single intraperitioneal injection of normal saline. After the model rats received intragastric administration of Qishen Yiqi droplet(QS) for 6 weeks, the contents of adenylate(ATP, ADP and AMP) in right myocardial tissues were measured by HPLC, and then the metabolism changes in myocardium of cor pulmonale rats with QS were investigated. The results showed that ATP, ADP, and AMP were well separated, with a good linearity within a certain range of concentration; and the recovery rates were within the range of 90%-108%. As compared with model group, the level of ATP was significantly elevated in high-dose treatment group; ADP contents showed an increasing trend and AMP contents showed a decreasing trend, indicating that QS could significantly improve energy metabolism system in myocardium. By using the HPLC, a qualitative and quantitative analysis method was given for the determination of ATP, ADP and AMP contents in myocardium, providing a method for energy metabolism measurement in biological samples. Copyright© by the Chinese Pharmaceutical Association.

  15. Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states.

    Science.gov (United States)

    Lad, P M; Reisinger, D M; Smiley, P A

    1983-06-21

    The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.

  16. 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 ...

  17. 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...

  18. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    Science.gov (United States)

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  19. A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

    Science.gov (United States)

    Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

    2010-10-01

    Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

  20. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

    Science.gov (United States)

    Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

    2010-01-01

    Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1–EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3′ addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3′ adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake. PMID:20719920

  1. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    , and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[ß,¿-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can...... of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine...... of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the ¿-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining...

  2. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

    Science.gov (United States)

    Sasaki, S; Watanabe, J; Ohtaki, H; Matsumoto, M; Murai, N; Nakamachi, T; Hannibal, J; Fahrenkrug, J; Hashimoto, H; Watanabe, H; Sueki, H; Honda, K; Miyazaki, A; Shioda, S

    2017-02-01

    Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. To investigate the effect of PACAP on eccrine sweat secretion. Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders. © 2016 British Association of Dermatologists.

  3. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  4. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    Directory of Open Access Journals (Sweden)

    Matthew M. Hurley

    2016-08-01

    Full Text Available While pituitary adenylate cyclase activating polypeptide (PACAP signaling in the hypothalamic ventromedial nuclei (VMN has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger and hedonic-related (palatability drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding; surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding. In contrast, inhibition of the nucleus accumbens (NAc, through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

  5. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

  6. Pituitary adenylate cyclase-activating polypeptide (PACAP) in fetal cord blood.

    Science.gov (United States)

    Winters, Stephen J; King, Jeffrey C; Brees, Carol K; Moore, Joseph P

    2014-09-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a centrally-acting peptide with highest concentration within the limbic area of the brain. PACAP is also expressed in and affects the functions of vascular and nervous tissues, endocrine glands, and the placenta. PACAP appears to be associated with the 'fight-or-flight' response to emergency partly through its effect on adrenal production of cortisol and catecholamines. We sought to explore the impact of labor as a stressor on the level of PACAP in the fetus, and hypothesized that PACAP levels would be increased when labor, abnormal fetal heart tracing, or fetal growth impairment was evident. Fetal cord venous blood samples were collected immediately after delivery from a random group of women undergoing either vaginal or Cesarean delivery. The blood was placed in chilled EDTA-aprotinin containing tubes, centrifuged, and stored at -80°C for PACAP immunoassay. Delivery data were abstracted for analysis. The level of PACAP in cord blood was similar in both males and females. There was a trend toward higher levels in the cord blood of fetuses delivered by Cesarean compared to those delivered vaginally. PACAP levels were unrelated to birth weight, Apgar scores, and the presence or absence of labor prior to delivery. While PACAP and its receptor are expressed in placenta, and PACAP protein is found in cord blood, no effect of labor stress on PACAP was found. Further research is needed to understand the role of PACAP in gestation and parturition. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    International Nuclear Information System (INIS)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-01-01

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R h ) and reduced thermal stability in the mutant complex. Taken together

  8. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  9. Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer

    Directory of Open Access Journals (Sweden)

    Kenyon Colin P

    2012-03-01

    Full Text Available Abstract Background The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. Results A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile. Conclusions The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the

  10. Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice

    Science.gov (United States)

    Roman, Brian B.; Meyer, Ronald A.; Wiseman, Robert W.

    2002-01-01

    Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.

  11. Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

    Science.gov (United States)

    Lu, Wan-Jung; Chang, Nen-Chung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Chou, Duen-Suey; Thomas, Philip Aloysius; Sheu, Joen-Rong

    2014-12-01

    CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and

  12. Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.

    Science.gov (United States)

    Misiek, Mathias; Braesel, Jana; Hoffmeister, Dirk

    2011-08-01

    The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  13. Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells.

    Science.gov (United States)

    Hernandez, A; Kimball, B; Romanchuk, G; Mulholland, M W

    1995-01-01

    The ability of PACAP-38 to stimulate morphological development was studied using rat pheochromocytoma PC12 cells. PACAP-38 produced concentration-dependent increases in percentage of cells exhibiting neurite extension. Similar increases were produced by forskolin (28 +/- 2% at 96 h) and 8-bromo cAMP (30 +/- 2%). Vasoactive intestinal peptide and alpha-calcitonin gene-related peptide were without effect. PACAP-38 produced significant increases in PC12 cell cAMP content and inositol phosphate turnover. Intracellular [Ca2+] increased from 169 +/- 14 nM to 560 +/- 58 nM in response to 1 microM PACAP-38. PACAP-stimulated neurite outgrowth was abolished by RpcAMPS, an inhibitor of cAMP-dependent kinases but was unaffected by the protein kinase C antagonist H7.

  14. Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

    Czech Academy of Sciences Publication Activity Database

    Novák, Jakub; Fabrik, I.; Linhartová, Irena; Link, M.; Černý, Ondřej; Stulík, J.; Šebo, Peter

    2017-01-01

    Roč. 7, NOV 24 (2017), č. článku 16298. ISSN 2045-2322 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S Institutional support: RVO:61388971 Keywords : GTPASE-ACTIVATING-PROTEIN * SALT-INDUCIBLE KINASES * GENE ONTOLOGY Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.259, year: 2016

  15. A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function

    DEFF Research Database (Denmark)

    Vuorenpää, Anne Elina; Ammendrup-Johnsen, Ina; Jorgensen, Trine N.

    2016-01-01

    cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein...... in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated...... in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated...

  16. Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

    Czech Academy of Sciences Publication Activity Database

    Bakal, Tomáš; Goo, K.-S.; Najmanová, Lucie; Plháčková, Kamila; Kadlčík, Stanislav; Ulanová, Dana

    2015-01-01

    Roč. 108, č. 5 (2015), s. 1267-1274 ISSN 0003-6072 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Nonribosomal peptide synthetase * Adenylation domain * Actinomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 1.944, year: 2015

  17. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.

    1994-01-01

    in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions...

  18. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  19. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, T. F.; Pávek, P.; Trejtnar, F.; Watts, V. J.; Janeba, Zlatko

    2015-01-01

    Roč. 10, č. 8 (2015), s. 1351-1364 ISSN 1860-7179 R&D Projects: GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : adenylate cyclase toxin * bisamidates * Bordetella pertussis * nucleosides * phosphonates Subject RIV: CC - Organic Chemistry Impact factor: 2.980, year: 2015

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP71-100 generates cAMP in vitro.

  6. Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity.

    Science.gov (United States)

    Fedele, Giorgio; Schiavoni, Ilaria; Adkins, Irena; Klimova, Nela; Sebo, Peter

    2017-09-21

    Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

  7. Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

    2017-09-01

    Full Text Available Adenylate cyclase toxin (CyaA is released in the course of B. pertussis infection in the host’s respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC, macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3′,5′-cyclic adenosine monophosphate (cAMP, which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

  8. Kinase signaling cascades in the mitochondrion: a matter of life or death.

    Science.gov (United States)

    Horbinski, Craig; Chu, Charleen T

    2005-01-01

    In addition to powering energy needs of the cell, mitochondria function as pivotal integrators of cell survival/death signals. In recent years, numerous studies indicate that each of the major kinase signaling pathways can be stimulated to target the mitochondrion. These include protein kinase A, protein kinase B/Akt, protein kinase C, extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. Although most studies focus on phosphorylation of pro- and antiapoptotic proteins (BAD, Bax, Bcl-2, Bcl-xL), kinase-mediated regulation of complex I activity, anion and cation channels, metabolic enzymes, and Mn-SOD mRNA has also been reported. Recent identification of a number of scaffold proteins (AKAP, PICK, Sab) that bring specific kinases to the cytoplasmic surface of mitochondria further emphasizes the importance of mitochondrial kinase signaling. Immunogold electron microscopy, subcellular fractionation and immunofluorescence studies demonstrate the presence of kinases within subcompartments of the mitochondrion, following diverse stimuli and in neurodegenerative diseases. Given the sensitivity of these signaling pathways to reactive oxygen and nitrogen species, in situ activation of mitochondrial kinases may represent a potent reverse-signaling mechanism for communication of mitochondrial status to the rest of the cell.

  9. cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2009-09-01

    The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

  10. Induction of rat hepatic zinc thionein by phorbol ester-mediated protein kinase C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Garrett, S.H.; Funk, A.E.; Brady, F.O.

    1986-05-01

    Metallothionein (MT) exists in rat liver mainly as a zinc protein. The levels of this protein fluctuate in response to a variety of internal and external stimuli. Among these inducers of MT are metals, glucocorticoids, catecholamines, and polypeptide hormones. Metals and glucocorticoids are primary inducers of MT, while the others operate either via adenylate cyclase/cAMP/cAMP-dependent protein kinase, or via phospholipase C/inositol 1,4,5-triphosphate, diacylglycerol/Ca/sup 2 +/-dependent protein kinase, protein kinase C. The authors have examined the role of the protein kinase C pathway in the induction of MT by using a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), to activate it. In vivo TPA is a good inducer of Zn/sub 7/-MT with an ED/sub 0.5/ of 26.5 nmoles/kg b.w. Maximal levels reached were about 7..mu..g Zn in MT/g liver, an induction increase of 8 to 10-fold. An inactive compound, 4..beta..-phorbol, and the vehicle (DMSO) did not stimulate the synthesis of Zn/sub 7/-MT. This induction by TPA requires de novo protein synthesis, as demonstrated by a cycloheximide/(/sup 35/S)-cysteine experiment. TPA stimulated Zn incorporation by 8.6-fold and (/sup 35/S)-cysteine incorporation by 4.8-fold during an 11h induction. These increases were blocked 100% by treatment with cycloheximide at -1 and +5h. These experiments have been repeated in cultured hepatocytes, using (/sup 35/S)-cysteine incorporation, slab SDS-PAGE, and autoradiography to quantitate MT levels.

  11. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

    Directory of Open Access Journals (Sweden)

    Radovan Fiser

    Full Text Available Bordetella adenylate cyclase toxin-hemolysin (CyaA penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

  12. Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity

    Directory of Open Access Journals (Sweden)

    Hayeß Katrin

    2005-08-01

    Full Text Available Abstract Background Creatine kinase (CK links phosphocreatine, an energy storage system, to cellular ATPases. CK activity serves as a temporal and spatial buffer for ATP content, particularly in fast-twitch skeletal muscles. The extraocular muscles are notoriously fast and active, suggesting the need for efficient ATP buffering. This study tested the hypotheses that (1 CK isoform expression and activity in rat extraocular muscles would be higher, and (2 the resistance of these muscles to fatigue would depend on CK activity. Results We found that mRNA and protein levels for cytosolic and mitochondrial CK isoforms were lower in the extraocular muscles than in extensor digitorum longus (EDL. Total CK activity was correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a fraction of CK activity is found, were downregulated in the extraocular muscles as was shown by immunocytochemistry and western blotting. CK inhibition significantly accelerated the development of fatigue in EDL muscle bundles, but had no major effect on the extraocular muscles. Searching for alternative ATP buffers that could compensate for the relative lack of CK in extraocular muscles, we determined that mRNAs for two adenylate kinase (AK isoforms were expressed at higher levels in these muscles. Total AK activity was similar in EDL and extraocular muscles. Conclusion These data indicate that the characteristic fatigue resistance of the extraocular muscles does not depend on CK activity.

  13. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

    Science.gov (United States)

    Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

    2016-01-01

    combined results indicate that glycolysis is regulated by the compartmental expression of hexokinase 2, pyruvate kinase M1, and pyruvate kinase M2 in photoreceptors, whereas the inner retinal neurons exhibit a lower capacity for glycolysis and aerobic glycolysis. Expression of nucleoside diphosphate kinase, mitochondria-associated adenylate kinase, and several mitochondria-associated creatine kinase isozymes was highest in the outer retina, whereas expression of cytosolic adenylate kinase and brain creatine kinase was higher in the cones, horizontal cells, and amacrine cells indicating the diversity of ATP-buffering strategies among retinal neurons. Based on the antibody intensities and the COX and LDH activity, Müller glial cells (MGCs) had the lowest capacity for glycolysis, aerobic glycolysis, and OXPHOS. However, they showed high expression of glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate thiokinase, GABA transaminase, and ~P transferring kinases. This suggests that MGCs utilize TCA cycle anaplerosis and cataplerosis to generate GTP and ~P transferring kinases to produce ATP that supports MGC energy requirements. Our comprehensive and integrated results reveal that the adult mouse retina expresses numerous isoforms of ATP synthesizing, regulating, and buffering genes; expresses differential cellular and compartmental levels of glycolytic, OXPHOS, TCA cycle, and ~P transferring kinase proteins; and exhibits differential layer-by-layer LDH and COX activity. New insights into cell-specific and compartmental ATP and GTP production, as well as utilization and buffering strategies and their relationship with known retinal and cellular functions, are discussed. Developing therapeutic strategies for neuroprotection and treating retinal deficits and degeneration in a cell-specific manner will require such knowledge. This work provides a platform for future research directed at identifying the molecular targets and proteins that regulate these processes.

  14. A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

    Science.gov (United States)

    Grammel, Nicolas; Pankevych, Kvitka; Demydchuk, Julia; Lambrecht, Klaus; Saluz, Hans-Peter; Krügel, Hans

    2002-01-01

    Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.

  15. Functional changes in the properties of the β-adrenoreceptors of pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1986-01-01

    The β-adrenoreceptors were solubilized from the plasma membranes of pigeon erythrocytes, treated with N-ethylmaleimide, using deoxycholate. The removal of the deoxycholate leads to incorporation of receptors into phospholipid vesicles and a restoration of their biological activity. After fusion of vesicles containing reconstituted receptors with vesicles containing the N/sub s/-protein and the catalytic component, a restoration of the hormonal activity of the enzyme was observed. If vesicles containing β-adrenoreceptors were incubated before fusion with the catalytic subunit of cAMP-dependent protein kinase, the hormonal activity of the preparation obtained was lowered by 45-50%. The decrease in activity occurred on account of an increase in the lag phase of activation of the enzyme in the presence of isoproterenol and GPP(NH)p, as well as on account of a decrease in the activity in the stationary phase of activation. Phosphorylation of the β-adrenoreceptors leads to a decrease in the content of the ternary isoproterenol-receptor-N/sub s/-protein complex, participating in the activation of adenylate cyclase. Thus, phosphorylation of the receptors leads to disruptions of the mechanism of transmission of the hormonal signal, analogous to those observed in the desensitization of adenylate cyclase

  16. Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase.

    Science.gov (United States)

    Sharp, G W; Le Marchand-Brustel, Y; Yada, T; Russo, L L; Bliss, C R; Cormont, M; Monge, L; Van Obberghen, E

    1989-05-05

    -dependent Ca2+ channels. One suggested mechanism for the action of galanin is inhibition of adenylate cyclase. However, it was found that galanin inhibits insulin release even in the presence of 8-Br-cAMP, an agent which effectively bypasses adenylate cyclase. Therefore, an additional mechanism for the inhibitory effect of galanin must be present. All of the effects of galanin were sensitive to pertussis toxin. These data suggest two G-protein-dependent actions of galanin, one to hyperpolarize the plasma membrane and one at a distal point in stimulus-secretion coupling, close to the exocytotic event.

  17. 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.

  18. Kynurenic Acid Inhibits the Electrical Stimulation Induced Elevated Pituitary Adenylate Cyclase-Activating Polypeptide Expression in the TNC

    Directory of Open Access Journals (Sweden)

    Tamás Körtési

    2018-01-01

    Full Text Available BackgroundMigraine is a primary headache of imprecisely known mechanism, but activation of the trigeminovascular system (TS appears to be essential during the attack. Intensive research has recently focused on pituitary adenylate cyclase-activating polypeptide (PACAP and the kynurenine systems as potential pathogenic factors.AimWe investigated the link between these important mediators and the effects of kynurenic acid (KYNA and its synthetic analog (KYNA-a on PACAP expression in the rat trigeminal nucleus caudalis (TNC in a TS stimulation model related to migraine mechanisms.MethodsAdult male Sprague-Dawley rats were pretreated with KYNA, KYNA-a, the NMDA receptor antagonist MK-801, or saline (vehicle. Next, the trigeminal ganglion (TRG was electrically stimulated, the animals were transcardially perfused following 180 min, and the TNC was removed. In the TNC samples, 38 amino acid form of PACAP (PACAP1–38-like radioimmunoactivity was measured by radioimmunoassay, the relative optical density of preproPACAP was assessed by Western blot analysis, and PACAP1–38 mRNA was detected by real-time PCR.Results and conclusionElectrical TRG stimulation resulted in significant increases of PACAP1–38-LI, preproPACAP, and PACAP1–38 mRNA in the TNC. These increases were prevented by the pretreatments with KYNA, KYNA-a, and MK-801. This is the first study to provide evidence for a direct link between PACAP and the kynurenine system during TS activation.

  19. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    Science.gov (United States)

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  20. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  1. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    International Nuclear Information System (INIS)

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-01-01

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 μM and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 μM and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 μM respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D 2 -dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 μM. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, 3 H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D 1 - and D 2 -dopamine receptors. 33 references, 3 figures, 2 tables

  2. Brain histamine H1- and H2-receptors and histamine-sensitive adenylate cyclase: effects of antipsychotics and antidepressants

    International Nuclear Information System (INIS)

    Coupet, J.; Szuches-Myers, V.A.

    1981-01-01

    Several classes of psychoactive compounds have been investigated for their effects on histamine-sensitive adenylate cyclase in cell-free preparations from the guinea-pig cerebral cortex. Their inhibitory actions on this enzyme system have been compared with their abilities to displace [ 3 H]pyrilamine and [ 3 H]cimetidine from histamine H 1 - and H 2 -receptor sites, respectively. The results of these studies show that compounds which inhibited the histamine-sensitive cyclase were also displacers of either [ 3 H]pyrilamine or [ 3 H]cimetidine or both 3 H-ligands from their binding sites. In spite of the lack of a correlation between binding and cyclase antagonism it was observed that compounds that displace both ligands showed greater inhibition of the cyclase than those that have affinities for sites labeled by one or the other ligand. It was concluded that antihistamines, the antipsychotics and the antidepressants share a common property through their antagonism of H 1 -receptors and that may be responsible for their sedative side effect. (Auth.)

  3. Mechanism of control of the turkey erythrocyte beta-adrenoceptor dependent adenylate cyclase by guanyl nucleotides: a minimum model.

    Science.gov (United States)

    Braun, S; Tolkovsky, A M; Levitzki, A

    1982-01-01

    Treatment of native turkey erythrocyte membranes with GMP and epinephrine produces a highly active but metastable form of adenylate cyclase which decays slowly to basal native state. The decay process is greatly facilitated by GTP and GDP beta S1, and is further enhanced by 1-epinephrine. This decay process is prevented reversibly by GMP. GppNHp, like GMP, prevents the decay process first reversibly, but with time stabilizes the highly active state in a persistently active state. The expression of the catalytic activity of the enzyme in the metastable state can also be inhibited reversibly by GTP, GDP beta S and GMP at all times during the decay process. The GppNHp stabilized form is not susceptible to nucleotide inhibition. Thus, two forms of the guanyl nucleotide unit are postulated to exist: an "open" and a "closed" form. In the presence of hormone and GTP, the enzyme shuttles between these two forms continuously. GMP and GppNHp favor the complete conversion to the "open" form in the presence of beta-agonist. Evidence is also presented for the existence of two GTP dependent processes which exhibit different apparent affinities towards the nucleotide: A high affinity GTP binding process is essential for the fruitful coupling between receptor and enzyme, and a low affinity GTPase site which is responsible for the termination of the hormonal signal.

  4. Kinetics of interaction between beta-receptors, GTP protein, and the catalytic unit of turkey erythrocyte adenylate cyclase.

    Science.gov (United States)

    Tolkovsky, A M; Braun, S; Levitzki, A

    1982-01-01

    The kinetics of turkey erythrocyte membrane adenylate cyclase activation by beta-agonists and guanyl-5'-yl imidodiphosphate is explored as a function of the concentration of the GTP regulatory protein and of the catalytic unit. It was found that the overall kinetics of activation is first order and is independent of the concentration of the GTP regulatory unit N, the catalytic unit C, and of hormone over a very wide concentration range. It was established that the rate-limiting step does not involve GDP dissociation from the inactive N unit or the association between activated N' and C. Also, it was found that guanyl-5'-yl imidodiphosphate binding occurs in a random fashion and is not hormone dependent. These results enable us to exclude models of the sequential type in which N in its inactive form is bound to receptor R, is released in an active form N' upon hormone activation, and then binds to C, activating the latter. An acceptable model that accounts for all of the data conforms to the original formulation of "collision coupling" in which N is tightly associated to C at all times.

  5. The effects of isatin (indole-2, 3-dione on pituitary adenylate cyclase-activating polypeptide-induced hyperthermia in rats

    Directory of Open Access Journals (Sweden)

    Tóth Gábor

    2002-02-01

    Full Text Available Abstract Background Previous studies have demonstrated that centrally administered natriuretic peptides and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38 have hyperthermic properties. Isatin (indole-2, 3-dione is an endogenous indole that has previously been found to inhibit hyperthermic effects of natriuretic peptides. In this study the aim was to investigate the effects of isatin on thermoregulatory actions of PACAP-38, in rats. Results One μg intracerebroventricular (icv. injection of PACAP-38 had hyperthermic effect in male, Wistar rats, with an onset of the effect at 2 h and a decline by the 6th h after administration. Intraperitoneal (ip. injection of different doses of isatin (25-50 mg/kg significantly decreased the hyperthermic effect of 1 μg PACAP-38 (icv., whereas 12.5 mg/kg isatin (ip. had no inhibiting effect. Isatin alone did not modify the body temperature of the animals. Conclusion The mechanisms that participate in the mediation of the PACAP-38-induced hyperthermia may be modified by isatin. The capability of isatin to antagonize the hyperthermia induced by all members of the natriuretic peptide family and by PACAP-38 makes it unlikely to be acting directly on receptors for natriuretic peptides or on those for PACAP in these hyperthermic processes.

  6. Comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

    International Nuclear Information System (INIS)

    Andersen, P.H.; Groenvald, F.C.; Jansen, J.A.

    1985-01-01

    Methods for measuring 3 H-SCH 23990 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3 H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the K/sub i/-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the K/sub i/-values obtained from competition curves of 3 H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in K/sub i/-values indicate the possibility of different binding sites for DA and 3 H-SCH 23390 or, that DA and 3 -SCH 23390 label different states of the same receptor. 19 references, 7 figures, 2 tables

  7. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    . The catalytic alpha subunits are distantly related to the CMGC subfamily of kinases, such as the Cdk kinases. There are some peculiarities associated with protein kinase CK2, which are not found with most other protein kinases: (i) the enzyme is constitutively active, (ii) it can use ATP and GTP and (iii...

  8. Crosstalk between G protein-coupled receptors (GPCRs and tyrosine kinase receptor (TXR in the heart after morphine withdrawal

    Directory of Open Access Journals (Sweden)

    Pilar eAlmela

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signalling, resulting in high expression of protein kinase (PK A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK, one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl-5-isoquinolinesulfonamide (HA-1004, a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  9. Citron kinase - renaissance of a neglected mitotic kinase.

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

    Cell division controls the faithful segregation of genomic and cytoplasmic materials between the two nascent daughter cells. Members of the Aurora, Polo and cyclin-dependent (Cdk) kinase families are known to regulate multiple events throughout cell division, whereas another kinase, citron kinase (CIT-K), for a long time has been considered to function solely during cytokinesis, the last phase of cell division. CIT-K was originally proposed to regulate the ingression of the cleavage furrow that forms at the equatorial cortex of the dividing cell after chromosome segregation. However, studies in the last decade have clarified that this kinase is, instead, required for the organization of the midbody in late cytokinesis, and also revealed novel functions of CIT-K earlier in mitosis and in DNA damage control. Moreover, CIT-K mutations have recently been linked to the development of human microcephaly, and CIT-K has been identified as a potential target in cancer therapy. In this Commentary, I describe and re-evaluate the functions and regulation of CIT-K during cell division and its involvement in human disease. Finally, I offer my perspectives on the open questions and future challenges that are necessary to address, in order to fully understand this important and yet unjustly neglected mitotic kinase. © 2017. Published by The Company of Biologists Ltd.

  10. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  11. Lack of adenylate cyclase 1 (AC1): consequences on corticospinal tract development and on locomotor recovery after spinal cord injury.

    Science.gov (United States)

    Nait Taleb Ali, H; Morel, M P; Doulazmi, M; Scotto-Lomassese, S; Gaspar, P; Dusart, I; Bennis, M

    2014-02-26

    Cyclic AMP (cAMP) signalling pathways are involved in axonal growth and regeneration. The calcium-calmodulin- stimulated adenylate cyclase 1 (AC1), a regulator of cAMP levels, is strongly expressed in the corticospinal motor neurons (CSMN) in cerebral cortex layer V during development, but its role in the development of the corticospinal tract (CST) is unknown. Here, we analyse the organization of the CST pathway using anterograde and retrograde tracers in the barrelless (brl) mouse that carries an inactivating mutation of the AC1 gene. We show that in brl mice the general organization of the CST is normal but there is an increase in the number of axons in the ipsilateral contingent in the dorsal and ventral medial funiculi of the cervical spinal cord. The density of CSMN in layer V of the motor cortex is increased in brl compared to wild-type mice. Thus, lack of AC1 likely perturbs late phases of CSMN and CST development. Next, we examine the motor recovery after a spinal cord injury (SCI). We find that brl mice show enhanced locomotor functions as assessed by the BMS (Basso mouse scale) as early as 6h and up to 6 weeks after SCI, indicating a smaller responsiveness of brl mice to SCI. It is therefore possible that developmental effects on motor systems might decrease the locomotor effects consecutive to a SCI. This point is particularly important with regards to the use of transgenic animals for testing SCI recovery. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Directory of Open Access Journals (Sweden)

    Laura B Duvall

    Full Text Available The neuropeptide Pigment Dispersing Factor (PDF is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+ LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  13. Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

    Science.gov (United States)

    Zor, U; Strulovici, B; Lamprecht, S A; Amsterdam, A; Oplatka, A; Lindner, H R

    1979-12-01

    Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0--10 microM), believed to stabilize the polymerized state of actin, in a dose-dependent manner. The spontaneous recovery of responsiveness to PGE2 upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 microgram/ml) or of the actin-binding myosin subfragment HMM S-1 (80 microgram/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D2O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 micrograms/ml) and by actinomycin D (10 micrograms/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action. These findings provide suggestive evidence for the hypothesis that cytoskeletal elements associated with the cell membrane take part in the modulation of the adenylate cyclase response to hormones.

  14. Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.

    Science.gov (United States)

    Arad, H; Rosenbusch, J P; Levitzki, A

    1984-11-01

    Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)5OH], which possesses a high critical micelle concentration (approximately equal to 6 mM) and forms small, dynamic micelles. Both the native enzyme Ns(GDP) X C and the p[NH]ppG-preactivated species N's X p[NH]ppG X C' were found to possess the same molecular mass of 215,000 +/- 17,000 daltons. Both enzyme species migrate as a tight complex between Ns and C on both gel permeation columns and on DEAE-Sephacel columns in detergent. The two functional units, Ns and C, remain associated even in dilute detergent solutions and throughout a 300- to 400-fold purification in octylpoly(oxyethylene). These results strongly support the view that Ns and C do not come apart during the process of enzyme activation by the beta-adrenergic receptor. Furthermore, these results strongly support our previous assertion that the beta-adrenergic receptor activation of adenylate cyclase is by a simple "collision coupling" between the receptor and NsC. These results are not compatible with shuttle mechanisms that postulate that Ns physically migrates from the receptor R to the catalytic unit C and back during the activation cycle, as suggested by Citri and Schramm [Citri, Y. & Schramm, M. (1980) Nature (London) 287, 297-300] and by De Lean et al. [De Lean, A., Stadel, J. M. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 5108-5117].

  15. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    International Nuclear Information System (INIS)

    Murayama, T.; Ui, M.

    1985-01-01

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45 Ca 2+ uptake into the cell monolayer, and (f) increased 86 Rb + uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca 2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca 2+ -mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca 2+ gating

  16. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  17. Excimer emission properties on pyrene-labeled protein surface: correlation between emission spectra, ring stacking modes, and flexibilities of pyrene probes.

    Science.gov (United States)

    Fujii, Akira; Sekiguchi, Yutaka; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Chung, Wen-Sheng; Hirota, Shun; Matsuo, Takashi

    2015-03-18

    The excimer emission of pyrene is popularly employed for investigating the association between pyrene-labeled biomolecules or between pyrene-labeled places in a biomolecule. The property of pyrene excimer emission is affected by the fluctuation in ring stacking modes, which originates from the structural flexibilities of pyrene probes and/or of labeled places. Investigations of the excimer emission in terms of dynamics of pyrene stacking modes provide the detailed spatial information between pyrene-labeled places. In order to evaluate the effects of probe structures and fluctuation in pyrene-pyrene association modes on their emission properties on protein surface, three types of pyrene probe with different linker lengths were synthesized and conjugated to two cysteine residues in the A55C/C77S/V169C mutant of adenylate kinase (Adk), an enzyme that shows a structural transition between OPEN and CLOSED forms. In the CLOSED form of Adk labeled by a pyrene probe with a short linker, excimer emission was found to be predominated by the ground-state association of pyrenes. The pyrene stacking structure on the protein surface was successfully determined by an X-ray crystallographic analysis. However, the emission decay in the protein suggested the existence of several stacking orientations in solution. With the increase in the linker length, the effect of fluctuation in pyrene association modes on the spectral properties distinctly emerged at both ground and excited states. The combination of steady-state and time-resolved spectroscopic analyses is useful for differentiation in the origin of the excimer emission, which is essential for precisely understanding the interaction fashions between pyrene-labeled biomolecules.

  18. The role of Tec family kinases in mononuclear phagocytes.

    Science.gov (United States)

    Koprulu, Afitap Derya; Ellmeier, Wilfried

    2009-01-01

    Mononuclear phagocytes, including monocytcs, macrophages, and dendritic cells, play an important role in innate and adaptive immune responses and are important regulators of the inflammatory response. Among these, monocytes/macrophages are involved in virtually all aspects of inflammation, ranging from the detection and phagocytosis of pathogens up to the resolution of inflammation and repair of tissue damage. The stimulation of cell surface receptors, such as Toll-like receptors, leads to the initiation of intracellular signal transduction pathways regulating macrophagc activation and effector functions. One group of signaling molecules stimulated on macrophage activation is formed by the Tec kinasc family, which consists of five members (Bmx, Btk, Itk, Rlk, and Tec) and constitutes the second-largest family of nonreceptor protein tyrosine kinases in the immune system. Some Tec kinases have been shown to be major regulators of antigen receptor signaling in lymphocytes, and deficiencies in Tec family kinases cause several immunological defects in humans and mice. Much less is known about the role of Tec family kinases in the myeloid branch of the hematopoietic system. In this review, we discuss the current knowledge about the role of Tec family kinases in monocytes/macrophages, in dendritic cells, and in osteoclasts.

  19. Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

    International Nuclear Information System (INIS)

    Jeffrey, W.H.; Paul, J.H.

    1990-01-01

    One assumption made in bacterial production estimates from [ 3 H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported the thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibro sp. strain DI9, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems

  20. Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Wald, Tomáš; Osičková, Adriana; Mašín, Jiří; Matyska Lišková, Petra; Petry-Podgorska, Inga; Matoušek, Tomáš; Šebo, Peter; Osička, Radim

    2016-01-01

    Roč. 74, č. 3 (2016), flw008 ISSN 2049-632X R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460; GA ČR GA13-14547S Institutional support: RVO:61388971 ; RVO:68081715 Keywords : adenylate cyclase toxin * ICP-MS * CD11b/CD18 Subject RIV: EE - Microbiology, Virology; CB - Analytical Chemistry, Separation (UIACH-O) Impact factor: 2.335, year: 2016

  1. 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....

  2. Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8(+) and CD4(+) T cells

    Czech Academy of Sciences Publication Activity Database

    Švédová, Martina; Mašín, Jiří; Fišer, Radovan; Černý, Ondřej; Tomala, Jakub; Freudenberg, M.; Tučková, Ludmila; Kovář, Marek; Dadaglio, G.; Adkins, Irena; Šebo, Peter

    2016-01-01

    Roč. 94, č. 4 (2016), s. 322-333 ISSN 0818-9641 R&D Projects: GA ČR(CZ) GA13-14547S; GA ČR(CZ) GAP302/12/0460 Institutional support: RVO:61388971 Keywords : N-TERMINAL KINASE * BORDETELLA- PERTUSSIS * MEMBRANE TRANSLOCATION Subject RIV: EE - Microbiology, Virology Impact factor: 4.557, year: 2016

  3. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes...

  4. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  5. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

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

    2016-01-01

    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...

  6. 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...

  7. Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability.

    Science.gov (United States)

    Tompkins, John D; Clason, Todd A; Buttolph, Thomas R; Girard, Beatrice M; Linden, Anne K; Hardwick, Jean C; Merriam, Laura A; May, Victor; Parsons, Rodney L

    2018-02-01

    Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

  8. Thyroid-stimulating immunoglobulins in Hashimoto's thyroiditis measured by radioreceptor assay and adenylate cyclase stimulation and their relationship to HLA-D alleles

    International Nuclear Information System (INIS)

    Bliddal, H.; Bech, K.; Feldt-Rasmussen, U.; Thomsen, M.; Ryder, L.P.; Hansen, J.M.; Siersbaek-Nielsen, K.; Friis, T.

    1982-01-01

    The relationship between thyroid-stimulating immunoglobulins, measured by both radioreceptor assay and adenylate cyclase stimulation, and the HLA alleles was studied in 41 patients with Hashimoto's thyroiditis. TSH binding-inhibiting immunoglobulins (TBII) were detected in 9 (22%) patients, and human thyroid adenylate cyclase-stimulating immunoglobulins (HTACS) were found in 21 (51%) patients. Only 2 patients were positive in both assays, and an inverse relationship was observed between TBII and HTACS. In the 21 HTACS-positive patients, HLA-Dw5 was found in 1 subject, compared to 8 of the 20 HTACS-negative patients (P < 0.01), while 4 of the 9 TBII-positive patients had HLA-Dw5 compared to 5 of the 32 TBII-negative subjects (P = 0.09).No significant relations were observed between the presence of HTACS or TBII and HLA-Dw3 or HLA-B8. It is concluded that TBII and HTACS are produced independently in Hashimoto's thyroiditis, and that the production of these autoantibodies seems to be related to the HLA-D region in this disease

  9. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  10. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    International Nuclear Information System (INIS)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-01-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism

  11. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. (Univ. of Missouri-Columbia (USA))

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  12. Multiple pertussis toxin substrates as candidates for regulatory G proteins of adenylate cyclase coupled to the somatostatin receptor in primary rat astrocytes.

    Science.gov (United States)

    Gebicke-Haerter, P J; Seregi, A; Wurster, S; Schobert, A; Allgaier, C; Hertting, G

    1988-10-01

    The involvement of G proteins in receptor mediated astroglial cAMP formation was studied. Isoproterenol or prostaglandin E2 stimulated adenylate cyclase of primary astroglial cells was inhibited by somatostatin. Preincubation of cells with increasing concentrations of islet activating protein (IAP) diminished somatostatin inhibition of adenylate cyclase. At an IAP concentration of 50 ng/ml somatostatin inhibition was completely abolished. Studies on IAP catalyzed 32P-ADP-ribosylation of astroglial cell particulate material revealed an incorporation of radiolabel into three polypeptides in the molecular weight range of 41,000-39,000 Dalton. Pretreatment of intact cells with IAP reduced radiolabeling of this molecular species in a concentration dependent manner. No further radiolabeling above background level was detectable after pretreatment of cultures with 10 ng IAP/ml or more. At present, the occurrence of at least three IAP substrates (G proteins) does not permit an identification of the somatostatin receptor coupled G protein. Rather, the finding reveals that astrocytes are endowed with multiple variants of GTP binding proteins likely to be coupled to different receptors.

  13. The alpha-kinase family: an exceptional branch on the protein kinase tree.

    NARCIS (Netherlands)

    Middelbeek, J.A.J.; Clark, K.; Venselaar, H.; Huynen, M.A.; Leeuwen, F.N. van

    2010-01-01

    The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in

  14. Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

    Science.gov (United States)

    Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

    2017-08-01

    The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure 95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in

  15. Growth factors and kinases in glioblastoma growth

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Peña-Ortiz

    2016-10-01

    Full Text Available Glioblastoma multiforme (GBM is the most aggressive type of brain cancer, having the highest invasion, migration, proliferation, and angiogenesis rates. Several signaling pathways are involved in the regulation of these processes including growth factors and their tyrosine kinase receptors, such as vascular endothelial growth factor (VEGF, transforming growth factor beta (TGFβ, fibroblast growth factor (FGF, platelet-derived growth factor (PDGF, and insulin-like growth factor–I (IGF–I. Different kinases and regulators also participate in signaling pathways initiated by growth factors, such as mitogen-activated kinases (MAPK, protein kinases C (PKC, phosphatidylinositol-3 kinases (PI3K, protein kinase B (PKB or Akt, glycogen synthase kinase 3β (GSK3β, the mTOR complex, and Bcl-2. In this review, we will focus on the role of these proteins as possible therapeutic targets in GBM.

  16. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  17. 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....

  18. Spinach Pyruvate Kinase Isoforms 1

    Science.gov (United States)

    Baysdorfer, Chris; Bassham, James A.

    1984-01-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide Km values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The Ki for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a Ka of 0.05 millimolar, and glutamate is an inhibitor with a Ki of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation. PMID:16663425

  19. 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.

  20. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    International Nuclear Information System (INIS)

    Liang, B.T.

    1989-01-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-diproylxanthine ([3H]CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that [3H] CPX is an antagonist radioligand. Competition curves for [3H] CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific [3H]CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid)

  1. Bioinformatic mining of kinase inhibitors that regulate autophagy through kinase signaling pathways.

    Science.gov (United States)

    Yang, Yang; Ma, Biao; Jin, Ye; Ben, Wei; Zhang, Dandan; Jiang, Keping; Feng, Shujun; Huang, Lu; Zheng, Jianhua

    2014-12-01

    The aim of this study was to predict the kinase inhibitors that may regulate autophagy. A total of 62 kinases were obtained through text mining by importing the keyword 'autophagy' and a 'protein kinase' Excel file to PubMed. Subsequently, 146 kinases were derivated through screening in the PubMed database by importing the 'autophagy‑associated gene' and 'protein kinase' files. Following intersection of the above two methods, 54 candidate autophagy‑associated kinases were obtained. Enrichment analysis indicated that these candidate autophagy‑associated kinases were mainly enriched in pathways such as the calcium, Wnt, HIF‑1 and mTOR signaling pathways. Among the 54 kinases, 24 were identified through text mining to have specific kinase inhibitors that regulate the corresponding functions; a total of 56 kinase inhibitors were found to be involved in the regulation of these 24 kinases. In total, nine of these 56 kinase inhibitors identified had been widely reported in autophagy regulation studies, 23 kinase inhibitors had been seldom reported and 24 had never been reported. Therefore, introducing these kinases into autophagy regulation analysis in subsequent studies may produce important results.

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-02

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

  4. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Škopová, Karolína; Tomalová, Barbora; Kanchev, Ivan; Rossmann, Pavel; Švédová, Martina; Adkins, Irena; Bíbová, Ilona; Tomala, Jakub; Mašín, Jiří; Guiso, N.; Osička, Radim; Sedláček, Radislav; Kovář, Marek; Šebo, Peter

    2017-01-01

    Roč. 85, č. 6 (2017), s. 1-22, č. článku e00937-16. ISSN 0019-9567 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S; GA ČR GA13-12885S; GA ČR GA15-09157S; GA ČR(CZ) GAP302/12/0460; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LM2015040 Institutional support: RVO:61388971 ; RVO:68378050 Keywords : Bordetella pertussis * adenylate cyclase toxin-hemolysin * cAMP intoxication Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (UMG-J) OBOR OECD: Microbiology; Microbiology (UMG-J) Impact factor: 3.593, year: 2016

  5. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    Science.gov (United States)

    Penzkofer, A.; Tanwar, M.; Veetil, S. K.; Kateriya, S.; Stierl, M.; Hegemann, P.

    2013-09-01

    The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr-Tyr cross-linking (o,o‧-ditysosine formation) and partial flavin cofactor reduction.

  6. Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes.

    Science.gov (United States)

    Hei, Y J; Farahbakhshian, S; Chen, X; Battell, M L; McNeill, J H

    1998-01-01

    To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.

  7. Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultaet fuer Physik, Universitaet Regensburg, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Stierl, M.; Hegemann, P. [Institut fuer Biologie/Experimentelle Biophysik, Humboldt Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2012-01-02

    Graphical abstract: Protein color center emissions were observed in the wavelength range from 340 nm to 900 nm from nano-clusters of the photo-activated adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi. Highlights: Black-Right-Pointing-Pointer Adenylyl cyclase nPAC in aqueous pH 7.5 buffer dissolved only to nano-clusters. Black-Right-Pointing-Pointer Nano-cluster size was determined by light attenuation (scattering) measurements. Black-Right-Pointing-Pointer The size of the nano-clusters was growing by coalescing during observation period. Black-Right-Pointing-Pointer In nPAC nano-clusters color centers were present in emission range of 360-900 nm. Black-Right-Pointing-Pointer The nPAC color center emission is compared with fluorescent protein emission. - Abstract: The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

  8. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    International Nuclear Information System (INIS)

    Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

    2010-01-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate

  9. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, Asim K.; Atanasova, Vesna [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States); Gamage, Swarna [Auckland Cancer Society Research Centre, School of Medicine, Faculty of Medical and Health Sciences, University of Auckland, Auckland (New Zealand); Robinson, Howard [Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (United States); Parsons, James F., E-mail: parsonsj@umbi.umd.edu [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States)

    2010-06-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  10. Adaptation of an L-Proline Adenylation Domain to Use 4-Propyl-L-Proline in the Evolution of Lincosamide Biosynthesis

    Science.gov (United States)

    Kadlčík, Stanislav; Kučera, Tomáš; Chalupská, Dominika; Gažák, Radek; Koběrská, Markéta; Ulanová, Dana; Kopecký, Jan; Kutejová, Eva; Najmanová, Lucie; Janata, Jiří

    2013-01-01

    Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accomodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin - but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit. PMID:24386435

  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. Conformational snapshots of Tec kinases during signaling.

    Science.gov (United States)

    Joseph, Raji E; Andreotti, Amy H

    2009-03-01

    The control of cellular signaling cascades is of utmost importance in regulating the immune response. Exquisitely precise protein-protein interactions and chemical modification of substrates by enzymatic catalysis are the fundamental components of the signals that alert immune cells to the presence of a foreign antigen. In particular, the phosphorylation events induced by protein kinase activity must be spatially and temporally regulated by specific interactions to maintain a normal and effective immune response. High resolution structures of many protein kinases along with supporting biochemical data are providing significant insight into the intricate regulatory mechanisms responsible for controlling cellular signaling. The Tec family kinases are immunologically important kinases for which regulatory details are beginning to emerge. This review focuses on bringing together structural insights gained over the years to develop an understanding of how domain interactions both within the Tec kinases and between the Tec kinases and other signaling molecules control immune cell function.

  13. Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

    Science.gov (United States)

    Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

    2018-03-15

    Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  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. Tec family kinases in inflammation and disease.

    Science.gov (United States)

    Horwood, Nicole J; Urbaniak, Ania M; Danks, Lynett

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis.

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

    Science.gov (United States)

    2017-01-01

    prostate cancer patients have abnormalities in the AKT signaling pathway. These abnormalities are driven by mutations in the PTEN and AKT proteins as...AWARD NUMBER: W81XWH-12-1-0560 TITLE: Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway...2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase

  17. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  18. Nonreceptor Tyrosine Kinases in Prostate

    Directory of Open Access Journals (Sweden)

    Cancer Yu-Ming Chang

    2007-02-01

    Full Text Available BACKGROUND: Carcinoma of the prostate (CaP is the most commonly diagnosed cancer in men in the United States. Signal transduction molecules such as tyrosine kinases play important roles in CaP. Src, a nonreceptor tyrosine kinase (NRTK and the first proto-oncogene discovered is shown to participate in processes such as cell proliferation and migration in CaP. Underscoring NRTK's and, specifically, Src's importance in cancer is the recent approval by the US Food and Drug Administration of dasatinib, the first commercial Src inhibitor for clinical use in chronic myelogenous leukemia (CML. In this review we will focus on NRTKs and their roles in the biology of CaP. MATERIALS AND METHODS: Publicly available literature from PubMed regarding the topic of members of NRTKs in CaP was searched and reviewed. RESULTS: Src, FAK, JaK1/2, and ETK are involved in processes indispensable to the biology of CaP: cell growth, migration, invasion, angiogenesis, and apoptosis. CONCLUSIONS: Src emerges as a common signaling and regulatory molecule in multiple biological processes in CaP. Src's relative importance in particular stages of CaP, however, required further definition. Continued investigation of NRTKs will increase our understanding of their biological function and potential role as new therapeutic targets.

  19. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  20. A multisubstrate deoxyribonucleoside kinase from plants

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Knecht, Wolfgang

    2008-01-01

    and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene...

  1. Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

    DEFF Research Database (Denmark)

    Andersen, Martin Nybo; Skibsbye, Lasse; Saljic, Arnela

    2018-01-01

    Over the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center......, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits....

  2. MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism.

    Directory of Open Access Journals (Sweden)

    Laura P Bernard

    Full Text Available The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs, are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer's disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA, through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.

  3. Kinase activity and specificity assay using synthetic peptides.

    Science.gov (United States)

    Wu, Xu Na; Schulze, Waltraud X

    2015-01-01

    Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.

  4. Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

    Full Text Available The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA, an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC, an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT or a mutant lacking CyaA (BpCyaA-, or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

  5. Mitotic regulation by NIMA-related kinases

    Directory of Open Access Journals (Sweden)

    Blot Joelle

    2007-08-01

    Full Text Available Abstract The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

  6. Role of phosphatidylinositol 3-kinase activation on insulin action and its alteration in diabetic conditions.

    Science.gov (United States)

    Asano, Tomoichiro; Fujishiro, Midori; Kushiyama, Akifumi; Nakatsu, Yusuke; Yoneda, Masayasu; Kamata, Hideaki; Sakoda, Hideyuki

    2007-09-01

    Inositol phospholipids phosphorylated on D3-position of their inositol rings (3-phosphoinositides) are known to play important roles in various cellular events. Activation of PI (phosphatidylinositol) 3-kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit and one of two widely-distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. Activation of PI 3-kinase and its downstream AKT has been demonstrated to be essential for almost all of the insulin-induced glucose and lipid metabolism such as glucose uptake, glycogen synthesis, suppression of glucose output and triglyceride synthesis as well as insulin-induced mitogenesis. Accumulated PI(3,4,5)P(3) activates several serine/threonine kinases containing a PH (pleckstrin homology) domain, including Akt, atypical PKCs, p70S6 kinase and GSK. In the obesity-induced insulin resistant condition, JNK and p70S6K are activated and phosphorylate IRS-proteins, which diminishes the insulin-induced tyrosine phosphorylation of IRS-proteins and thereby impairs the PI 3-kinase/AKT activations. Thus, the drugs which restore the impaired insulin-induced PI 3-kinase/AKT activation, for example, by suppressing JNK or p70S6K, PTEN or SHIP2, could be novel agents to treat diabetes mellitus.

  7. Protein Kinase A in Cancer

    Directory of Open Access Journals (Sweden)

    Antonio Caretta

    2011-02-01

    Full Text Available 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.

  8. 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

  9. 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.

  10. Plasmonic ruler on field-effect devices for kinase drug discovery applications.

    Science.gov (United States)

    Bhalla, Nikhil; Formisano, Nello; Miodek, Anna; Jain, Aditya; Di Lorenzo, Mirella; Pula, Giordano; Estrela, Pedro

    2015-09-15

    Protein kinases are cellular switches that mediate phosphorylation of proteins. Abnormal phosphorylation of proteins is associated with lethal diseases such as cancer. In the pharmaceutical industry, protein kinases have become an important class of drug targets. This study reports a versatile approach for the detection of protein phosphorylation. The change in charge of the myelin basic protein upon phosphorylation by the protein kinase C-alpha (PKC-α) in the presence of adenosine 5'-[γ-thio] triphosphate (ATP-S) was detected on gold metal-insulator-semiconductor (Au-MIS) capacitor structures. Gold nanoparticles (AuNPs) can then be attached to the thio-phosphorylated proteins, forming a Au-film/AuNP plasmonic couple. This was detected by a localized surface plasmon resonance (LSPR) technique alongside MIS capacitance. All reactions were validated using surface plasmon resonance technique and the interaction of AuNPs with the thio-phosphorylated proteins quantified by quartz crystal microbalance. The plasmonic coupling was also visualized by simulations using finite element analysis. The use of this approach in drug discovery applications was demonstrated by evaluating the response in the presence of a known inhibitor of PKC-α kinase. LSPR and MIS on a single platform act as a cross check mechanism for validating kinase activity and make the system robust to test novel inhibitors. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study.

    Science.gov (United States)

    Li, Min; Maderdrut, Jerome L; Lertora, Juan J L; Batuman, Vecihi

    2007-09-01

    We have recently shown significant renoprotective effects with the administration of pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney. PACAP markedly inhibited the production of proinflammatory cytokines stimulated by immunoglobulin light chains in human renal proximal tubule epithelial cells and in the kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of 4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood rapidly declined after the cessation of administration with a half-life of about 5-10 min. The continuous infusion did not significantly alter the basal glucose level, blood gases or blood pressure. There was a large reduction in free lambda light chains in urine after the start of the treatment with PACAP. These studies show that PACAP can be safely used in humans and suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney.

  12. ePAT: A simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications

    Science.gov (United States)

    Jänicke, Amrei; Vancuylenberg, John; Boag, Peter R.; Traven, Ana; Beilharz, Traude H.

    2012-01-01

    The addition of a poly(A)-tail to the 3′ termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3′-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3′ UTR usage in 3′ RACE applications. PMID:22543866

  13. Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor.

    Science.gov (United States)

    Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

    2016-02-29

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1(+) were large in size, had oval nuclei, and merged with CD34(+) cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1(-/-) mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1(-/-) and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.

  14. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

  15. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg (Germany); Tanwar, M.; Veetil, S.K.; Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Stierl, M.; Hegemann, P. [Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany)

    2013-09-23

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

  16. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    International Nuclear Information System (INIS)

    Penzkofer, A.; Tanwar, M.; Veetil, S.K.; Kateriya, S.; Stierl, M.; Hegemann, P.

    2013-01-01

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction

  17. 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 ...

  18. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    During cell-matrix adhesion, both tyrosine and serine/threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface...

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

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... including PKA, PKG, ribosomal S6 kinase, and epidermal growth factor receptor kinase (EGFRK) in an ATP- competitive manner, while having no effect on other kinases such as CK1, CK2, MAP kinase, and CSK (Meggio et al. 1995). The necessity for more specific inhibitors of PKC led to the discovery of ...

  20. 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.

  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. MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

    OpenAIRE

    Gardner, A M; Vaillancourt, R R; Lange-Carter, C A; Johnson, G L

    1994-01-01

    MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphory...

  4. Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Shimotohno, A.; Ohno, R.; Bišová, Kateřina; Sakaguchi, N.; Huang, J.; Koncz, C.; Uchimiya, H.; Umeda, M.

    2006-01-01

    Roč. 47, - (2006), s. 701-710 ISSN 0960-7412 Institutional research plan: CEZ:AV0Z50200510 Keywords : cyclin -dependent kinase * cdk-activating kinase * cyclin Subject RIV: EE - Microbiology, Virology Impact factor: 6.565, year: 2006

  5. 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

  6. 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

  7. 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 ...

  8. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    Directory of Open Access Journals (Sweden)

    Martin Siepmann

    2010-10-01

    Full Text Available Neprilysin (NEP is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1 stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.

  9. Giant hub Src and Syk tyrosine kinase thermodynamic profiles recapitulate evolution

    Science.gov (United States)

    Phillips, J. C.

    2017-10-01

    Thermodynamic scaling theory, previously applied mainly to small proteins, here analyzes quantitative evolution of the titled functional network giant hub enzymes. The broad domain structure identified homologically is confirmed hydropathically using amino acid sequences only. The most surprising results concern the evolution of the tyrosine kinase globular surface roughness from avians to mammals, which is first order, compared to the evolution within mammals from rodents to humans, which is second order. The mystery of the unique amide terminal region of proto oncogene tyrosine protein kinase is resolved by the discovery there of a rare hydroneutral septad targeting cluster, which is paralleled by an equally rare octad catalytic cluster in tyrosine kinase in humans and a few other species (cat and dog). These results, which go far towards explaining why these proteins are among the largest giant hubs in protein interaction networks, use no adjustable parameters.

  10. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  11. 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.

  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. Mechanism of polyphosphate kinase from Propionibacterium shermanii

    Energy Technology Data Exchange (ETDEWEB)

    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 approx.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 (/sup 32/P) short-chain polyphosphate incorporation into long chain polyphosphate by the kinase.

  14. Mining protein kinases regulation using graphical models.

    Science.gov (United States)

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2011-03-01

    Abnormal kinase activity is a frequent cause of diseases, which makes kinases a promising pharmacological target. Thus, it is critical to identify the characteristics of protein kinases regulation by studying the activation and inhibition of kinase subunits in response to varied stimuli. Bayesian network (BN) is a formalism for probabilistic reasoning that has been widely used for learning dependency models. However, for high-dimensional discrete random vectors the set of plausible models becomes large and a full comparison of all the posterior probabilities related to the competing models becomes infeasible. A solution to this problem is based on the Markov Chain Monte Carlo (MCMC) method. This paper proposes a BN-based framework to discover the dependency correlations of kinase regulation. Our approach is to apply the MCMC method to generate a sequence of samples from a probability distribution, by which to approximate the distribution. The frequent connections (edges) are identified from the obtained sampling graphical models. Our results point to a number of novel candidate regulation patterns that are interesting in biology and include inferred associations that were unknown.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  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

    2017-11-08

    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. © 2017 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. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    Science.gov (United States)

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

  19. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    Directory of Open Access Journals (Sweden)

    Barzan I Khayatt

    Full Text Available There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs and polyketide synthases (PKSs of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A and acyl-transferase (AT domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively. Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

  20. Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions.

    Science.gov (United States)

    Botz, Bálint; Imreh, András; Sándor, Katalin; Elekes, Krisztián; Szolcsányi, János; Reglődi, Dóra; Quinn, John P; Stewart, James; Zimmer, Andreas; Hashimoto, Hitoshi; Helyes, Zsuzsanna

    2013-05-01

    Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK1 tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP(-/-), Tac1(-/-), and Tacr1(-/-)) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30-40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1(-/-) and Tacr1(-/-) mice, but was absent in PACAP(-/-) animals. Motor coordination of the PACAP(-/-) and Tac1(-/-) groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1(-/-) mice. Basal postoperative microcirculation on the plantar skin of PACAP(-/-) mice did not differ from the wildtypes, but was significantly lower in Tac1(-/-) and Tacr1(-/-) ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP(-/-) mice, but not in Tacr1(-/-) and Tac1(-/-) animals. Both PACAP and SP/NKA, but not NK1 receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats.

    Science.gov (United States)

    Meloni, Edward G; Venkataraman, Archana; Donahue, Rachel J; Carlezon, William A

    2016-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 μg) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7 days) or following a delay (7, 10, and 13 days) after conditioning. PACAP (1.5 μg) produced a bi-phasic response in freezing behavior across test days: relative to controls, PACAP-treated rats showed a reduction in freezing when tested 1 or 7 days after fear conditioning that evolved into a significant elevation in freezing by the third test session in the early, but not delayed, group. Corticosterone (CORT) levels were significantly elevated in PACAP-treated rats following fear conditioning, but not at the time of testing (Day 1). Brain c-Fos expression revealed PACAP-dependent alterations within, as well as outside of, areas typically implicated in fear conditioning. Our findings raise the possibility that PACAP disrupts fear memory consolidation by altering synaptic plasticity within neurocircuits normally responsible for encoding fear-related cues, producing a type of dissociation or peritraumatic amnesia often seen in people early after exposure to a traumatic event. However, fear memories are retained such that repeated testing and memory reactivation (e.g., re-experiencing) causes the freezing response to emerge and persist at elevated levels. PACAP systems may represent an axis on which stress and exposure to trauma converge to promote maladaptive behavioral responses characteristic of psychiatric illnesses such as post-traumatic stress disorder (PTSD). Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats.

    Science.gov (United States)

    Kirry, Adam J; Herbst, Matthew R; Poirier, Sarah E; Maskeri, Michelle M; Rothwell, Amy C; Twining, Robert C; Gilmartin, Marieke R

    2018-05-01

    A genetic polymorphism within the gene encoding the pituitary adenylate cyclase- activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Pituitary adenylate cyclase-activating polypeptide (PACAP has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    Directory of Open Access Journals (Sweden)

    Gabor Maasz

    2017-02-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin, metabolic enzyme (S-COMT, MB-COMT and MAO-B and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  4. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

    Science.gov (United States)

    Pirger, Zsolt; Laszlo, Zita; Hiripi, Laszlo; Hernadi, Laszlo; Toth, Gabor; Lubics, Andrea; Reglodi, Dora; Kemenes, Gyorgy; Mark, Laszlo

    2010-11-01

    PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

  5. Crystal Structure of Human Nicotinamide Riboside Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  6. Crystal structure of human nicotinamide riboside kinase.

    Science.gov (United States)

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  7. 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.

  8. The preparation of nucleotides uniformly labelled with carbon-14 by biosynthetic methods. Isolation of adenylic, uridylic, cytidylic,and guanylic acids, from the alkaline hydrolysate of escherichia coli RNA

    International Nuclear Information System (INIS)

    Garcia Pineda, M. D.; Pacheco Lopez, J.

    1978-01-01

    A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14 C . Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14 C as their only source of carbon. RNA is isolated from the cells, and after hydrolysis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the Isolated nucleotides is determined, and also its specific radioactivity. (Author) 30 refs

  9. X-ray crystal structure of bone marrow kinase in the x chromosome: a Tec family kinase.

    Science.gov (United States)

    Muckelbauer, Jodi; Sack, John S; Ahmed, Nazia; Burke, James; Chang, Chiehying Y; Gao, Mian; Tino, Joseph; Xie, Dianlin; Tebben, Andrew J

    2011-11-01

    Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 Å resolution and PP2 at 1.9 Å resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-ordered protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors. © 2011 John Wiley & Sons A/S.

  10. 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

  11. Production of Protein Kinases in E. coli.

    Science.gov (United States)

    Dodson, Charlotte A

    2017-01-01

    Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies.

  12. Rational design of protein kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Yarmoluk S. M.

    2013-07-01

    Full Text Available Modern methodological approaches to rational design of low molecular weight compounds with specific activity in relation to predetermined biomolecular targets are considered by example of development of high effective protein kinase inhibitors. The application of new computational methods that allow to significantly improve the quality of computational experiments (in, particular, accuracy of low molecular weight compounds activity prediction without increase of computational and time costs are highlighted. The effectiveness of strategy of rational design is demonstrated by examples of several own investigations devoted to development of new inhibitors that are high effective and selective towards protein kinases CK2, FGFR1 and ASK1.

  13. Protein Kinase C (PKC Isozymes and Cancer

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

    2014-01-01

    Full Text Available Protein kinase C (PKC is a family of phospholipid-dependent serine/threonine kinases, which can be further classified into three PKC isozymes subfamilies: conventional or classic, novel or nonclassic, and atypical. PKC isozymes are known to be involved in cell proliferation, survival, invasion, migration, apoptosis, angiogenesis, and drug resistance. Because of their key roles in cell signaling, PKC isozymes also have the potential to be promising therapeutic targets for several diseases, such as cardiovascular diseases, immune and inflammatory diseases, neurological diseases, metabolic disorders, and multiple types of cancer. This review primarily focuses on the activation, mechanism, and function of PKC isozymes during cancer development and progression.

  14. Treatment of vitiligo with the topical Janus kinase inhibitor ruxolitinib.

    Science.gov (United States)

    Rothstein, Brooke; Joshipura, Deep; Saraiya, Ami; Abdat, Rana; Ashkar, Huda; Turkowski, Yana; Sheth, Vaneeta; Huang, Victor; Au, Shiu Chung; Kachuk, Courtney; Dumont, Nicole; Gottlieb, Alice B; Rosmarin, David

    2017-06-01

    Existing therapies for vitiligo are limited in efficacy and can be associated with undesirable side effects. Topical Janus kinase inhibitors may offer a new therapeutic option for vitiligo. We sought to assess the role of topical ruxolitinib 1.5% cream, a Janus kinase inhibitor, in vitiligo treatment. This 20-week, open-label, proof-of-concept trial of twice-daily topical ruxolitinib 1.5% cream was conducted in 12 patients with a minimum of 1% affected body surface area of vitiligo. The primary outcome was percent improvement in Vitiligo Area Scoring Index from baseline to week 20. Of 12 patients screened, 11 were enrolled and 9 completed the study (54.5% men; mean age, 52 years). Four patients with significant facial involvement at baseline had a 76% improvement in facial Vitiligo Area Scoring Index scores at week 20 (95% confidence interval, 53-99%; P = .001). A 23% improvement in overall Vitiligo Area Scoring Index scores was observed in all enrolled patients at week 20 (95% confidence interval, 4-43%; P = .02). Three of 8 patients responded on body surfaces and 1 of 8 patients responded on acral surfaces. Adverse events were minor, including erythema, hyperpigmentation, and transient acne. Limitations of the study include the small sample size and open-label study design. Topical ruxolitinib 1.5% cream provided significant repigmentation in facial vitiligo and may offer a valuable new treatment for vitiligo. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  15. 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.

  16. 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.

  17. Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Samantha J.; Parthasarathy, Gopal; Darke, Paul L.; Diehl, Ronald E.; Ford, Rachael E.; Hall, Dawn L.; Johnson, Scott A.; Reid, John C.; Rickert, Keith W.; Shipman, Jennifer M.; Soisson, Stephen M.; Zuck, Paul; Munshi, Sanjeev K.; Lumb, Kevin J. (Merck)

    2015-07-01

    G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.

  18. Identification of an allosteric signaling network within Tec family kinases.

    Science.gov (United States)

    Joseph, Raji E; Xie, Qian; Andreotti, Amy H

    2010-10-22

    The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the 'regulatory spine', which assembles and disassembles as a kinase switches between its active and inactive states, has been identified. Here, we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the Src homology 2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine, resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant, rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Bruton's tyrosine kinase R544 on the activation loop and Bruton's tyrosine kinase E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Receptor-like kinase complexes in plant innate immunity.

    Directory of Open Access Journals (Sweden)

    Christiaan eGreeff

    2012-08-01

    Full Text Available Receptor-like kinases (RLKs are surface localized, transmembrane receptors comprising a large family of well-studied kinases. RLKs signal through their transmembrane and juxtamembrane domains with the aid of various interacting partners and downstream components. The N-terminal extracellular domain defines ligand specificity, and RLK families are sub-classed according to this domain. The most studied of these subfamilies include those with 1 leucine rich repeat (LRR domains, 2 LysM domains (LYM and 3 the Catharanthus roseus RLK1-like (CrRLK1L domain. These proteins recognize distinct ligands of microbial origin or ligands derived from intracellular protein/carbohydrate signals. For example, the pattern recognition receptor (PRR AtFLS2 recognizes flg22 from flagellin, and the PRR AtEFR recognizes elf18 from elongation factor (EF-Tu. Upon binding of their cognate ligands, the aforementioned RLKs activate generic immune responses termed pattern triggered immunity (PTI. RLKs can form complexes with other family members and engage a variety of intracellular signaling components and regulatory pathways upon stimulation. This review focuses on interesting new data about how these receptors form protein complexes to exert their function.

  20. 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

  1. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    Science.gov (United States)

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. 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...

  3. Inhibitors of Cyclin-dependent Kinases

    Czech Academy of Sciences Publication Activity Database

    Kryštof, V.; Strnad, Miroslav

    2001-01-01

    Roč. 95, č. 5 (2001), s. 295-300 ISSN 0009-2770 R&D Projects: GA ČR GA301/02/0475; GA MŠk VS96154 Institutional research plan: CEZ:AV0Z5038910 Keywords : Cyclin-dependent Kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.317, year: 2001

  4. 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

  5. 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

  6. Protein kinase CK1 from Trypanosoma cruzi.

    Science.gov (United States)

    Calabokis, Maritza; Kurz, Liliana; Gonzatti, Mary I; Bubis, José

    2003-08-01

    A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.

  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. 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...

  9. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    Science.gov (United States)

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

  10. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Science.gov (United States)

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  11. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Directory of Open Access Journals (Sweden)

    Westa Domanova

    Full Text Available In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds, making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same

  12. Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation.

    Science.gov (United States)

    Staswick, Paul E; Tiryaki, Iskender; Rowe, Martha L

    2002-06-01

    Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.

  13. TPX2 Protein of Arabidopsis Activates Aurora Kinase 1, But Not Aurora Kinase 3 In Vitro

    Czech Academy of Sciences Publication Activity Database

    Tomaštíková, Eva; Demidov, D.; Jeřábková, Hana; Binarová, Pavla; Houben, A.; Doležel, Jaroslav; Petrovská, Beáta

    2015-01-01

    Roč. 33, č. 6 (2015), s. 1988-1995 ISSN 0735-9640 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204; GA ČR GAP501/12/2333 Institutional support: RVO:61389030 ; RVO:61388971 Keywords : Aurora kinase * Targeting protein for Xklp2 * In vitro kinase assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.304, year: 2015

  14. Circadian and pharmacological regulation of casein kinase I in the ...

    Indian Academy of Sciences (India)

    2008-12-31

    Dec 31, 2008 ... [Agostino P. V., Plano S. A. and Golombek D. A. 2008 Circadian and pharmacological regulation of casein kinase I in the hamster suprachi- asmatic nucleus. ... The protein kinases, casein kinase I epsilon (CKIε) and delta (CKIδ) ... 1 mM EGTA, 50 mM sodium fluoride, 200 mM sodium vana- date and a ...

  15. Molecular Pathways : Targeting the Protein Kinase Wee1 in Cancer

    NARCIS (Netherlands)

    Geenen, Jill J.; Schellens, Jan H M

    2017-01-01

    Wee1 is a protein kinase that regulates the G2checkpoint and prevents entry into mitosis in response to DNA damage. Cyclin-dependent kinases (CDK) are a family of 14 serine/threonine protein kinases that coordinate the progression through the cell cycle. The Cdc2/cyclin B complex controls the

  16. 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…

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

    Indian Academy of Sciences (India)

    Protein kinases are one of the largest gene families and have regulatory roles in all aspects of eukaryotic cell function. Modulation of protein kinase activity is a desirable therapeutic approach for a number of human diseases associated with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders.

  18. Clinical heterogeneity and novel mutations in the glycerol kinase gene in three families with isolated glycerol kinase deficiency

    NARCIS (Netherlands)

    Sjarif, D. R.; Sinke, R. J.; Duran, M.; Beemer, F. A.; Kleijer, W. J.; Ploos van Amstel, J. K.; Poll-The, B. T.

    1998-01-01

    Isolated glycerol kinase deficiency (GKD) is an X linked recessive disorder. The clinical and biochemical picture may vary from a childhood metabolic crisis to asymptomatic adult "pseudohypertriglyceridaemia", the result of hyperglycerolaemia. We performed glycerol kinase (GK) gene analysis to study

  19. Computational analysis of ABL kinase mutations allows predicting drug sensitivity against selective kinase inhibitors.

    Science.gov (United States)

    Kamasani, Swapna; Akula, Sravani; Sivan, Sree Kanth; Manga, Vijjulatha; Duyster, Justus; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

    2017-05-01

    The ABL kinase inhibitor imatinib has been used as front-line therapy for Philadelphia-positive chronic myeloid leukemia. However, a significant proportion of imatinib-treated patients relapse due to occurrence of mutations in the ABL kinase domain. Although inhibitor sensitivity for a set of mutations was reported, the role of less frequent ABL kinase mutations in drug sensitivity/resistance is not known. Moreover, recent reports indicate distinct resistance profiles for second-generation ABL inhibitors. We thus employed a computational approach to predict drug sensitivity of 234 point mutations that were reported in chronic myeloid leukemia patients. Initial validation analysis of our approach using a panel of previously studied frequent mutations indicated that the computational data generated in this study correlated well with the published experimental/clinical data. In addition, we present drug sensitivity profiles for remaining point mutations by computational docking analysis using imatinib as well as next generation ABL inhibitors nilotinib, dasatinib, bosutinib, axitinib, and ponatinib. Our results indicate distinct drug sensitivity profiles for ABL mutants toward kinase inhibitors. In addition, drug sensitivity profiles of a set of compound mutations in ABL kinase were also presented in this study. Thus, our large scale computational study provides comprehensive sensitivity/resistance profiles of ABL mutations toward specific kinase inhibitors.

  20. Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultaet fuer Physik, Universitaet Regensburg, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Stierl, M.; Hegemann, P. [Institut fuer Biologie/Experimentelle Biophysik, Humboldt Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Kateriya, Suneel [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2011-08-25

    Graphical abstract: The photoactivated adenylyl cyclase (nPAC) from Naegleria gruberi was expressed heterologously in Escherichia coli and its photo-cycling dynamics was studied by optical absorption and fluorescence spectroscopy. Highlights: {yields} Photo-activated adenylyl cyclase (nPAC) from Naegleria gruberi NEG-M was expressed. {yields} Photodynamics of BLUF domain in BLUF sensor - cyclase actuator protein was studied. {yields} Photo-excitation caused BLUF photo-cycling and permanent protein re-conformation. {yields} Re-conformed protein enabled photo-induced flavin reduction by proton transfer. {yields} Fluorescence of flavin in dark- and light-adapted state of nPAC was characterized. - Abstract: The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF (blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl{sub ox}) to semi-reduced flavin (FlH{sup {center_dot}}) occurred. Furthermore, photo-excitation of FlH{sup {center_dot}} caused irreversible electron transfer to fully reduced anionic flavin (FlH{sup -}). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr{sup {center_dot}+}-Fl{sup {center_dot}-} or Trp{sup {center_dot}+}-Fl{sup {center_dot}-} radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl{sub ox} to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.

  1. RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice.

    Science.gov (United States)

    Khaibullina, Alfia; Adjei, Elena A; Afangbedji, Nowah; Ivanov, Andrey; Kumari, Namita; Almeida, Luis E F; Quezado, Zenaide M N; Nekhai, Sergei; Jerebtsova, Marina

    2018-03-08

    Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of renal disease development in sickle cell disease. Copyright © 2018, Ferrata Storti Foundation.

  2. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J. (Merck)

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  3. Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis

    Directory of Open Access Journals (Sweden)

    Thanikkal Edvin J

    2012-09-01

    Full Text Available Abstract Background The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis. Results CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the λcI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain. Conclusions This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway

  4. 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

    2016-12-24

    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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway

    NARCIS (Netherlands)

    Arico, S.; Petiot, A.; Bauvy, C.; Dubbelhuis, P. F.; Meijer, A. J.; Codogno, P.; Ogier-Denis, E.

    2001-01-01

    The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic

  6. Purine-Metabolizing Ectoenzymes Control IL-8 Production in Human Colon HT-29 Cells

    Directory of Open Access Journals (Sweden)

    Fariborz Bahrami

    2014-01-01

    Full Text Available Interleukin-8 (IL-8 plays key roles in both chronic inflammatory diseases and tumor modulation. We previously observed that IL-8 secretion and function can be modulated by nucleotide (P2 receptors. Here we investigated whether IL-8 release by intestinal epithelial HT-29 cells, a cancer cell line, is modulated by extracellular nucleotide metabolism. We first identified that HT-29 cells regulated adenosine and adenine nucleotide concentration at their surface by the expression of the ectoenzymes NTPDase2, ecto-5′-nucleotidase, and adenylate kinase. The expression of the ectoenzymes was evaluated by RT-PCR, qPCR, and immunoblotting, and their activity was analyzed by RP-HPLC of the products and by detection of Pi produced from the hydrolysis of ATP, ADP, and AMP. In response to poly (I:C, with or without ATP and/or ADP, HT-29 cells released IL-8 and this secretion was modulated by the presence of NTPDase2 and adenylate kinase. Taken together, these results demonstrate the presence of 3 ectoenzymes at the surface of HT-29 cells that control nucleotide levels and adenosine production (NTPDase2, ecto-5′-nucleotidase and adenylate kinase and that P2 receptor-mediated signaling controls IL-8 release in HT-29 cells which is modulated by the presence of NTPDase2 and adenylate kinase.

  7. Structure and Assembly of the PI3K-like Protein Kinases (PIKKs Revealed by Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Angel Rivera-Calzada

    2015-03-01

    Full Text Available The phosphatidylinositol 3-kinase-like kinases (PIKKs are large serine-threonine protein kinases with a catalytic domain homologous to the phosphatidylinositol 3-kinase (PI3K. All PIKK family members share a general organization comprising a conserved C-terminus that contains the PI3K domain, which is preceded by a large N-terminal region made of helical HEAT repeats. In humans, the PIKK family includes six members, which play essential roles in various processes including DNA repair and DNA damage signalling (ATM, ATR, DNA-PKcs, control of cell growth (mTOR, nonsense-mediated mRNA decay (SMG1 and transcriptional regulation (TRRAP. High-resolution structural information is limited due to the large size (approx. 280-470 kDa and structural complexity of these kinases. Adding further complexity, PIKKs work as part of larger assemblies with accessory subunits. These complexes are dynamic in composition and protein-protein and protein-DNA interactions regulate the kinase activity and functions of PIKKs. Moreover, recent findings have shown that the maturation and correct assembly of the PIKKs require a large chaperon machinery, containing RuvBL1 and RuvBL2 ATPases and the HSP90 chaperon. Single-particle electron microscopy (EM is making key contributions to our understanding of the architecture of PIKKs and their complex regulation. This review summarizes the findings on the structure of these kinases, focusing mainly on medium-low resolution structures of several PIKKs obtained using EM, combined with X-ray crystallography of DNA-PKcs and mTOR. In addition, EM studies on higher-order complexes have revealed some of the mechanisms regulating the PIKKs, which will also be addressed. The model that emerges is that PIKKs, through their extensive interacting surfaces, integrate the information provided by multiple accessory subunits and nucleic acids to regulate their kinase activity in response to diverse stimuli.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. PI-3 kinase-PTEN signaling node: an intercept point for the control of angiogenesis.

    Science.gov (United States)

    Castellino, R C; Muh, C R; Durden, D L

    2009-01-01

    Angiogenesis is tightly regulated by opposing mechanisms in mammalian cells and is controlled by the angiogenic switch. Other review articles have described a central role for the PTEN/PI-3 kinase/AKT signaling node in the coordinate control of cell division, tumor growth, apoptosis, invasion and cellular metabolism [1, 2]. In this review, we focus on literature that supports the PTEN/PI-3 kinase/AKT signaling node as a major control point for the angiogenic switch in both the on and off positions. We also discuss the rationale for designing small molecule drugs that target the PTEN/PI-3 kinase/AKT signaling node for therapeutic intervention. Our hypothesis is that, instead of inhibiting one cell surface receptor, such as VEGFR2 with bevacizumab (Avastin), thereby leaving a significant number of receptors free to pulse angiogenic signals, a more effective strategy may be to regulate signaling through an intercept node where redundant cell surface receptor signals converge to transmit important signaling events within the cell. This therapeutic configuration brings coordinate control over multiple cell surface receptors in concert with a physiologic response which may combine arrest of cell cycle progression with growth inhibition and the induction of genes involved in specialized functions such as movement, which are all required for the complex process of angiogenesis to occur in a temporal-spatial paradigm.

  16. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.

    NARCIS (Netherlands)

    Ritzerfeld, J.; Remmele, S.; Wang, T.; Temmerman, K.; Brugger, B.; Wegehingel, S.; Tournaviti, S.; Strating, J.R.P.M.; Wieland, F.T.; Neumann, B.; Ellenberg, J.; Lawerenz, C.; Hesser, J.; Erfle, H.; Pepperkok, R.; Nickel, W.

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary

  17. 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...... that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.......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...

  18. The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

    Energy Technology Data Exchange (ETDEWEB)

    Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S. (Oregon HSU); (FSU)

    2012-05-29

    Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

  19. 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.

  20. 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

  1. 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

  2. Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase

    NARCIS (Netherlands)

    van den Blink, B.; Branger, J.; Weijer, S.; Deventer, S. H.; van der Poll, T.; Peppelenbosch, M. P.

    2001-01-01

    All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to

  3. 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...... DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization....

  4. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  5. Phosphatidylinositol 3-Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes

    DEFF Research Database (Denmark)

    Christ, Bruno; Yazici, Emine; Nath, Annegret

    2000-01-01

    Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase......Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase...

  6. 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.

  7. eIF2α Kinases Control Chalone Production in Dictyostelium discoideum ▿

    Science.gov (United States)

    Bowman, Robert L.; Xiong, Yanhua; Kirsten, Janet H.; Singleton, Charles K.

    2011-01-01

    Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α. PMID:21278229

  8. 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.

  9. A Dual Non-ATP Analogue Inhibitor of Aurora Kinases A and B, Derived from Resorcinol with a Mixed Mode of Inhibition.

    Science.gov (United States)

    Karthigeyan, Dhanasekaran; Surabhi, Sudhevan; Mizar, Pushpak; Soumik, Siddhanta; Banerjee, Amrita; Sinha, Sarmistha Halder; Dasgupta, Dipak; Narayana, Chandrabhas; Kundu, Tapas K

    2016-06-01

    Aurora kinases are the most commonly targeted mitotic kinases in the intervention of cancer progression. Here, we report a resorcinol derivative, 5-methyl-4-(2-thiazolylazo) resorcinol (PTK66), a dual inhibitor of Aurora A and Aurora B kinases. PTK66 is a surface binding non-ATP analogue inhibitor that shows a mixed pattern of inhibition against both of Aurora A and B kinases. The in vitro IC50 is approximately 47 and 40 μm for Aurora A and Aurora B kinases, respectively. In cellular systems, PTK66 exhibits a substantially low cytotoxicity at micromolar concentrations but it can induce aneuploidy under similar dosages as a consequence of Aurora kinase inhibition. This result was corroborated by a drop in the histone H3 (S10) phosphorylation level detected via Western blot analysis using three different cell types. Altogether, our findings indicate that the ligand containing resorcinol backbone is one of the novel scaffolds targeting the Aurora family of kinases, which could be a target for antineoplastic drug development. © 2016 John Wiley & Sons A/S.

  10. 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...

  11. 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...

  12. 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.

  13. 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...

  14. 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 deposite...

  15. 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

  16. Polymeric immunoglobulin receptor-mediated invasion of Streptococcus pneumoniae into host cells requires a coordinate signaling of SRC family of protein-tyrosine kinases, ERK, and c-Jun N-terminal kinase.

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M; Dierdorf, Nina I; Hauck, Christof R; Hammerschmidt, Sven

    2010-11-12

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.

  17. Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin......, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions....

  18. 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.

  19. Identification of a fungi-specific lineage of protein kinases closely related to tyrosine kinases.

    Science.gov (United States)

    Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2014-01-01

    Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

  20. Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Richard L. Schroeder

    2014-09-01

    Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

  1. Kinase pathway dependence in primary human leukemias determined by rapid inhibitor screening

    NARCIS (Netherlands)

    J.W. Tyner (Jeffrey); W.F. Yang (Wayne); A. Bankhead III (Armand); G. Fan (Guang); L.B. Fletcher (Luke); J. Bryant (Jade); J.M. Glover (Jason); B.H. Chang (Bill); S.E. Spurgeon (Stephen); W.H. Fleming (William); T. Kovacsovics; J. Gotlib (Jason); S.T. Oh (Stephen); M.W.N. Deininger (Michael W.); C.M. Zwaan (Christian Michel); M.L. den Boer (Monique); M.M. van den Heuvel-Eibrink (Marry); T. O'Hare (Thomas); B.J. Druker (Brian); M.M. Loriaux (Marc)

    2013-01-01

    textabstractKinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway

  2. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

  3. 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.

  4. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    Directory of Open Access Journals (Sweden)

    Raouf A. Khalil

    2013-03-01

    Full Text Available Blood pressure (BP is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN. In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i, which forms a complex with calmodulin, activates myosin light chain (MLC kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC. PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK and MAPK kinase (MEK, a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in

  5. Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Sandro R. Valentini

    2011-01-01

    Full Text Available The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1 was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM. In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly and growth time (t were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946. Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB, 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

  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

    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...... of T antigen by the associated kinase is reduced whereas a p34cdc2-kinase-specific peptide has no influence. In addition, the T-antigen-associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated...

  7. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases.

    Science.gov (United States)

    Marcotte, Douglas J; Liu, Yu-Ting; Arduini, Robert M; Hession, Catherine A; Miatkowski, Konrad; Wildes, Craig P; Cullen, Patrick F; Hong, Victor; Hopkins, Brian T; Mertsching, Elisabeth; Jenkins, Tracy J; Romanowski, Michael J; Baker, Darren P; Silvian, Laura F

    2010-03-01

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 A resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 A resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  8. The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

    Energy Technology Data Exchange (ETDEWEB)

    Bakszt, R.; Wernimont, A; Allali-Hassani, A; Mok, M; Hills, T; Hui, R; Pizarro, J

    2010-01-01

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  9. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72......syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...

  10. 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.

  11. Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).

    OpenAIRE

    Mouton, Veronique; Toussaint, Louise; Vertommen, Didier; Gueuning, Marie-Agnes; Maisin, Liliane; Havaux, Xavier; Sanchez-Canedo, Cossette; Bertrand, Luc; Dequiedt, Franck; Hemmings, Brian A; Hue, Louis; Rider, Mark H

    2010-01-01

    On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [gamma-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in H...

  12. Effect of adenosine 3':5'-cyclic monophosphate and inhibition of protein kinase a on heat sensitivity in H35 hepatoma cells

    International Nuclear Information System (INIS)

    Rijn, Johannes van; Berg, Jaap van den

    1996-01-01

    Purpose: To investigate the role of the cyclic adenosine 3':5'-monophosphate (AMP) signal transduction pathway in heat-induced cell death and the development of thermotolerance. Methods and Materials: Reuber H35 rat hepatoma cells were heated after preincubation with various compounds known to modulate the cyclic AMP signal transduction pathway. Cell survival was determined by colony-forming ability. Results: Preincubation of H35 cells with forskolin, a stimulator of adenylate cyclase, in combination with IBMX (3-isobutyl-1-methylxanthine), an inhibitor of cyclic AMP phosphodiesterase, results in thermosensitization. Similar results are obtained with various cyclic AMP analogs. Maximum thermosensitization occurs with 0.5 mM dibutyryl cyclic AMP (DBcAMP) after a preincubation period of 5 h and heating in the presence of the drug. The same relative degree of thermosensitization is found with 8-Cl-cAMP, but at a 10-fold lower concentration. Thermosensitization by DBcAMP is prevented by H89, a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Without additional cyclic AMP-inducing factors, H89 induces thermoprotection. None of the drug treatments are cytotoxic at 37 deg. C. DBcAMP does not affect the development of heat-induced thermotolerance but it reduces its expression to an extent similarly found in the observed thermosensitization in nonthermotolerant cells. Conclusion: The results strongly indicate that the cyclic AMP signal transduction pathway is involved in the process of heat-induced cell death. DBcAMP reduces the expression of thermotolerance, but does not affect its induction

  13. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-01-01

    Research highlights: → cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. → Pka1 phosphorylation is further induced by physiological stresses. → Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. → Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1 + or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  14. The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

    Directory of Open Access Journals (Sweden)

    Roberts-Thomson Sarah J

    2006-07-01

    Full Text Available Abstract Background The vanilloid receptor 1 (TRPV1 is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described. Results In the present studies we investigated the hypothesis that the μ opioid receptor (MOP agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+ responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1. Conclusion The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

  15. Long-acting β2-agonists increase fluticasone propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1 in airway smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Melanie Manetsch

    Full Text Available Mitogen-activated protein kinase phosphatase 1 (MKP-1 represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol, alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone in primary human airway smooth muscle (ASM cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6 and interleukin 8 (IL-8 secretion. As IL-6 expression (like MKP-1 is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα. Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.

  16. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    Science.gov (United States)

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.

  17. 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

  18. 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 ...

  19. Brd4 Shields Chromatin from ATM Kinase Signaling Storms

    OpenAIRE

    Choi, Serah; Bakkenist, Christopher J.

    2013-01-01

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin.

  20. Purine inhibitors of protein kinases, G proteins and polymerases

    Science.gov (United States)

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  1. dependent/calmodulin- stimulated protein kinase from moss

    Indian Academy of Sciences (India)

    Unknown

    lin-dependent protein kinase homolog; Planta 203 S91–. S97. Lu Y-T, Hidaka H and Feldman L J 1996 Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism; Planta. 199 18–24. Mitra D and Johri M M 2000 Enhanced expression of a cal-.

  2. One for all: the receptor-associated kinase BAK1.

    NARCIS (Netherlands)

    Chinchilla, D.; Shan, L.; He, P.; Vries, de S.C.; Kemmerling, B.

    2009-01-01

    The plant receptor kinase BAK1/SERK3 has been identified as a partner of ligand-binding leucine-rich repeat receptor kinases, in particular the brassinosteroid receptor BRI1 and the immune receptor FLS2. BAK1 positively regulates BRI1 receptor function via physical interaction and

  3. 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...

  4. Enhanced expression of a calcium-dependent protein kinase from ...

    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. 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

  6. 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 ...

  7. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Unknown

    Calcium-dependent protein kinase; degenerate primer; Funaria hygrometrica; nutrient; polymerase chain reaction; starvation ... Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an inter- esting class of ..... Saunders M J and Hepler P K 1983 Calcium antagonists and.

  8. Over-expression of NAD kinase in Corynebacterium crenatum and ...

    African Journals Online (AJOL)

    Purpose: To improve the biosynthesis of L-arginine by overexpressing homologous NAD kinase (ppnk) in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) and low oxygen supply (LOS). Methods: A recombinant plasmid (pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk) was ...

  9. 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 ...

  10. 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

  11. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

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

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... 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...

  12. Homing in: Mechanisms of Substrate Targeting by Protein Kinases.

    Science.gov (United States)

    Miller, Chad J; Turk, Benjamin E

    2018-03-12

    Protein phosphorylation is the most common reversible post-translational modification in eukaryotes. Humans have over 500 protein kinases, of which more than a dozen are established targets for anticancer drugs. All kinases share a structurally similar catalytic domain, yet each one is uniquely positioned within signaling networks controlling essentially all aspects of cell behavior. Kinases are distinguished from one another based on their modes of regulation and their substrate repertoires. Coupling specific inputs to the proper signaling outputs requires that kinases phosphorylate a limited number of sites to the exclusion of hundreds of thousands of off-target phosphorylation sites. Here, we review recent progress in understanding mechanisms of kinase substrate specificity and how they function to shape cellular signaling networks. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

    Science.gov (United States)

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-04-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I.

  14. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    Science.gov (United States)

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  15. In this issue: Tec kinases in the crosshairs.

    Science.gov (United States)

    Faris, Mary; Bot, Adrian

    2012-04-01

    Tec family kinases are the second largest family of non-receptor tyrosine kinases, of critical importance to the biology of cells derived from the bone marrow. Significant progress in this area during the last decade revealed a role for several of these kinases in inflammatory and oncologic disorders. This led to the discovery and development of molecular targeted therapies against Tec kinases, most notably to date Btk, already showing promising clinical activity in B cell lymphoma and autoimmunity. As Btk does not carry oncogenic modifications, this also establishes a novel therapeutic paradigm, based on targeting ancillary pathways recruited by the pathogenic process to become pivotal to cancer cell's biology. This issue focuses on biology and translation of Tec kinases, at the cross-roads between immunology and cancer.

  16. MAP Kinase Cascades in Plant Innate Immunity

    Directory of Open Access Journals (Sweden)

    Magnus Wohlfahrt Rasmussen

    2012-07-01

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

  17. 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.

  18. 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

  19. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    Science.gov (United States)

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  20. The role of MAP2 kinases and p38 kinase in acute murine liver injury models

    Science.gov (United States)

    Zhang, Jun; Min, Robert W M; Le, Khanh; Zhou, Sheng; Aghajan, Mariam; Than, Tin A; Win, Sanda; Kaplowitz, Neil

    2017-01-01

    c-Jun N-terminal kinase (JNK) mediates hepatotoxicity through interaction of its phospho-activated form with a mitochondrial outer membrane protein, Sh3bp5 or Sab, leading to dephosphorylation of intermembrane Src and consequent impaired mitochondrial respiration and enhanced ROS release. ROS production from mitochondria activates MAP3 kinases, such as MLK3 and ASK1, which continue to activate a pathway to sustain JNK activation, and amplifies the toxic effect of acetaminophen (APAP) and TNF/galactosamine (TNF/GalN). Downstream of MAP3K, in various contexts MKK4 activates both JNK and p38 kinases and MKK7 activates only JNK. The relative role of MKK4 versus 7 in liver injury is largely unexplored, as is the potential role of p38 kinase, which might be a key mediator of toxicity in addition to JNK. Antisense oligonucleotides (ASO) to MKK4, MKK7 and p38 (versus scrambled control) were used for in vivo knockdown, and in some experiments PMH were used after in vivo knockdown. Mice were treated with APAP or TNF/GalN and injury assessed. MKK4 and MKK7 were expressed in liver and each was efficiently knocked down with two different ASOs. Massive liver injury and ALT elevation were abrogated by MKK4 but not MKK7 ASO pretreatment in both injury models. The protection was confirmed in PMH. Knockdown of MKK4 completely inhibited basal P-p38 in both cytoplasm and mitochondria. However, ALT levels and histologic injury in APAP-treated mice were not altered with p38 knockdown versus scrambled control. p38 knockdown significantly increased P-JNK levels in cytoplasm but not mitochondria after APAP treatment. In conclusion, MKK4 is the major MAP2K, which activates JNK in acute liver injury. p38, the other downstream target of MKK4, does not contribute to liver injury from APAP or TNF/galactosamine. PMID:28661486

  1. 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...

  2. 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...

  3. Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

    Directory of Open Access Journals (Sweden)

    Sun K

    2017-03-01

    Full Text Available Kai Sun, Yong Chang, Binbin Zhou, Xiaojin Wang, Lin Liu Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People’s Republic of China Abstract: This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. Keywords: electrochemical biosensor, colorimetric assay, gold nanoparticle, aptameric peptide, protein kinase A, signal amplification 

  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. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    Directory of Open Access Journals (Sweden)

    Adam J. Johnson

    2016-12-01

    Full Text Available The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport.

  6. In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.

    Science.gov (United States)

    Cable, Jennifer; Prutzman, Kirk; Gunawardena, Harsha P; Schaller, Michael D; Chen, Xian; Campbell, Sharon L

    2012-03-20

    Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

  7. Structure of protein O-mannose kinase reveals a unique active site architecture.

    Science.gov (United States)

    Zhu, Qinyu; Venzke, David; Walimbe, Ameya S; Anderson, Mary E; Fu, Qiuyu; Kinch, Lisa N; Wang, Wei; Chen, Xing; Grishin, Nick V; Huang, Niu; Yu, Liping; Dixon, Jack E; Campbell, Kevin P; Xiao, Junyu

    2016-11-23

    The 'pseudokinase' SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg 2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome.

  8. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Science.gov (United States)

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-09-01

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  10. GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein.

    Science.gov (United States)

    Clark, Margaret J; Miduturu, Chandra; Schmidt, Aaron G; Zhu, Xuling; Pitts, Jared D; Wang, Jinhua; Potisopon, Supanee; Zhang, Jianming; Wojciechowski, Amy; Hann Chu, Justin Jang; Gray, Nathanael S; Yang, Priscilla L

    2016-04-21

    Dengue virus infects more than 300 million people annually, yet there is no widely protective vaccine or drugs against the virus. Efforts to develop antivirals against classical targets such as the viral protease and polymerase have not yielded drugs that have advanced to the clinic. Here, we show that the allosteric Abl kinase inhibitor GNF-2 interferes with dengue virus replication via activity mediated by cellular Abl kinases but additionally blocks viral entry via an Abl-independent mechanism. To characterize this newly discovered antiviral activity, we developed disubstituted pyrimidines that block dengue virus entry with structure-activity relationships distinct from those driving kinase inhibition. We demonstrate that biotin- and fluorophore-conjugated derivatives of GNF-2 interact with the dengue glycoprotein, E, in the pre-fusion conformation that exists on the virion surface, and that this interaction inhibits viral entry. This study establishes GNF-2 as an antiviral compound with polypharmacological activity and provides "lead" compounds for further optimization efforts. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.

    Science.gov (United States)

    Ebert, Antje D; Laussmann, Mareike; Wegehingel, Sabine; Kaderali, Lars; Erfle, Holger; Reichert, Jürgen; Lechner, Johannes; Beer, Hans-Dietmar; Pepperkok, Rainer; Nickel, Walter

    2010-06-01

    Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

  12. Diacylglycerol kinase α regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling.

    Science.gov (United States)

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2014-11-14

    Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Stress-Kinase Regulation of TASK-1 and TASK-3

    Directory of Open Access Journals (Sweden)

    Susanne Rinné

    2017-11-01

    Full Text Available Background/Aims: TASK channels belong to the two-pore-domain potassium (K2P channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs which are known to modify many cellular functions by modulating ion channels. Methods: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments. Results: SGKs and proteinkinase B (PKB induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis. Conclusion: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.

  14. 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.

  15. Arginine kinase in Phytomonas, a trypanosomatid parasite of plants.

    Science.gov (United States)

    Canepa, Gaspar E; Carrillo, Carolina; Miranda, Mariana R; Sayé, Melisa; Pereira, Claudio A

    2011-09-01

    Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. 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.

  17. Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

    Science.gov (United States)

    Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

    2013-06-14

    The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

  18. Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation*

    Science.gov (United States)

    Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

    2013-01-01

    The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [35S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL. PMID:23629654

  19. PI-3 kinase activity is required for epithelial-mesenchymal transformation during palate fusion.

    Science.gov (United States)

    Kang, Pei; Svoboda, Kathy K H

    2002-11-01

    Epithelial-mesenchymal transformation (EMT) is the primary mechanism for the disappearance of medial edge epithelia (MEE) during palate fusion. This phenotype transition is highly regulated by growth factors, extracellular matrix, cell surface receptors, and a variety of intracellular signaling. Phosphatidylinositol-3 (PI-3) kinase regulates cytoskeleton reorganization, cell migration, and transforming growth factor (TGF) beta-regulated EMT. Therefore, we investigated the role of PI-3 kinase in EMT during palatal fusion in vitro. Palatal shelves from embryonic (E) 13.5 day mouse embryos were collected and cultured for up to 72 hr. A specific PI-3 kinase inhibitor, LY294002, was added to the medium at concentrations of 100 etaM, 1 microM, and 10 microM. The fate of midline epithelia was traced by carboxyfluorescence labeling and analyzed by confocal microscopy. Harvested tissues were also processed for immunohistochemical analysis of a specific marker for basal lamina (laminin). Palatal fusion stages were scored on a scale of 1 to 5, with 1 equal to complete nonfusion and 5 equal to complete fusion. The mean fusion score (MFS) was calculated for each treatment group. Palatal shelves fused after 72 hr of culture in control and 100 etaM LY294002 inhibitor-treated groups, with MFS of 4.67 and 4.5, respectively. Laminin was absent in the midline and epithelia transformed into mesenchyme. However, when cultured palates were treated with 1 and 10 microM LY294002, MEE persisted in the midline and the basal lamina remained intact after 72 hr. The MFS was significantly less in the 1 and 10 microM LY294002-treated tissues at 2.08 and 1.33, respectively. Our results demonstrate that EMT during palatal fusion in vitro is dependent on PI-3 kinase activity. Copyright 2002 Wiley-Liss, Inc.

  20. Profiling bacterial kinase activity using a genetic circuit

    DEFF Research Database (Denmark)

    van der Helm, Eric; Bech, Rasmus; Lehning, Christina Eva

    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....

  1. Targeting the Synthetic Essential Kinases of Breast Cancers

    Science.gov (United States)

    2016-05-01

    kinases in mitosis and tumorigenesis. Mol Cancer Res 2007, 5(1):1-10. 12. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA: Human TPX2 is...AWARD NUMBER: W81XWH-15-1-0027 TITLE: Targeting the synthetic essential kinases of breast cancers PRINCIPAL INVESTIGATOR: Jen-Tsan...Targeting the synthetic essential kinases of breast cancers 5b. GRANT NUMBER W81XWH-15-1-0027 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

  2. 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.

  3. Second-generation inhibitors of Bruton tyrosine kinase.

    Science.gov (United States)

    Wu, Jingjing; Liu, Christina; Tsui, Stella T; Liu, Delong

    2016-09-02

    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.

  4. Organizing signal transduction through A-kinase anchoring proteins (AKAPs).

    Science.gov (United States)

    Logue, Jeremy S; Scott, John D

    2010-11-01

    A fundamental role for protein-protein interactions in the organization of signal transduction pathways is evident. Anchoring, scaffolding and adapter proteins function to enhance the precision and directionality of these signaling events by bringing enzymes together. The cAMP signaling pathway is organized by A-kinase anchoring proteins. This family of proteins assembles enzyme complexes containing the cAMP-dependent protein kinase, phosphoprotein phosphatases, phosphodiesterases and other signaling effectors to optimize cellular responses to cAMP and other second messengers. Selected A-kinase anchoring protein signaling complexes are highlighted in this minireview. © 2010 The Authors Journal compilation © 2010 FEBS.

  5. Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Amit K Gandhi

    Full Text Available Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase, resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP. Pyridoxal (PL, an inactive form of vitamin B₆ is converted to PLP by PL kinase. PLP is the B₆ vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B₆, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.

  6. 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.

  7. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

    Science.gov (United States)

    Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

    2005-08-01

    OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

  8. Prostate-derived sterile 20-like kinase 2 (PSK2) regulates apoptotic morphology via C-Jun N-terminal kinase and Rho kinase-1.

    Science.gov (United States)

    Zihni, Ceniz; Mitsopoulos, Costas; Tavares, Ignatius A; Ridley, Anne J; Morris, Jonathan D H

    2006-03-17

    We have reported previously that human prostate-derived sterile 20-like kinase (PSK) 1 alters actin cytoskeletal organization and binds to microtubules, regulating their organization and stability. We have shown a structurally related protein kinase PSK2, which lacks a microtubule-binding site, activated c-Jun N-terminal kinase (JNK), and induced apoptotic morphological changes that include cell contraction, membrane blebbing, and apoptotic body formation. Apoptotic stimuli increased the catalytic activity of endogenous PSK2 and JNK, and dominant negative JNK or a physiological inhibitor of JNK blocked these apoptotic morphological responses to PSK2, demonstrating a requirement for JNK. PSK2 also stimulated the cleavage of Rho kinase-1 (ROCK-I), and the activity of ROCK-I was required for PSK2 to induce cell contraction and membrane blebbing. The activation of caspases was also needed for the induction of membrane blebbing by PSK2, which was itself a substrate for caspase 3. PSK2 therefore regulates apoptotic morphology associated with the execution phase of apoptosis, which involves dynamic reorganization of the actin cytoskeleton, via downstream targets that include JNK and ROCK-I. Our findings suggest that PSKs form a subgroup of sterile 20 (STE20)-like kinases that regulate different cytoskeletal processes.

  9. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

    Science.gov (United States)

    Ard, Ryan; Mulatz, Kirk; Pomoransky, Julia L; Parks, Robin J; Trinkle-Mulcahy, Laura; Bell, John C; Gee, Stephen H

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

  10. Targeting Sphingosine Kinase-1 To Inhibit Melanoma

    Science.gov (United States)

    Madhunapantula, SubbaRao V.; Hengst, Jeremy; Gowda, Raghavendra; Fox, Todd E.; Yun, Jong K; Robertson, Gavin P.

    2012-01-01

    SUMMARY Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient’s tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage dependent and independent growth as well as sensitized melanoma cells to apoptosis inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I, decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents. PMID:22236408

  11. Hybrid histidine kinases in pathogenic fungi.

    Science.gov (United States)

    Defosse, Tatiana A; Sharma, Anupam; Mondal, Alok K; Dugé de Bernonville, Thomas; Latgé, Jean-Paul; Calderone, Richard; Giglioli-Guivarc'h, Nathalie; Courdavault, Vincent; Clastre, Marc; Papon, Nicolas

    2015-03-01

    Histidine kinases (HK) sense and transduce via phosphorylation events many intra- and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N-terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape-function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs. © 2015 John Wiley & Sons Ltd.

  12. Cyclin-dependent kinases in C. elegans

    Directory of Open Access Journals (Sweden)

    Boxem Mike

    2006-05-01

    Full Text Available Abstract Cell division is an inherent part of organismal development, and defects in this process can lead to developmental abnormalities as well as cancerous growth. In past decades, much of the basic cell-cycle machinery has been identified, and a major challenge in coming years will be to understand the complex interplay between cell division and multicellular development. Inevitably, this requires the use of more complex multicellular model systems. The small nematode Caenorhabditis elegans is an excellent model system to study the regulation of cell division in a multicellular organism, and is poised to make important contributions to this field. The past decade has already seen a surge in cell-cycle research in C. elegans, yielding information on the function of many basic cell-cycle regulators, and making inroads into the developmental control of cell division. This review focuses on the in vivo roles of cyclin-dependent kinases in C. elegans, and highlights novel findings implicating CDKs in coupling development to cell-cycle progression.

  13. Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

    Directory of Open Access Journals (Sweden)

    Soltis Pamela S

    2006-02-01

    Full Text Available Abstract Background The glycogen synthase kinase 3 (GSK3/SHAGGY-like kinases (GSKs are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences. Results We found that the structure of GSK genes is generally conserved in Arabidopsis and rice, although we documented examples of exon expansion and intron loss. Our phylogenetic analyses of 139 sequences revealed four major clades of GSK genes that correspond to the four subgroups initially recognized in Arabidopsis. ESTs from basal angiosperms were represented in all four major clades; GSK homologs from the basal angiosperm Persea americana (avocado appeared in all four clades. Gymnosperm sequences occurred in clades I, III, and IV, and a sequence of the red alga Porphyra was sister to all green plant sequences. Conclusion Our results indicate that (1 the plant-specific GSK gene lineage was established early in the history of green plants, (2 plant GSKs began to diversify prior to the origin of extant seed plants, (3 three of the four major clades of GSKs present in Arabidopsis and rice were established early in the evolutionary history of extant seed plants, and (4 diversification into four major clades (as initially reported in Arabidopsis occurred either just prior to the origin of the angiosperms or very early in angiosperm history.

  14. A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

    Science.gov (United States)

    Torada, Atsushi; Koike, Michiya; Ogawa, Taiichi; Takenouchi, Yu; Tadamura, Kazuki; Wu, Jianzhong; Matsumoto, Takashi; Kawaura, Kanako; Ogihara, Yasunari

    2016-03-21

    Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

    NARCIS (Netherlands)

    Bem, A.E.; Velikova, N.R.; Pellicer, M.T.; Baarlen, van P.; Marina, A.; Wells, J.M.

    2015-01-01

    Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In

  17. Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome.

    NARCIS (Netherlands)

    Mayr, J.A.; Haack, T.B.; Graf, E.; Zimmermann, F.A.; Wieland, T.; Haberberger, B.; Superti-Furga, A.; Kirschner, J.; Steinmann, B.; Baumgartner, M.R.; Moroni, I.; Lamantea, E.; Zeviani, M.; Rodenburg, R.J.T.; Smeitink, J.; Strom, T.M.; Meitinger, T.; Sperl, W.; Prokisch, H.

    2012-01-01

    Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in

  18. Emerging roles of protein kinases in microglia-mediated neuroinflammation.

    Science.gov (United States)

    Lee, Sun-Hwa; Suk, Kyoungho

    2017-12-15

    Neuroinflammation is mediated by resident central nervous system glia, neurons, peripherally derived immune cells, blood-brain barrier, and inflammatory mediators secreted from these cells. Neuroinflammation has been implicated in stroke and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. Protein kinases have been one of the most exploited therapeutic targets in the current pharmacological research, especially in studies on cancer and inflammation. To date, 32 small-molecule protein kinase inhibitors have been approved by the United States Food and Drug Administration for the treatment of cancer and inflammation. However, there is no drug effectively targeting neuroinflammation and/or neurodegenerative diseases. Recent studies have advanced several protein kinases as important drug targets in neuroinflammation and/or neurodegenerative diseases. Here, we review emerging protein kinases potentially involved in neuroinflammation and subsequent neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Inhibitors caveolin-1 and protein kinase G show differential ...

    African Journals Online (AJOL)

    protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells ...

  20. 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 ...

  1. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Hochachka and Somero 2002). Therefore, some animals have to initiate anaerobic metabolism to meet part of energy needs (Costanzo et al. 2004; Colson-Proch et al. 2009). Adenosine monophosphate-activated protein kinase.

  2. 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

  3. 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)

  4. 21 CFR 862.1650 - Pyruvate kinase test system.

    Science.gov (United States)

    2010-04-01

    ...). Measurements obtained by this device are used in the diagnosis and treatment of various inherited anemias due to pyruvate kinase deficiency or of acute leukemias. (b) Classification. Class I (general controls...

  5. The Snf1 Protein Kinase in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Usaite, Renata

    2008-01-01

    . Failure in the AMPK regulatory cascade leads to metabolic disorders, such as obesity or type 2 diabetes. The knowledge about the Snf1 protein kinase remains to be of much interest in studying yeast carbon metabolism and human biology. To investigate the effect of Snf1 kinase and its regulatory subunit Snf......In yeast, Saccharomyces cerevisiae, the Snf1 protein kinase is primarily known as a key component of the glucose repression regulatory cascade. The Snf1 kinase is highly conserved among eukaryotes and its mammalian homolog AMPK is responsible for energy homeostasis in cells, organs and whole bodies...... proteome datasets (2388 proteins) to date was generated using Multidimensional Protein Identification Technology followed by quantitation using stable isotope labeling approach (chapter 3). The stable isotope labeling was compared to the spectral counting quantitative approach and the study showed...

  6. Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

    International Nuclear Information System (INIS)

    Hussain, Alamdar; Faryal, Rani; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C.I. Edvard

    2009-01-01

    Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

  7. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

    Science.gov (United States)

    Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

    2002-08-15

    Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.

  8. Cyclin-dependent kinase Inhibitors Inspired by Roscovitine: Purine Bioisosteres

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Paruch, K.; Kryštof, Vladimír

    2012-01-01

    Roč. 18, č. 20 (2012), s. 2974-2980 ISSN 1381-6128 R&D Projects: GA ČR GAP305/12/0783 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cancer * cyclin-dependent kinase * inhibitor Subject RIV: ED - Physiology Impact factor: 3.311, year: 2012 http://www.benthamdirect.org/pages/article/1/3177382/cyclin-dependent-kinase-inhibitors-inspired-by-roscovitine-purine-bioisosteres.html

  9. Targeting SRC Family Kinases in HSP90 in Lung Cancer

    Science.gov (United States)

    2015-10-01

    Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited Targeting SRC Family Kinases and HSP90 in Lung Cancer...COVERED Annual 30 Sep 2014 - 29 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting SRC Family Kinases in HSP90 in Lung Cancer 5b. GRANT...AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT 15. SUBJECT TERMS Nedd9, Src , HSP90

  10. 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

  11. 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.

  12. 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...

  13. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj

    2009-01-01

    As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase......-substrate specificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms...

  14. 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

  15. Auto-thiophosphorylation activity of Src tyrosine kinase.

    Science.gov (United States)

    Cabail, M Zulema; Chen, Emily I; Koller, Antonius; Miller, W Todd

    2016-07-07

    Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low. Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase. Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.

  16. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    In this study we report that phosphatidylinositol 3-kinase (PI 3-kinase), a lipid kinase which participates in downstream signalling events of heterotrimeric G protein-coupled receptors and receptor tyrosine kinases, contains a high affinity binding site for calmodulin (CaM). The putative Ca......M. Furthermore, a sequence comparison among different PI 3-kinase isoforms revealed that the sequence which can bind CaM is highly conserved within different PI 3-kinase isoforms. These results indicate a novel mechanism for regulating PI 3-kinase and provide a new direct link between Ca2+ and phospholipid...

  17. 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.

  18. A protein kinase C inhibitor attenuates cyanide toxicity in vivo.

    Science.gov (United States)

    Maduh, E U; Nealley, E W; Song, H; Wang, P C; Baskin, S I

    1995-06-26

    We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs (6-10 kg). Brain high energy phosphates were analyzed using a 31P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Telsa horizontal bore magnet. H-7, 1 mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN-). Prior to NaCN, H-7, or H-7 + CN- administration, baseline 31P resonance spectra of 1-min duration were acquired for 5-10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the 31P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN- antagonism, underscoring the role of PKC in cyanide intoxication.

  19. Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Guanggan Hu

    2007-03-01

    Full Text Available A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP-dependent protein kinase A (PKA is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential

  20. 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.

  1. Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

    Directory of Open Access Journals (Sweden)

    K. Pollock

    1993-01-01

    Full Text Available The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK and protein kinase C (PKC, respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP induced generation of superoxide anion and thromboxane B2 (TXB2 in guinea-pig alveolar macrophages (AM. Genistein (3–100 μM dose dependently inhibited FMLP (3 nM induced superoxide generation in non-primed AM and TXB2 release in non-primed or in lipopolysaccharide (LPS (10 ng/ml primed AM to a level > 80% but had litle effect up to 100 μM on phorbol myristate acetate (PMA (10 nM induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC50 0.21 ± 0.10 μM but had no effect on or potentiated (at 3 and 10 μM FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 μM inhibited primed TXB2 release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.

  2. Rapamycin Induces Mitogen-activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Expression through Activation of Protein Kinase B and Mitogen-activated Protein Kinase Kinase Pathways*

    Science.gov (United States)

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

    2013-01-01

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition. PMID:24126911

  3. 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.

  4. Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis.

    Science.gov (United States)

    Jongstra-Bilen, Jenny; Puig Cano, Adrianet; Hasija, Manvi; Xiao, Haiyan; Smith, C I Edvard; Cybulsky, Myron I

    2008-07-01

    Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.

  5. Activation of p38 and Erk Mitogen-Activated Protein Kinases Signaling in Ocular Rosacea.

    Science.gov (United States)

    Wladis, Edward J; Swamy, Supraja; Herrmann, Alyssa; Yang, Jinhong; Carlson, J Andrew; Adam, Alejandro P

    2017-02-01

    Rosacea-related cutaneous inflammation is a common cause of ocular surface disease. Currently, there are no specific pharmacologic therapies to treat ocular rosacea. Here, we aimed at determining the differences in intracellular signaling activity in eyelid skin from patients with and without ocular rosacea. This was an observational, comparative case series including 21 patients undergoing lower lid ectropion surgery at one practice during 2013 and 2014 (18 patients with rosacea, 13 control patients), and 24 paraffin-embedded archival samples from Albany Medical Center, selected randomly (12 patients with rosacea, 12 control patients). Cutaneous biopsies resulting from elective lower lid ectropion surgery were analyzed by Proteome Profiler Human Phospho-Kinase Array, Western blot, and/or immunohistochemistry. Samples derived from ocular rosacea patients showed increased levels of phosphorylated (active) p38 and Erk kinases. Phosphoproteins were mainly localized to the epidermis of affected eyelids. This finding provides a novel potential therapeutic target for treatment of ocular rosacea and possibly other forms of rosacea. Further testing is required to determine if p38 and Erk activation have a causal role in ocular rosacea. The selective activation of keratinocytes in the affected skin suggests that topical pathway inhibition may be an effective treatment that will ultimately prevent ocular surface damage due to ocular rosacea.

  6. Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Puja; Wang, Benlian; Maeda, Tadao; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2008-10-08

    G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1{center_dot}(Mg{sup 2+}){sub 2} {center_dot}ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

  7. Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

    Science.gov (United States)

    Duverger, Alexandra; Wolschendorf, Frank; Anderson, Joshua C; Wagner, Frederic; Bosque, Alberto; Shishido, Takao; Jones, Jennifer; Planelles, Vicente; Willey, Christopher; Cron, Randall Q; Kutsch, Olaf

    2014-01-01

    Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation.

  8. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L.; Spiller, Benjamin W. (Vanderbilt)

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

  9. Cloning and sequence analysis of lily and tobacco guanylate kinases.

    Science.gov (United States)

    Kumar, V

    2000-03-01

    Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phosphoryl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74-84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.

  10. Thrombin generation in abdominal sepsis is Rho-kinase-dependent.

    Science.gov (United States)

    Wang, Yongzhi; Braun, Oscar Ö; Zhang, Su; Norström, Eva; Thorlacius, Henrik

    2015-05-08

    Sepsis causes severe derangements of the coagulation system. However, the signaling mechanisms regulating sepsis-induced thrombin generation remain elusive. Herein, we hypothesized that Rho-kinase might be an important regulator of thrombin generation in abdominal sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. Thrombin generation, coagulation factors, lung histology and myeloperoxidase (MPO) activity were determined 6 h and 24 h after induction of CLP. Induction of CLP triggered a systemic inflammatory response characterized by neutrophil accumulation and tissue injury in the lung as well as thrombocytopenia and leukocytopenia. Administration of Y-27632, a Rho-kinase inhibitor, attenuated these markers of systemic inflammation in CLP animals. Moreover, peak thrombin formation was decreased by 77% and 81% in plasma from mice 6 h and 24 h after induction of CLP. Total thrombin generation was reduced by 64% and 67% 6 h and 24 h after CLP induction, respectively. Notably, administration of Y-27632 increased peak formation by 99% and total thrombin generation by 66% in plasma from septic animals. In addition, CLP markedly decreased plasma levels of prothrombin, factor V and factor X at 6 h and 24 h. Interestingly, Rho-kinase inhibition significantly enhanced levels of prothrombin, factor V and factor X in plasma from septic mice. In addition, inhibition of Rho-kinase decreased CLP-induced elevations of CXCL2 by 36% and interleukin-6 by 38%. These novel findings suggest that sepsis-induced thrombin generation is regulated by Rho-kinase. Moreover, inhibition of Rho-kinase reverses sepsis-evoked consumption of coagulation factors. Thus, our results show that targeting Rho-kinase signaling might protect against coagulation dysfunction in abdominal sepsis. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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.

  13. Overexpression of Populus trichocarpa Mitogen-Activated Protein Kinase Kinase4 Enhances Salt Tolerance in Tobacco

    Directory of Open Access Journals (Sweden)

    Chengjun Yang

    2017-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK is one of the factors of cascade reactions affecting responses to signal pathway of environmental stimuli. Throughout the life of plants, MAPK family members participate in signal transduction pathways and regulate various intracellular physiological and metabolic reactions. To gain insights into regulatory function of MAPK kinase (MAPKK in Populus trichocarpa under salt stress, we obtained full-length cDNA of PtMAPKK4 and analyzed different expression levels of PtMAPKK4 gene in leaves, stems, and root organs. The relationship between PtMAPKK4 and salt stress was studied by detecting expression characteristics of mRNA under 150 mM NaCl stress using real-time quantitative polymerase chain reaction. The results showed that expression of PtMAPKK4 increased under salt (NaCl stress in leaves but initially reduced and then increased in roots. Thus, salt stress failed to induce PtMAPKK4 expression in stems. PtMAPKK4 possibly participates in regulation of plant growth and metabolism, thereby improving its salt tolerance. We used Saccharomyces cerevisiae strain INVScI to verify subcellular localization of PtMAPKK4 kinase. The yeast strains containing pYES2-PtMAPKK4-GFP plasmid expressed GFP fusion proteins under the induction of d-galactose, and the products were located in nucleus. These results were consistent with network prediction and confirmed location of PtMAPKK4 enzyme in the nucleus. We tested NaCl tolerance in transgenic tobacco lines overexpressing PtMAPKK4 under the control of 35S promoter at germination stage to detect salt tolerance function of PtMAPKK4. Compared withK326 (a wild-type tobacco, lines overexpressing PtMAPKK4 showed a certain degree of improvement in tolerance, germination, and growth. NaCl inhibited growth of overexpressed line and K326 at the seedling stage. However, statistical analysis showed longer root length, higher fresh weight, and lower MDA content in transgenic lines in

  14. 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...

  15. 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

    Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that ...

  16. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  17. Enhancement of HIV-1-induced syncytium formation in T cells by the tyrosyl kinase p56lck.

    Science.gov (United States)

    Briand, G; Barbeau, B; Corbeil, J; Tremblay, M

    1997-04-28

    The CD4 glycoprotein is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1) and has also been reported to be physically associated with p56lck, a tyrosyl protein kinase p56lck is a member of the src family of nonreceptor protein-tyrosine kinases and is expressed predominantly in T lymphocytes. Our objective was to study the effect of p56lck on the biology of HIV-1. For this purpose, we have stably transfected two human p56lck negative T cell lines (C8166-45 and MT-2) with plasmids encoding for this cellular protein. Following coculture with HIV-1-infected cells or infection with cell-free virus, p56lck-expressing cell lines showed a greater propensity for virus-mediated syncytium formation than parental p56lck-negative cells. The enhancement of HIV-1-induced syncytium formation was not associated with the kinase activity of p56lck, as demonstrated by experiments using a kinase-deficient mutant. However, the physical interaction between CD4 and p56lck was shown to be necessary to obtain the enhancement of syncytium formation since a mutated version of p56lck, which is deficient in its capacity to associate with CD4, did not lead to an increase in virus-mediated cell-to-cell fusion events. Finally, we determined that cells transfected with wild-type and kinase-negative mutant p56lck showed a reduced rate of CD4 endocytosis compared to parental p56lck-negative cells. Together, these results suggest that p56lck can be seen as an accessory molecule facilitating HIV-1-mediated syncytium formation in T cells by a mechanism involving the stabilization of the CD4 molecule at the cell surface.

  18. Minimal surfaces

    CERN Document Server

    Dierkes, Ulrich; Sauvigny, Friedrich; Jakob, Ruben; Kuster, Albrecht

    2010-01-01

    Minimal Surfaces is the first volume of a three volume treatise on minimal surfaces (Grundlehren Nr. 339-341). Each volume can be read and studied independently of the others. The central theme is boundary value problems for minimal surfaces. The treatise is a substantially revised and extended version of the monograph Minimal Surfaces I, II (Grundlehren Nr. 295 & 296). The first volume begins with an exposition of basic ideas of the theory of surfaces in three-dimensional Euclidean space, followed by an introduction of minimal surfaces as stationary points of area, or equivalently

  19. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  20. Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.

    Directory of Open Access Journals (Sweden)

    Shuangxi Li

    2016-06-01

    Full Text Available Hedgehog (Hh signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR-family protein Smoothened (Smo. Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail. We find that CL-II phosphorylation is promoted by protein kinase A (PKA-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.