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Sample records for adenylate kinase activity

  1. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    Trung Thanh Thach

    2014-01-01

    Full Text Available Streptococcus pneumoniae (pneumococcus infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S. pneumoniae (SpAdK serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A. Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

  2. Long-Term Exposure to High Corticosterone Levels Inducing a Decrease of Adenylate Kinase 1 Activity

    ZHAO Yu'nan; SHEN Jia; SU Hui; HUANG Yufang; XING Dongming; DU Lijun

    2009-01-01

    Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex, can be cumula-tively toxic to hippocampal neurons, the cause of which is not known. The present study determined whether the cytosol adenylate kinase (AK) system was involved in the neuronal damage induced by long-term exposure to high corticosterone levels. We investigated the effects of long-term exposure to high corticosterone levels on AK1 activity, AK1 mRNA expression, and energy levels in cultured hippocampal neurons. The results show that long-term exposure to high corticosterone levels induces a reduction of the cultured hippocampal neuron viability, significantly reduces energy levels, and causes a time-dependant re-duction of the AK1 activity. These findings indicate that changes in the AK system might be the mechanism underlying neuronal damage induced by long-term exposure to high corticosterone levels.

  3. Structural studies of Schistosoma mansoni adenylate kinases

    Marques, I.A. [Universidade Federal de Goias (UFG), Goiania, GO (Brazil); Pereira, H.M.; Garrat, R.C. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil)

    2012-07-01

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

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

    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. A Continuous Kinetic Assay for Adenylation Enzyme Activity and Inhibition

    Daniel J. Wilson; Aldrich, Courtney C.

    2010-01-01

    Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PPi). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hy...

  6. Correlated inter-domain motions in adenylate kinase.

    Santiago Esteban-Martín

    2014-07-01

    Full Text Available Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK, using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs measured under steric alignment by nuclear magnetic resonance (NMR. We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

  7. Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

    Oliver, S.; Tiessen, A.; Fernie, A.; Geigenberger, P.

    2008-01-01

    Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial ade...

  8. Pituitary adenylate cyclase activating polypeptide and migraine

    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...

  9. Interconversion of functional motions between mesophilic and thermophilic adenylate kinases.

    Michael D Daily

    2011-07-01

    Full Text Available Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK, for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō simulation of mesophilic AK from E. coli (AKmeso to simulations of thermophilic AK from Aquifex aeolicus (AKthermo. In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A "heated" simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the "corresponding states" hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.

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

    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.

  11. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  12. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

    Drakou, Christina E.; Malekkou, Anna; Hayes, Joseph M.; Carsten W Lederer; Leonidas, Demetres D.; Oikonomakos, Nikos G.; Lamond, Angus I.; Santama, Niovi; Zographos, Spyros E.

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. D...

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

    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.

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

    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

  15. Glucagon and adenylate cyclase: binding studies and requirements for activation.

    Levey, G S; Fletcher, M A; Klein, I

    1975-01-01

    Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase. PMID:165684

  16. Fluorescence and NMR investigations in the ligand binding properties of adenylate kinases

    A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand α,ω-di[(3' or 2')-O-(N-methyl-anthraniloyl)adenosine-5'] pentaphosphate (MAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 x 10-9 to 5 x 10-2 M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. Together with the results obtained by Tian et al on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity

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

    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

  18. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, ...

  19. Path ensembles for conformational transitions in adenylate kinase using weighted--ensemble path sampling

    Bhatt, Divesh

    2009-01-01

    We perform first path sampling simulations of conformational transitions of semi--atomistic protein models. We generate an ensemble of pathways for conformational transitions between open and closed forms of adenylate kinase using weighted ensemble path sampling method. Such an ensemble of pathways is critical in determining the important regions of configuration space sampled during a transition. To different semi--atomistic models are used: one is a pure Go model, whereas the other includes level of residue specificity via use of Miyajawa--Jernigan type interactions and hydrogen bonding. For both the models, we find that the open form of adenylate kinase is more flexible and the the transition from open to close is significantly faster than the reverse transition. We find that the transition occurs via the AMP binding domain snapping shut at a fairly fast time scale. On the other hand, the flexible lid domain fluctuates significantly and the shutting of the AMP binding domain does not depend upon the positi...

  20. Down-regulation of adenylate kinase 5 in temporal lobe epilepsy patients and rat model.

    Lai, Yujie; Hu, Xiaotong; Chen, Guojun; Wang, Xuefeng; Zhu, Binglin

    2016-07-15

    Adenylate kinase 5 (AK5) is one member of the AK family and plays a critical role in maintaining cellular homeostasis. Different from the other AKs, AK5 is almost exclusively expressed in the brain. However, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of AK5 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic rat model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that AK5 protein was mainly expressed in neurons, demonstrated by colocalization with the dendritic marker, MAP2, which were similar to the corresponding controls. However, the expression of AK5 decreased remarkably in epileptic patients and experimental rats. Furthermore, immunoprecipitation analysis showed that the interaction of AK5 with copine VI (CPNE6, a brain specific protein) increased in epileptic patients and rat models. Our results are the first to indicate that the expression of AK5 in epileptic brain tissue may play important roles in epilepsy, especially refractory epilepsy. PMID:27288770

  1. Phylogenetic relationships of 18 passerines based on Adenylate Kinase Intron 5 sequences

    GUO Hui-yan; YU Hui-xin; BAI Su-ying; MA Yu-kun

    2008-01-01

    The 18 species of bird studied originally are known to belong to muscicapids, robins and sylviids of passerines, but some disputations are always present in their classification systems. In this experiment, phylogenetic relationships of 18 species of passerines were studied using Adenylate Kinase Intron 5 (AK5) sequences and DNA techniques. Through sequences analysis in comparison with each other, phylogenetic tree figures of 18 species of passerines were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods . The results showed that sylviids should be listed as an independent family, while robins and flycatchers should be listed into Muscicapidae. Since the phylogenetic relationships between long-tailed tits and old world warblers are closer than that between long-tailed tits and parids, the long-tailed tits should be independent of paridae and be categorized into aegithalidae. Muscicapidae and Paridae are known to be two monophylitic families, but Sylviidae is not a monophyletic group. AK5 sequences had better efficacy in resolving close relationships of interspecies among intrageneric groups.

  2. Oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes in various diseases.

    Fujii, N; Kotake, S.; Hirose, S; Ohno, S; Yasuda, I.; Sagawa, A; Ishikawa, K.; Minagawa, T

    1984-01-01

    Interferon induces oligo-2',5'-adenylate synthetase in cells. In various diseases, interferon was detectable in the circulation or was produced spontaneously from peripheral blood mononuclear leukocytes. The oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes was examined in various diseases, including systemic lupus erythematosus, sarcoidosis, Vogt-Koyanagi-Harada disease, and Behcet's disease. The activity of this enzyme was significantly increased in system...

  3. An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells.

    Schulze, D.U.; Dufer, M.; Wieringa, B.; Krippeit-Drews, P.; Drews, G.

    2007-01-01

    AIMS/HYPOTHESIS: In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic intera

  4. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: structural and functional studies.

    Drakou, Christina E; Malekkou, Anna; Hayes, Joseph M; Lederer, Carsten W; Leonidas, Demetres D; Oikonomakos, Nikos G; Lamond, Angus I; Santama, Niovi; Zographos, Spyros E

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells. PMID:22038794

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

    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, [125I]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 [125I]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

  6. A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

    Nathan J. Lanning

    2014-05-01

    Full Text Available Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4 that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

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

    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 [125I]iodocyanopindolol. Binding sites had the characteristics of mixed β1- and β2-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 β1-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

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

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. (Veterans Administration Medical Center, Los Angeles, CA (USA) Univ. of California, Los Angeles (USA))

    1987-12-01

    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 {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 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 {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-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 {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  9. Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

    Bouhss, A; Krin, E; Munier, H; Gilles, A M; Danchin, A; Glaser, P; Bârzu, O

    1993-01-25

    The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme. PMID:8420945

  10. Glucose Repression of Fbp1 Transcription in Schizosaccharomyces Pombe Is Partially Regulated by Adenylate Cyclase Activation by a G Protein α Subunit Encoded by Gpa2 (Git8)

    Nocero, M.; Isshiki, T.; Yamamoto, M.; Hoffman, C. S.

    1994-01-01

    In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, a...

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

    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.

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

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

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

    Koichi Tanda; Norihito Shintani; Akemichi Baba; Hitoshi Hashimoto; Tsuyoshi Miyakawa

    2012-01-01

    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 backcrosse...

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

    Hass, Mathias A. S.; Liu, Wei-Min [Leiden University, Leiden Institute of Chemistry (Netherlands); Agafonov, Roman V.; Otten, Renee; Phung, Lien A. [Brandeis University, Department of Biochemistry, Howard Hughes Medical Institute (United States); Schilder, Jesika T. [Leiden University, Leiden Institute of Chemistry (Netherlands); Kern, Dorothee [Brandeis University, Department of Biochemistry, Howard Hughes Medical Institute (United States); Ubbink, Marcellus, E-mail: m.ubbink@chem.leidenuniv.nl [Leiden University, Leiden Institute of Chemistry (Netherlands)

    2015-02-15

    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.

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

    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

  16. Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, local binding energy, and the catalytic mechanism?

    Adenylyl (β,γ-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the β,γ-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (τc) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the τc values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. The authors have found that the Ki values for the mixture, the Δ isomer, and the Λ isomer of CrATP are 16, 11, and 20 μM, respectively, which suggest that ground-state binding by AK is stereochemically permissive. The results of both problems fully support the conclusion that the phosphonate chain of AK-MgAMPPCP possesses considerable local mobility and illuminate the relationship between the dynamics of bound substrates and the catalytic mechanism

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

    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)

  18. Pituitary adenylate cyclase activating peptide (PACAP participates in adipogenesis by activating ERK signaling pathway.

    Tatjana Arsenijevic

    Full Text Available Pituitary adenylate cyclase activating peptide (PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2, strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ.

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

    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.

  20. Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase.

    Ratner, V; Amir, D; Kahana, E; Haas, E

    2005-09-23

    The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo

  1. 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

    Adenylate kinase (AK) from D. gigas was purified and crystallized in three different metal-bound forms: Zn2+–AK, Co2+–AK and Fe2+–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. Zn2+–AK and Fe2+–AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+–AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+–AK and Fe2+–AK forms and a dimer was present for the Co2+–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

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

    The incorporation of [14C] 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.)

  3. Activation of oocyte phosphatidylinositol kinase by polyamines

    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 [32ATP]. 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 H1 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

  4. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of [125]Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10-5 M) suggesting predominate beta2-type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta-2-type BAR coupled to adenylate cyclase in rat brown fat

  5. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

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

    1986-03-01

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of (/sup 125/)Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10/sup -5/ M) suggesting predominate beta/sub 2/-type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta-/sub 2/-type BAR coupled to adenylate cyclase in rat brown fat.

  6. BIOTIC STRESS IMPACT ON ACTIVITY OF VARIOUS FORMS OF ADENYLATE CYCLASE IN ORGANELLES OF POTATO PLANT CELLS

    Lomovatskaya L.A.

    2006-12-01

    Full Text Available Notwithstanding significant interest towards study of adenylate cyclase plant signal system, there is still no complete picture of functioning and regulation mechanisms of this signal system in plants under biotic stress. With this in view, our study was aimed at identification of various forms of adenylate cyclase (transmembrane and “soluble” in the nucleus and chloroplasts of potato cells and modulation of their activity under the impact of exopolysaсcharides ofpotato ring rot pathogen. The investigations conducted allowed to conclude that two forms of adenylate cyclase function in nuclei and chloroplasts of potato plants: transmembrane and “soluble”. Activity of these forms of the enzyme extracted from plant cells of the two potato varieties contrasted by resistance to potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus, changed in the reverse manner with the mediated impact of exopolysaсcharides secreted by virulent and mucinous strain of bacterial pathogen: in the plants of resistant сultivar it increased, in the plants of sensitive сultivar it was oppressed. It was concluded that activity of both forms of adenylate cyclase directly depended on the degree of resistance of a particular potato variety to given pathogen.

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

    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.

  8. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  9. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  10. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

    Zhang, Wenjun; Heemstra, John R.; Walsh, Christopher T.; Imker, Heidi J.

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated n...

  11. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    Mounira Tlili

    2015-01-01

    Full Text Available The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP, we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC and cytokines (IL-1α and TNF-α in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders.

  12. Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

    Vaudry, David; Rousselle, Cécile; Basille, Magali; Falluel-Morel, Anthony; Pamantung, Tommy F.; Fontaine, Marc; Fournier, Alain; Vaudry, Hubert; Gonzalez, Bruno J

    2002-01-01

    Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoke...

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

    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 [3H]-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 [3H]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

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

    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

  15. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

    Zhang, Wenjun; Heemstra, John R.; Walsh, Christopher T.; Imker, Heidi J.

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

  16. Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

    Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

    2010-11-23

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

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

    Hurley, Matthew M; Maunze, Brian; Block, Megan E; Frenkel, Mogen M; Reilly, Michael J; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    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. PMID:27597817

  18. Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

    Adrenalectomy caused a large increase in the number of β-adrenergic binding sites on liver plasma membranes as measured by 125I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for 3H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in β-adrenergic mediated action was much less than what may be expected as a result of the increase in the β-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 μM) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory α2-adrenergic receptors in adrenalectomy is responsible for the muted β-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 μM), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The α-adrenergic antagonists had no significant effect on the β-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the β-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of α-adrenergic receptors

  19. The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation.

    Landry, S; Pettit, M T; Apolinario, E; Hoffman, C. S.

    2000-01-01

    Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gbeta subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway. While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet...

  20. Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

    Ono, Junichi [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); Takada, Shoji [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan); Saito, Shinji, E-mail: shinji@ims.ac.jp [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); The Graduate University for Advanced Studies, Okazaki 444-8585 (Japan)

    2015-06-07

    An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.

  1. Examination of the relationship of substrate dynamics to enzymic structure, binding energy, and catalysis: NMR studies of adenosine 5'-triphosphate and adenylate kinase

    By measuring the deuterium NMR-relaxation rates of adenylyl (β, λ-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring [8-2H]AMPPCP and upon the phosphonate chain (AMPPCD2P) free in solution and bound to the MgATP site of adenylate kinases (AK) the local motional dynamics of AMPPCP and MgAMPPCP in the two environments were established. The analysis of the experimental data involved the rigorous experimental verification that the systems studied were in the fast exchange limit on an NMR timescale. In addition analysis required careful examination of the equations describing quadrupolar relaxation, particularly the spectral density equations which contain information on molecular motion. Having determined the local dynamics of the nucleotides and their complexes with Mg + 2 free in solution and bound to AK and observing that MgAMPPCP is an excellent model for the natural substrate of AK, MgATP, we examined the relationship of local substrate dynamics to enzyme structure, binding energy, and catalysis

  2. Fast closure of N-terminal long loops but slow formation of β strands precedes the folding transition state of Escherichia coli adenylate kinase.

    Orevi, Tomer; Ben Ishay, Eldad; Gershanov, Sivan Levin; Dalak, Mayan Ben; Amir, Dan; Haas, Elisha

    2014-05-20

    The nature of the earliest steps of the initiation of the folding pathway of globular proteins is still controversial. To elucidate the role of early closure of long loop structures in the folding transition, we studied the folding kinetics of subdomain structures in Escherichia coli adenylate kinase (AK) using Förster type resonance excitation energy transfer (FRET)-based methods. The overall folding rate of the AK molecule and of several segments that form native β strands is 0.5 ± 0.3 s(-1), in sharp contrast to the 1000-fold faster closure of three long loop structures in the CORE domain. A FRET-based "double kinetics" analysis revealed complex transient changes in the initially closed N-terminal loop structure that then opens and closes again at the end of the folding pathway. The study of subdomain folding in situ suggests a hierarchic ordered folding mechanism, in which early and rapid cross-linking by hydrophobic loop closure provides structural stabilization at the initiation of the folding pathway. PMID:24787383

  3. 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.

    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.

  4. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes.

    Ahmad, Jawid Nazir; Cerny, Ondrej; Linhartova, Irena; Masin, Jiri; Osicka, Radim; Sebo, Peter

    2016-03-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host. PMID:26334669

  5. Degradation of Activated Protein Kinases by Ubiquitination

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  6. Event Detection and Sub-state Discovery from Bio-molecular Simulations Using Higher-Order Statistics: Application To Enzyme Adenylate Kinase

    Ramanathan, Arvind; Savol, Andrej J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

    2012-01-01

    Biomolecular simulations at milli-second and longer timescales can provide vital insights into functional mechanisms. Since post-simulation analyses of such large trajectory data-sets can be a limiting factor in obtaining biological insights, there is an emerging need to identify key dynamical events and relating these events to the biological function online, that is, as simulations are progressing. Recently, we have introduced a novel computational technique, quasi-anharmonic analysis (QAA) (PLoS One 6(1): e15827), for partitioning the conformational landscape into a hierarchy of functionally relevant sub-states. The unique capabilities of QAA are enabled by exploiting anharmonicity in the form of fourth-order statistics for characterizing atomic fluctuations. In this paper, we extend QAA for analyzing long time-scale simulations online. In particular, we present HOST4MD - a higher-order statistical toolbox for molecular dynamics simulations, which (1) identifies key dynamical events as simulations are in progress, (2) explores potential sub-states and (3) identifies conformational transitions that enable the protein to access those sub-states. We demonstrate HOST4MD on micro-second time-scale simulations of the enzyme adenylate kinase in its apo state. HOST4MD identifies several conformational events in these simulations, revealing how the intrinsic coupling between the three sub-domains (LID, CORE and NMP) changes during the simulations. Further, it also identifies an inherent asymmetry in the opening/closing of the two binding sites. We anticipate HOST4MD will provide a powerful and extensible framework for detecting biophysically relevant conformational coordinates from long time-scale simulations. PMID:22733562

  7. Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

    Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 104, increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. (iv) Qualitative comparison of the NOESY cross peaks between aliphatic side chains and aromatic protons indicates that the patterns are almost identical between free WT and free R138K. (v) The above kinetic and structural results led to the conclusion that Arg-138 stabilizes the ternary complexes by 1.4-1.8 kcal/mol and stabilizes the transition state by at least 7 kcal/mol and that the critical functional role of Arg-138 cannot be replaced by lysine. (vi) Since Arg-138 is distant from the substrate sites proposed from previous NMR studies serious revision will be required for this model

  8. A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Suzuki, Takehiro; Dohmae, Naoshi; Kakeya, Hideaki

    2015-12-01

    Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments. PMID:26467472

  9. Molecular cloning and amplification of the adenylate cyclase gene.

    Wang, J Y; Clegg, D O; Koshland, D E

    1981-01-01

    A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage lambda gt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight 81,000 that gives rise to adenylate cyclase activity. This protein represents a functional mutant of the ...

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

    Tamás Juhász; Eszter Szentléleky; Csilla Szűcs Somogyi; Roland Takács; Nóra Dobrosi; Máté Engler; Andrea Tamás; Dóra Reglődi; Róza Zákány

    2015-01-01

    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 cho...

  11. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  12. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems.

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-07-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexia produced by PACAP in the central nucleus of the amygdala (CeA), a limbic structure implicated in the emotional components of ingestive behavior. Male rats were microinfused with PACAP (0-1 μg per rat) into the CeA and home-cage food intake, body weight change, microstructural analysis of food intake, and locomotor activity were assessed. Intra-CeA (but not intra-basolateral amygdala) PACAP dose-dependently induced anorexia and body weight loss without affecting locomotor activity. PACAP-treated rats ate smaller meals of normal duration, revealing that PACAP slowed feeding within meals by decreasing the regularity and maintenance of feeding from pellet-to-pellet; postprandial satiety was unaffected. Intra-CeA PACAP-induced anorexia was blocked by coinfusion of either the melanocortin receptor 3/4 antagonist SHU 9119 or the tyrosine kinase B (TrKB) inhibitor k-252a, but not the CRF receptor antagonist D-Phe-CRF(12-41). These results indicate that the CeA is one of the brain areas through which the PACAP system promotes anorexia and that PACAP preferentially lessens the maintenance of feeding in rats, effects opposite to those of palatable food. We also demonstrate that PACAP in the CeA exerts its anorectic effects via local melanocortin and the TrKB systems, and independently from CRF. PMID:25649277

  13. Regulation of brain adenylate cyclase by calmodulin

    This thesis examined the interaction between the Ca2+-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-[125I]-CaM-diazopyruvamide (125I-CAM-DAP) behaved like native CaM with respect to Ca2+-enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca2+-dependent stimulation of adenylate cyclase. 125I-CaM-DAP cross-linked to CaM-binding proteins in a Ca2+-dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

  14. Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1

    Frödin, M; Sekine, N; Roche, E; Filloux, C; Prentki, M; Wollheim, C B; Van Obberghen, E

    1995-01-01

    of this kinase is not sufficient for secretion. In the presence of glucose, however, nerve growth factor potentiated insulin secretion. In INS-1 cells, activation of 44-kDa MAP kinase was partially correlated with the induction of early response genes junB, nur77, and zif268 but not with stimulation......The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways...... glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK...

  15. Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe

    Susan M. Byrne; Hoffman, Charles S.

    1993-01-01

    An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produ...

  16. The influence of low-level radiation and gangliosides on adenylate cyclase activity in thymus and thyroid glands of chicks in ontogenesis

    Adenylate cyclase (AC) activity was studied in thymus and thyroid gland of intact chick embryos and those irradiated with a dose of 0.029 Gy prior to incubation, and newly hatched chocks in the presence of total ganglioside fractions extracted from the same organs. Gangliosides were shown to increase the enzyme activity of thymocytes and thyreocytes during the postnatal development. It is suggested that small radiation doses potentiate the stimulatory effect of ganglioside fractions on AC

  17. Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice

    Rat, Dorothea; Schmitt, Ulrich; Tippmann, Frank; Dewachter, Ilse; Theunis, Clara; Wieczerzak, Ewa; Postina, Rolf; Van Leuven, Fred Van; Fahrenholz, Falk; Kojro, Elzbieta

    2011-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent alpha-secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimer's disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long-term intranasal da...

  18. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

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

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

  19. The Role of Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptide in the Neural Pathways Controlling the Lower Urinary Tract

    Yoshiyama, Mitsuharu; de Groat, William C.

    2008-01-01

    Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in the neural pathways regulating the lower urinary tract. VIP-immunoreactivity (IR) is present in afferent and autonomic efferent neurons innervating the bladder and urethra, whereas PACAP-IR is present primarily in afferent neurons. Exogenously applied VIP relaxes bladder and urethral smooth muscle and excites parasympathetic neurons in bladder ganglia. PACAP relaxes bladder ...

  20. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-01-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexi...

  1. Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient Mice

    Otto, Christiane; Kovalchuk, Yury; Wolfer, David Paul; Gass, Peter; Mart??n, Miguel; Zuschratter, Werner; Gr??ne, Hermann Josef; Kellendonk, Christoph; Tronche, Fran??ois; Maldonado, Rafael; Lipp, Hans-Peter; Konnerth, Arthur; Sch??tz, G??nter

    2001-01-01

    The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, har...

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

    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.

  3. {beta}-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure

    Chang, Huoy-Rou [I-Shou University, Department of Biomedical Engineering, Dashu Shiang, Kaohsiung County (Taiwan); Tsao, Der-An [Fooyin University of Technology, Department of Medical Technology (Taiwan); Yu, Hsin-Su [Taiwan University, Department of Dermatology, College of Medicine (Taiwan); Ho, Chi-Kung [Kaohsiung Medical University, Occupational Medicine (Taiwan); Kaohsiung Medical University, Graduate Institute of Medicine, Research Center for Occupational Disease (Taiwan)

    2005-01-01

    To understanding the reversible or irreversible harm to the {beta}-adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain {beta}-adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and {beta}-adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of {beta}-adrenergic receptor using the ligand [{sup 125}I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain {beta}-adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the {beta}-adrenergic system of the brain is a partly reversible condition. (orig.)

  4. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity. PMID:26851652

  5. Mitogen-activated protein kinases in atherosclerosis

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  6. [Mitogen-activated protein kinases in atherosclerosis].

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  7. Protein kinase A activity is associated with metacyclogenesis in Leishmania amazonensis.

    Genestra, Marcelo; Cysne-Finkelstein, Léa; Leon, Leonor

    2004-01-01

    Because of the importance of cell signalling processes in proliferation and differentiation, the adenylate cyclase pathway was studied, specifically the protein kinase A (PKA) in Leishmania amazonensis. The PKAs of soluble (SF) and enriched membrane fractions (MF) from infective/non-infective promastigotes and axenic amastigotes were assayed. In order to purify the PKA molecule, fractions were chromatographed on DEAE-cellulose columns and the phosphorylative activity was evaluated using [gamma(32)P]-ATP as the phosphate source. These experiments were performed in the presence of cyclic adenosine monophosphate (cAMP) and an inhibitor of PKA. Our data demonstrated that the PKA activity was significantly higher (about two times) in SF from promastigotes with a high concentration of metacyclic forms, when compared with the non-infective promastigotes, suggesting an association of this activity and the metacyclogenesis process. A discrete phosphorylative activity in axenic amastigotes was observed. As the adenylate cyclase/cAMP pathway would be involved in the parasite-host interiorization, the PKA activity may constitute a good intracellular target for studies of leishmanicidal drugs. PMID:15338471

  8. Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

    Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

    2015-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

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

    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

  10. [Dependence of creatine kinase and glycogen synthetase activities of skeletal muscles on state of adenine nucleotide phosphorylation and cAMP metabolism].

    Iakovlev, N N; Chagovets, N R; Maksimova, L V

    1980-01-01

    Changes in the contents of adenine nucleotides, creatine phosphate, inorganic phosphate, creatine, glucose-6-phosphate and glycogen and the activity of adenylate cyclase, creatine kinase, glycogen phosphorylase 31:51-AMP-phosphodiesterase and glycogen synthetase in muscles and of blood catecholamines were studied in adult rats before loading, immediately after the cessation of the muscular activity, and at rest. Adenine nucleotides are established to play a regulatory role in catabolic and anabolic processes nucleotides are established to play a regulatory role in catabolic and anabolic processes related to the muscular activity. It is established that compensation and supercompensation of the working losses of muscular creatine phosphate and glycogen are due to activation of anabolic processes under conditions of higher phosphorylation of the adenylic system. PMID:6247797

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

    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 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ 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 Ca2+ 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 Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating

  12. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

  13. Urinary cyclic adenosine 3',5'-monophosphate response in McCune-Albright syndrome: clinical evidence for altered renal adenylate cyclase activity.

    Zung, A; Chalew, S A; Schwindinger, W F; Levine, M A; Phillip, M; Jara, A; Counts, D R; Kowarski, A A

    1995-12-01

    The recent finding of an activating mutation in the Gs alpha protein, the protein that couples receptors to stimulation of adenylate cyclase, from endocrine and nonendocrine tissues of patients with McCune-Albright syndrome (MAS) suggests that alterations in adenylate cyclase activity may account for the clinical abnormalities in these patients. Many patients with MAS have hypophosphatemia. This may result from the presence of the activating Gs alpha mutation in proximal renal tubules or the elaboration of a phosphaturic factor from fibrous dysplasia. We, therefore, sought to characterize renal cAMP generation and phosphate handling in MAS patients. Intravenous infusion of PTH is a classic clinical test used to evaluate hormonal responsiveness of renal proximal tubule adenylate cyclase and examine PTH-dependent phosphate clearance. We performed PTH infusion in 6 MAS patients, 10 normal subjects, and 6 patients with pseudohypoparathyroidism (PHP). The basal urinary cAMP (UcAMP) level in the MAS group [5.5 +/- 2.6 nmol/dL glomerular filtration (GF)] was elevated (P PHP (1.9 +/- 0.6 nmol/dL GF). However, PTH-stimulated peak UcAMP (15.0 +/- 7.0 nmol/dL GF) and the peak/basal UcAMP ratio (3.1 +/- 1.7) in MAS were significantly lower than the respective values in normal subjects (30.8 +/- 16.9 nmol/dL GF and 9.3 +/- 2.9; P PHP (respectively, 3.1 +/- 1.5 nmol/dL GF and 2.0 +/- 1.7). By contrast, the PTH-induced phosphaturic response in MAS patients was similar to that in the normal subjects. Our study provides clinical evidence that MAS patients have altered renal adenylate cyclase activity, manifested by an elevated basal UcAMP, but a blunted UcAMP response to PTH stimulation. These observations are presumably due to a mutation in the Gs alpha protein in the renal tubules. Despite the blunted UcAMP excretion, the phosphaturic response to PTH in MAS patients is intact. PMID:8530601

  14. Molecular Imaging of the ATM Kinase Activity

    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

  15. Molecular Imaging of the ATM Kinase Activity

    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.

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

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

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses 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...... remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction....

  17. Janus Activated Kinase inhibition in Myelofibrosis

    H Malhotra

    2012-01-01

    Full Text Available Janus Activated Kinase (JAK 2 plays an important role in the pathogenesis of myelofibrosis (MF. Ruxolitinib (INCB018424, Jakafi is a potent dual JAK1 and JAK2 inhibitor. In November 2011, it became approved by the US FDA for the treatment of intermediate or high-risk MF. This review shall outline the role of Ruxolitinib in the current management of MF and its potential future.

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

    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.

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

    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: ► Adenylyl cyclase nPAC in aqueous pH 7.5 buffer dissolved only to nano-clusters. ► Nano-cluster size was determined by light attenuation (scattering) measurements. ► The size of the nano-clusters was growing by coalescing during observation period. ► In nPAC nano-clusters color centers were present in emission range of 360–900 nm. ► 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.

  20. Creatine kinase activity is associated with blood pressure

    L.M. Brewster; G. Mairuhu; N.R. Bindraban; R.P. Koopmans; J.F. Clark; G.A. van Montfrans

    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 cardiovascula

  1. Evaluation of Kinase Activity Profiling Using Chemical Proteomics.

    Ruprecht, Benjamin; Zecha, Jana; Heinzlmeir, Stephanie; Médard, Guillaume; Lemeer, Simone; Kuster, Bernhard

    2015-12-18

    Protein kinases are important mediators of intracellular signaling and are reversibly activated by phosphorylation. Immobilized kinase inhibitors can be used to enrich these often low-abundance proteins, to identify targets of kinase inhibitors, or to probe their selectivity. It has been suggested that the binding of kinases to affinity beads reflects a kinase's activation status, a concept that is under considerable debate. To assess the merits of the idea, we performed a series of experiments including quantitative phosphoproteomics and purification of kinases by single or mixed affinity matrices from signaling activated or resting cancer cells. The data show that mixed affinity beads largely bind kinases independent of their activation status, and experiments using individual immobilized kinase inhibitors show mixed results in terms of preference for binding the active or inactive conformation. Taken together, activity- or conformation-dependent binding to such affinity resins depends (i) on the kinase, (ii) on the affinity probe, and (iii) on the activation status of the lysate or cell. As a result, great caution should be exercised when inferring kinase activity from such binding data. The results also suggest that assaying kinase activity using binding data is restricted to a limited number of well-chosen cases. PMID:26378887

  2. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  3. A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.

    Lawrence, Anthony J; Coote, John G; Kazi, Yasmin F; Lawrence, Paul D; MacDonald-Fyall, Julia; Orr, Barbara M; Parton, Roger; Riehle, Mathis; Sinclair, James; Young, John; Price, Nicholas C

    2002-06-21

    Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. PMID:11934879

  4. Uridylation and adenylation of RNAs.

    Song, JianBo; Song, Jun; Mo, BeiXin; Chen, XueMei

    2015-11-01

    The posttranscriptional addition of nontemplated nucleotides to the 3' ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3' ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. PMID:26563174

  5. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

    GilLevkowitz

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  6. Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress

    Shuang Liang; Renbin Huang; Xing Lin; Jianchun Huang; Zhongshi Huang; Huagang Liu

    2012-01-01

    The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression were significantly elevated, and depression-like behaviors were improved. Open-field and novelty-suppressed feeding tests showed that mouse activity levels were increased and feeding latency was shortened following treatment. Our results indicate that YLSPS inhibits depression by upregulating monoamine neurotransmitters, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression.

  7. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2003-01-01

    contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the...

  8. High quality, small molecule-activity datasets for kinase research.

    Sharma, Rajan; Schürer, Stephan C; Muskal, Steven M

    2016-01-01

    Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR) data. Bioactivity databases such as the Kinase Knowledgebase (KKB), WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note. PMID:27429748

  9. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

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

    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.

  11. Mitogen-activated protein kinase signaling in plants

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

    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.

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

    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

  14. Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress☆

    Liang, Shuang; Huang, Renbin; Lin, Xing; Huang, Jianchun; Huang, Zhongshi; Liu, Huagang

    2012-01-01

    The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity ...

  15. [Determination of riboflavin kinase activity in yeast].

    Shavlovsky, G M; Kashchenko, V E

    1975-01-01

    It is established that the main reason of the riboflavin kinase (RFK, EC 2.7.1.26) low specific activity in the cell-free extracts of the yeast Pichia guillermondii Wickerham ATCC 9058 is the presence of alkaline phosphatase (EC 3.1.3.1), effectively destructing flaven mononucleotide. By chromatography of the cell-free extracts of P. guillermondii on DEAE-Sephadex A-50, CM-Sphadex C-50, CM-cellulose, Sephadexes G-75 and G-100 RFK and alkaline phosphatase may be separated completely. Any of these procedures results in a several times increase of the RFK activity as compared with the initial preparation. One failed to obtain a similar effect by fractionation of the extracts with amminium sulphate and by hydroxylapatite chromatography. A simple method is developed for determining the activity of RFK in the cell-free extracts of yeast on the basis of negative adsorption of this enzyme on DEAE-Sephadex A-50. A selective inhibition of alkaline phosphatase by ions Be2+ and F- yields a less satisfactory result. The data are presented on the PFK activity of certain species of flavinogenic (Pichia guillermondii, Torulopsis camdida) and non-flavinogenic (Pichia ohmeri, Candida utilis, Saccharomyces cervisiae) yeast. PMID:174262

  16. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  17. Pituitary Adenylate Cyclase Activating Peptide (1-38 and its analog (Acetyl-[Ala15, Ala20] PACAP 38-polyamide reverse methacholine airway hyperresponsiveness in rats

    Mounira Tlili

    2015-09-01

    Full Text Available The aim of this study was to investigate both functionally and structurally bronchodilator effects of Pituitary adenylate cyclase activating peptide (PACAP38 and acetyl-[Ala15, Ala20] PACAP38-polyamide, a potent PACAP38 analog, in rats challenged by methacholine (MeCh. Male Wistar rats were divided randomly into five groups. Groups 1 and 2 inhaled respectively aerosols of saline or increasing doses of MeCh (0.5, 1, 2.12, 4.25, 8.5, 17, 34 and 68mg/L. The other groups received terbutaline (Terb (250 µg/rat (10-6 M, PACAP38 (50 µg/rat (0.1 mM or PACAP38 analog (50 µg/rat associated to MeCh from the dose of 4.25 mg/L. Total lung resistances (RL were recorded before and 2 min after MeCh administration by pneumomultitest equipment. MeCh administration induced a significant and a dose-dependent increase (p<0.05 of RL compared to control rats. Terb, PACAP38 and PACAP38 analog reversed significantly the MeCh-induced bronchial constriction, smooth muscle (SM layer thickness and bronchial lumen mucus abundance. PACAP38 analog prevents effectively bronchial smooth muscle layer thickness, mucus hypersecretion and lumen decrease. Therefore, it may constitute a potent therapeutic bronchodilator.

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

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

    2016-01-01

    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. PMID:26925806

  19. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

    Egri, P; Fekete, C; Dénes, Á; Reglődi, D; Hashimoto, H; Fülöp, B D; Gereben, Balázs

    2016-06-01

    The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes. PMID:27046436

  20. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Bernhard Roppenser

    Full Text Available SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4 P2/PI(3-5 P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4 P2/PI(3-5 P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  1. Structural and functional identification of the pituitary adenylate cyclase-activating polypeptide receptor VPAC2 from the frog Rana tigrina rugulosa.

    Hoo, R L; Alexandre, D; Chan, S M; Anouar, Y; Pang, R T; Vaudry, H; Chow, B K

    2001-10-01

    Recently, a frog pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor (fPVR) has been characterized, and interestingly, this receptor exhibits characteristics of both mammalian PACAP type II receptors VPAC(1)R and VPAC(2)R. In order to investigate the receptors responsible for mediating the actions of VIP and PACAP in amphibians, in this report, a frog VPAC(2) receptor (fVPAC(2)R) cDNA was isolated. fVPAC(2)R shares 47.7, 46.9 and 62.5% amino acid sequence identity with fPVR, human VPAC(1)R and human VPAC(2)R respectively. Functionally, fVPAC(2)R, when expressed in CHO cells, was responsive to both frog peptides including VIP, PACAP38 and PACAP27 where the EC(50) values of these peptides in intracellular cAMP production were 0.15, 0.18 and 0.16 microM respectively. The pharmacological profiles of human peptides (VIP, PACAP38 and peptide histidine methionine) to stimulate frog and human VPAC(2)Rs were compared, and it was found that these peptides could only activate the frog receptor at micromolar concentrations. fVPAC(2)R was found to be widely distributed in various peripheral tissues as well as several regions of the brain. The presence of the receptor transcripts suggests the functional roles of the receptor in mediating the actions of PACAP and/or VIP in these tissues. As VIP and particularly PACAP27 are highly conserved peptides in vertebrate evolution, comparative studies of these peptides and their receptors in non-mammalian vertebrates should provide clues to better understand the physiology of these important peptides in human and other vertebrates. PMID:11564605

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

    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

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

    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

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

    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

  5. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title Receptor tyrosine kinases

  6. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  7. Enhanced casein kinase II activity in human tumour cell cultures

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...... and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated....

  8. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    Sandrini, Michael; Shannon, O.; Clausen, A.R.;

    2007-01-01

    kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug...... gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising...

  9. Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos

    Testerink, C.; Vennik, M.; Kijne, J.W.; Wang, M.; Heimovaara-Dijkstra, S.

    2000-01-01

    We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti-MAPK (mitogen-activated protein kinase) antib

  10. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4IRAK-1-K239S). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4IRAK-1-K239S cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate

  11. A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion

    Yi Ma; Tianjie Luo; Wenna Xu; Zulu Ye; An Hong

    2012-01-01

    The recombinant peptide,DBAYL,a promising therapeutic peptide for type 2 diabetes,is a new,potent,and highly selective agonist for VPAC2 generated through sitedirected mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP),vasoactive intestinal peptide (VIP),and related analogs.The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization.As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 I of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro.The bioactivity assay of DBAYL showed that it displaced [125I]PACAP38 and [125I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 ± 6.9 and 47.1 ± 4.9 nM,respectively,which were significantly lower than that of BAY55-9837,one established VPAC2 agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC5o) of 0.68 nM,whereas the receptor potency of DBAYL at human VPAC1 (ECso of 737 nM) was only 1/1083of that at human VPAC2,and DBAYL had no activity toward human PAC1 receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin receptor substrate 1 (IRS-1) and glucose transporter 4(GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes.Compared with BAY55-9837 and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice.These results suggested that DBAYL could efficiently improve glucose uptake and glucose-dependent insulin

  12. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    Ngo, HT; Pham, Long; Kim, JW;

    2013-01-01

    , approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...

  13. Conservation, variability and the modeling of active protein kinases.

    James D R Knight

    Full Text Available The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease. Understanding the structural biology of protein kinases in detail, including their commonalities, differences and modes of substrate interaction, is necessary for designing high quality inhibitors that will be of true use for cell biology and disease therapy. To this end, we here report on a structural analysis of all available active-conformation protein kinases, discussing residue conservation, the novel features of such conservation, unique properties of atypical kinases and variability in the context of substrate binding. We also demonstrate how this information can be used for structure prediction. Our findings will be of use not only in understanding protein kinase function and evolution, but they highlight the flaws inherent in kinase drug design as commonly practiced and dictate an appropriate strategy for the sophisticated design of specific inhibitors for use in the laboratory and disease therapy.

  14. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-05-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

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

    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

  16. Is kinase activity essential for biological functions of BRI1?

    Weihui Xu; Juan Huang; Baohua Li; Jiayang Li; Yonghong Wang

    2008-01-01

    Brassinosteroids (BRs) are a major group of plant hormones that regulate plant growth and development. BRI1, a protein localized to the plasma membrane, functions as a BR receptor and it has been proposed that its kinase activity has an essential role in BR-regulated plant growth and development. Here we report the isolation and molecular characterization of a new allele of bril, bril-301, which shows moderate morphological phenotypes and a reduced response to BRs under normal growth conditions. Sequence analysis identified a two-base alteration from GG to AT, resulting in a conversion of 989G to 9891 in the BRI1 kinase domain. An in vitro assay of kinase activity showed that bril-301 has no detectable autophosphorylation activity or phosphorylation activity towards the BRI1 substrates TTL and BAK1. Furthermore, our results suggest that bril-301, even with extremely impaired kinase activity, still retains partial function in regulating plant growth and development, which raises the question of whether BRI1 kinase activity is essential for BR-mediated growth and development in higher plants.

  17. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    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.

  18. The cystathionine-β-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels.

    Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian; Chatterjee, Abhishek; Ait-Tihyaty, Maria; Ullman, Buddy; Jardim, Armando

    2016-06-01

    The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools. PMID:26853689

  19. The molecular regulation of Janus kinase (JAK) activation.

    Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M; Nicola, Nicos A; Varghese, Leila N

    2014-08-15

    The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  20. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

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

    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

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

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

  3. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  4. Overinhibition of Mitogen-Activated Protein Kinase Inducing Tau Hyperphosphorylation

    LI Hong-lian; CHEN Juan; LIU Shi-jie; ZHANG Jia-yu; WANG Qun; WANG Jian-zhi

    2005-01-01

    To reveal the relationship between mitogen-activated protein kinase (MAPK) and tau phosphorylation, we used different concentration of PD98059, an inhibitor of MEK (MAPK kinase), to treat mice neuroblastma (N2a) cell line for 6 h. It showed that the activity of MAPK decreased in a dose-dependent manner. But Western blot and immunofluorescence revealed that just when the cells were treated with 16 μmol/L PD98059, tau was hyperphosphorylated at Ser396/404 and Ser199/202 sites. We obtained the conclusion that overinhibited MAPK induced tau hyperphosphorylation at Ser396/404 and Ser199/202 sites.

  5. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

    Pinkosky, Stephen L; Filippov, Sergey; Srivastava, Rai Ajit K; Hanselman, Jeffrey C; Bradshaw, Cheryl D; Hurley, Timothy R; Cramer, Clay T; Spahr, Mark A; Brant, Ashley F; Houghton, Jacob L; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  6. Redox Regulation of the AMP-Activated Protein Kinase

    Yingying Han; Qilong Wang; Ping Song; Yi Zhu; Ming-Hui Zou

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dos...

  7. Mitogen activated protein kinases: a role in inflammatory bowel disease?

    Broom, O J; Widjaya, B; Troelsen, J;

    2009-01-01

    Since their discovery more than 15 years ago, the mitogen activated protein kinases (MAPK) have been implicated in an ever-increasingly diverse array of pathways, including inflammatory signalling cascades. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, are...... their related signalling proteins in influencing the progression of IBD....

  8. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  9. Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

    Hauge, Camilla; Antal, Torben L; Hirschberg, Daniel; Doehn, Ulrik; Thorup, Katrine; Idrissova, Leila; Hansen, Klaus; Jensen, Ole N; Jørgensen, Thomas J; Biondi, Ricardo M; Frödin, Morten

    2007-01-01

    The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We invest...

  10. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  11. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdkl activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chkl and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  12. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

    Sayed, M; Kim, S O; Salh, B S;

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...

  13. Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

    Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field. PMID:23745726

  14. Emerging Roles of AMP-Activated Protein Kinase

    Fritzen, Andreas Mæchel

    The cellular energy sensor AMP-activated protein kinase (AMPK) is activated, when the energy balance of the cell decreases. AMPK has been proposed to regulate multiple metabolic processes. However, much of the evidence for these general effects of AMPK relies on investigations in cell systems or...... exercise appears to inhibit pyruvate dehydrogenase (PDH) activity by an immediate up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) protein content. Consequently, this may inhibit glucose oxidation and thereby generate conditions for increased FA oxidation and glycogen resynthesis in skeletal muscle...... importance for prioritising energy dissipation, inhibition of lipid storage pathways and regulation of mitochondrial and metabolic proteins, but this needs further investigations. In addition, we provide evidence that AMPK is regulating autophagic signalling in skeletal muscle. Thus, in skeletal muscle AMPK...

  15. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  16. Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia.

    Junji Kawashima

    Full Text Available Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+/Calmodulin-dependent protein kinase kinase (CaMKK, which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO, a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV in rats 30 min before 2DG ICV injection (40 µmol to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.

  17. Creatine kinase activity in dogs with experimentally induced acute inflammation

    Dimitrinka Zapryanova

    2013-01-01

    Full Text Available The main purpose of this study was to investigate the effect of acute inflammation on total creatine kinase (CK activity in dogs. In these animals, CK is an enzyme found predominantly in skeletal muscle and significantly elevated serum activity is largely associated with muscle damage. Plasma increases in dogs are associated with cell membrane leakage and will therefore be seen in any condition associated with muscular inflammation. The study was induced in 15 mongrel male dogs (n=9 in experimental group and n=6 in control group at the age of two years and body weight 12-15 kg. The inflammation was reproduced by inoculation of 2 ml turpentine oil subcutaneously in lumbar region. The plasma activity of creatine kinase was evaluated at 0, 6, 24, 48, 72 hours after inoculation and on days 7, 14 and 21 by a kit from Hospitex Diagnostics. In the experimental group, the plasma concentrations of the CK-activity were increased at the 48th hour (97.48±6.92 U/L and remained significantly higher (p<0.05 at the 72 hour (97.43±2.93 U/L compared to the control group (77.08±5.27 U/L. The results of this study suggest that the evaluation of creatine kinase in dogs with experimentally induced acute inflammation has a limited diagnostic value. It was observed that the creatine kinase activity is slightly affected by the experimentally induced acute inflammation in dogs.

  18. Effects of cadmium on canine renal cortical adenylate cyclase

    The present studies examine the effects of cadmium (Cd2+) on adenylate cyclase activity in basolateral renal cortical membranes from normal dogs. Cd2+, in the dose range of 1 to 200 μM caused a dose-dependent inhibition of adenylate cyclase activity due to competitive inhibition with respect to the allosteric activator Mg2+. In addition, increasing Cd2+ concentrations from 0 to 25 μM resulted in a purely competitive inhibition with respect to ATP. In the absence of other divalent cations Cd2+ was a potent stimulator of basal adenylate cyclase activity, far more potent than the physiological activator of the system Mg2+. It is concluded that Cd2+ behaves as a partial agonist in this system, due to its ability to form a new enzymatic substrate complex: Cd-ATP, which competes with the physiological substrate Mg-ATP at the catalytic site of the enzyme. In addition, Cd2+ in the absence of other divalent cation stimulates basal enzyme activity, presumably through interaction at an additional site, closely related to the allosteric metal regulatory site of this enzyme system

  19. Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

    Ting-Ting Li; Hong Qiao; Hui-Xin Tong; Tian-Wei Zhuang; Tong-Tong Wang

    2016-01-01

    Background:A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) gene for type 2 diabetes mellitus (T2DM) within the German population.Among the variants,five single nucleotide polymorphisms (SNPs) of MAP4K4 (rs1003376,rs11674694,rs2236935,rs2236936,and rs6543087) showed significant association with T2DM or diabetes-related quantitative traits.We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population.Methods:Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects,using the SNPscanTM method.All subjects were recruited from the Second Affiliated Hospital,Harbin Medical University from October 2010 to September 2013.We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis,and the association between the five SNPs and metabolic traits in the subgroups.Results:Of the five variants,SNP rs2236935T/C was significantly associated with T2DM in this study population (odds ratio =1.293;95% confidence interval:1.034-1.619,P =0.025).In addition,among the controls,rs 1003376 was significantly associated with an increased body mass index (P =0.045) and homeostatic model assessment-insulin resistance (P =0.037).Conclusions:MAP4K4 gene is associated with T2DM in a Chinese Han population,and MAP4K4 gene variants may contribute to the risk toward the development of T2DM.

  20. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Xiangpei Kong

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  1. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer;

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

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

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

    2005-01-01

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

  3. A mathematical model of human thymidine kinase 2 activity

    Radivoyevitch, Tom; Munch-Petersen, Birgitte; Wang, Liya;

    2011-01-01

    _ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative cooperati......_ The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ∼0.5 (apparent negative...

  4. Activation and signaling of the p38 MAP kinase pathway

    Tyler ZARUBIN; Jiahuai HAN

    2005-01-01

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

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

    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. PMID:26948880

  6. Training volume, androgen use and serum creatine kinase activity.

    Häkkinen, K; Alén, M

    1989-01-01

    Serum creatine kinase (CK) activities were investigated in elite male strength athletes (n = 20) during normal weight training and bodybuilding training (one training session per day), during high volume strength training (two sessions per day) and during strength training (one session per day) with the use of high dose synthetic androgens (five athletes in each subgroup). The findings demonstrated that the increase in serum CK was highest in the subgroup using androgens. These results sugges...

  7. Mitogen-activated protein kinases in the acute diabetic myocardium

    Strnisková, M.; Barančík, M.; Neckář, Jan; Ravingerová, T.

    2003-01-01

    Roč. 249, 1-2 (2003), s. 59-65. ISSN 0300-8177 R&D Projects: GA MŠk LN00A069 Grant ostatní: VEGA(SK) 2/2063/22 Institutional research plan: CEZ:AV0Z5011922 Keywords : experimental diabetes * ischemia * mitogen-activated protein kinases (MAPK) Subject RIV: ED - Physiology Impact factor: 1.763, year: 2003

  8. Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal†

    Katherine D McMahon; Dojka, Michael A.; Pace, Norman R.; Jenkins, David; Keasling, Jay D.

    2002-01-01

    A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria kn...

  9. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties. PMID:21774788

  10. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol 32P/mol calcineurin. Analysis by SDS-PAGE revealed that only the α subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn2+ or Ni2+ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn2+ or Ni2+ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni2+ was a more potent activator of phosphorylated calcineurin compared to Mn2+. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni2+ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca2+

  11. Subtype activation and interaction of protein kinase C and mitogen-activated protein kinase controlling receptor expression in cerebral arteries and microvessels after subarachnoid hemorrhage

    Ansar, Saema; Edvinsson, Lars

    2008-01-01

    BACKGROUND AND PURPOSE: The pathogenesis of cerebral ischemia associated with subarachnoid hemorrhage (SAH) still remains elusive. The aim of this study was to examine the involvement of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) subtypes in the pathophysiology of cerebral...... ischemia after SAH in cerebral arteries and microvessels and to examine temporal activation of the kinases. We hypothesize that treatment with a MAPK or PKC inhibitor will prevent the SAH-induced kinase activation in brain vessels. METHODS: SAH was induced by injecting 250 microL blood into the......: Among the 8 investigated PKC isoforms, only PKC delta was activated at 1 hour and at 48 hours, whereas PKC alpha was activated at 48 hours after SAH. For the MAPKs, there was early phosphorylation at 1 hour of extracellular signal-regulated kinase 1/2, whereas c-jun N-terminal kinase and p38 showed...

  12. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.J. (Univ. of Miami School of Medicine, FL (USA))

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

  13. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase

  14. Modulation of the protein kinase activity of mTOR.

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

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

    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.

  16. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  17. Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

    Anshuman Dixit

    2009-08-01

    Full Text Available Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition

  18. Cellular reprogramming through mitogen-activated protein kinases

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  19. Protein kinase activity associated with the nuclear lamina.

    Dessev, G; Iovcheva, C; Tasheva, B; R. Goldman

    1988-01-01

    A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on...

  20. Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.

    Park, S.; Rasmussen, H

    1985-01-01

    The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g.,...

  1. Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model.

    Lamine, Asma; Létourneau, Myriam; Doan, Ngoc Duc; Maucotel, Julie; Couvineau, Alain; Vaudry, Hubert; Chatenet, David; Vaudry, David; Fournier, Alain

    2016-09-01

    Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases. PMID:26006268

  2. Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

    Uno, I.; Mitsuzawa, H.; Matsumoto, K.; Tanaka, K; Oshima, T.; Ishikawa, T

    1985-01-01

    Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 pr...

  3. P21-activated kinase 1 and breast cancer

    Jun-Xiang Zhang; Da-Qiang Li; Rakesh Kumar

    2010-01-01

    @@ The p21 activated kinase 1 (PAK1) belongs to PAKs family, a group of highly evolutionarily conserved protein family of serine/threonine kinases, which acts as a downstream effector of the small GTPases Cdc42 and Rac1, firstly reported in 1994[1]. As a serine/threonine kinase, PAK1 plays an important role in many cellular functions including cell morphogenesis, motility, survival, mitosis, angiogenesis, and tumorigenesis. More than 40 proteins have been reported to be phosphorylated by PAK1[2]. Accumulating experimental data in multiple experimental systems provide compelling evidence that PAK1 plays an important role in breast cancer promotion and progression. PAK1 is overexpressed and/or hyperactivated in more than 50% of breast cancers[3]. On the other hand, PAK1 overexpression in estrogen receptor alpha (ER α) positive breast cancer is also closely associated with a reduced responsiveness to tamoxifen therapy[4]. Since PAK1 plays such a vital role in breast cancer, PAK1 targeted therapeutic approaches are likely to be useful in breast cancer treatment as well as in other human cancers with PAK1 upregulation and/or hyperactivation[5].

  4. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila

    Straarup, EM; Schousboe, P; Hansen, HQ;

    1997-01-01

    Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium. At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation. The protein...... kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleyl 2-acetate glycerol (OAG) when added to 250 cells ml-1 supported cell survival and proliferation. In the presence of the serine and threonine kinase inhibitor staurosporine the cells died both at 250 cells ml-1 in cultures supplemented...... with either PMA or OAG, or at 2,500 cells ml-1. At 500 cells ml-1 PMA induced the in vivo phosphorylation of at least six proteins. The myelin basic protein fragment 4-14 was phosphorylated in vitro in crude extracts of a culture of 250,000 cells ml-1. Both the in vivo and the in vitro phosphorylation...

  5. Activation of the Antiviral Kinase PKR and Viral Countermeasures

    Bianca Dauber

    2009-10-01

    Full Text Available The interferon-induced double-stranded (dsRNA-dependent protein kinase (PKR limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5’-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR.

  6. Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

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

    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.

  8. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine

    Leonardi, Roberta; Rock, Charles O.; Jackowski, Suzanne; Zhang, Yong-Mei

    2007-01-01

    The human isoform 2 of pantothenate kinase (PanK2) is localized to the mitochondria, and mutations in this protein are associated with a progressive neurodegenerative disorder. PanK2 inhibition by acetyl-CoA is so stringent (IC50 < 1 μM) that it is unclear how the enzyme functions in the presence of intracellular CoA concentrations. Palmitoylcarnitine was discovered to be a potent activator of PanK2 that functions to competitively antagonize acetyl-CoA inhibition. Acetyl-CoA was a competitive...

  9. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  10. Mitogen-activated protein kinase (MAPK) in cardiac tissues.

    Page, C; Doubell, A F

    Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. PMID:8739228

  11. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen;

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically...... transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed. RESULTS Even though...

  12. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

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

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

  14. Linked decreases in Liver Kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

    Petursson, Freyr; Husa, Matt; June, Ron; Lotz, Martin; Terkeltaub, Robert; Liu-Bryan, Ru

    2013-01-01

    Abstract Introduction AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and p...

  15. Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.

    Jung, Se-Hui; Kong, Deok-Hoon; Jeon, Hye-Yoon; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Min-Soo; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-08-15

    Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34μg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions. PMID:27040940

  16. Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish.

    Canosa, Luis Fabián; Stacey, Norm; Peter, Richard Ector

    2008-12-01

    In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain. PMID:18815210

  17. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    Kolkova, K; Novitskaya, V; Pedersen, N;

    2000-01-01

    ), protein kinase C (PKC), and the Ras-mitogen-activated protein (MAP) kinase pathway. This was done using a coculture system consisting of PC12-E2 cells grown on fibroblasts, with or without NCAM expression, allowing NCAM-NCAM interactions resulting in neurite outgrowth. PC12-E2 cells were transiently......The signal transduction pathways associated with neural cell adhesion molecule (NCAM)-induced neuritogenesis are only partially characterized. We here demonstrate that NCAM-induced neurite outgrowth depends on activation of p59(fyn), focal adhesion kinase (FAK), phospholipase Cgamma (PLCgamma...... transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor...

  18. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    Kopper, F.; Bierwirth, C.; Schon, M.;

    2013-01-01

    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (gamma H2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on transiesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

  19. Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk

    Debnath Jayanta

    2001-05-01

    Full Text Available Abstract Background The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. Results We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. Conclusions Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.

  20. Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

    杨仲安; 曹丹; 桂建芳

    2000-01-01

    The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

  1. Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development

    1991-01-01

    Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protei...

  2. High quality, small molecule-activity datasets for kinase research [version 1; referees: 2 approved

    Rajan Sharma

    2016-06-01

    Full Text Available Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR data. Bioactivity databases such as the Kinase Knowledgebase (KKB, WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note.

  3. AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte

    AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with agents...

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

    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: 1.656, year: 2014

  5. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism[S

    Pinkosky, Stephen L.; Filippov, Sergey; Srivastava, Rai Ajit K.; Hanselman, Jeffrey C.; Bradshaw, Cheryl D.; Hurley, Timothy R.; Cramer, Clay T.; Spahr, Mark A.; Brant, Ashley F.; Houghton, Jacob L.; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S.

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  6. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  7. H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

    Yong-Chang Chen; Ying Wang; Jing-Yan Li; Wen-Rong Xu; You-Li Zhang

    2006-01-01

    AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.METHODS: A VacA (+) and CagA (+) standard Hpyloriline NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.RESULTS: Incubation with Hpylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with Hpylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pyloriextract increased c-Fos expression and SRE-dependentgene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

  8. Estrus cycle effect on muscle tyrosine kinase activity in bitches.

    Gomes Pöppl, Álan; Costa Valle, Sandra; Hilário Díaz González, Félix; de Castro Beck, Carlos Afonso; Kucharski, Luiz Carlos; Silveira Martins Da Silva, Roselis

    2012-03-01

    Estrus cycle is a well recognized cause of insulin resistance in bitches. The insulin receptor (IR) as well as the insulin-like growth factor-I receptor belong to the same subfamily of tyrosine kinase (TK) receptors. The objective of this study was to evaluate basal TK activity in muscle tissue of bitches during the estrus cycle. Twenty-four bitches were used in the study (7 in anestrus, 7 in estrus, and 10 in diestrus). Muscle samples, taken after spaying surgery to determine TK activity, were immediately frozen in liquid nitrogen and then stored at -80°C until the membranes were prepared by sequential centrifugation after being homogenized. TK activity was determined by Poly (Glu 4:Tyr 1) phosphorylation and expressed in cpm/μg of protein. TK activity was significantly lower (P < 0.001) in the animals in estrus (104.5 ± 11.9 cpm/μg of protein) and diestrus (94.5 ± 16.9 cpm/μg of protein) when compared with bitches in anestrus (183.2 ± 39.2 cpm/μg of protein). These results demonstrate, for the first time, lower basal TK activity in the muscle tissue of female dogs during estrus and diestrus, which may represent lower insulin signaling capacity, opening a new field of investigation into the molecular mechanisms of insulin resistance in dogs. PMID:22139063

  9. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    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 ...... on histidine residues, however, only the B isoform appeared to be serine phosphorylated....

  10. Parkin Regulates the Activity of Pyruvate Kinase M2.

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-05-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  11. Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

    José Manuel Galán-Caridad

    2004-12-01

    Full Text Available Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA for casein kinase (CK1 and P2 (RRRADDSDDDDD for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22, a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II

  12. The casein kinase II beta subunit binds to Mos and inhibits Mos activity.

    Chen, M.; D. Li; Krebs, E G; Cooper, J. A.

    1997-01-01

    Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation. Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK). We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos. The Mos-interacting region of CKII beta was mapped to the C terminus. Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta a...

  13. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

    Bøgebo, Rikke; Horn, Heiko; Olsen, Jesper V;

    2014-01-01

    -arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data...... developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets....

  14. Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

    Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

    1997-01-01

    Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these

  15. Elevated serum thymidine kinase activity in canine splenic hemangiosarcoma*.

    Thamm, D H; Kamstock, D A; Sharp, C R; Johnson, S I; Mazzaferro, E; Herold, L V; Barnes, S M; Winkler, K; Selting, K A

    2012-12-01

    Thymidine kinase 1 (TK1) is a soluble biomarker associated with DNA synthesis. This prospective study evaluated serum TK1 activity in dogs presenting with hemoabdomen and a splenic mass. An ELISA using azidothymidine as a substrate was used to evaluate TK1 activity. Sixty-two dogs with hemoabdomen and 15 normal controls were studied. Serum TK1 activity was significantly higher in dogs with hemangiosarcoma (HSA) than in normal dogs (mean ± SEM = 17.0 ± 5.0 and 2.01 ± 0.6, respectively), but not dogs with benign disease (mean ± SEM = 10.0 ± 3.3). Using a cut-off of 6.55 U/L, TK activity demonstrated a sensitivity of 0.52, specificity of 0.93, positive predictive value of 0.94 and negative predictive value of 0.48 for distinguishing HSA versus normal. When interval thresholds of 7.95 U/L were used together, diagnostic utility was increased. Serum TK1 evaluation may help to discriminate between benign disease and HSA in dogs with hemoabdomen and a splenic mass. PMID:22236280

  16. Mitogen-activated protein kinase signaling in plants under abiotic stress.

    Sinha, Alok Krishna; Jaggi, Monika; Raghuram, Badmi; Tuteja, Narendra

    2011-02-01

    Mitogen-activated protein kinase cascade is evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade consists essentially of three components, a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK connected to each other by the event of phosphorylation. These kinases play various roles in intra- and extra-cellular signaling in plants by transferring the information from sensors to responses. Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as responses to various stresses. MAPK signaling has also been associated with hormonal responses. In plants, MAP kinases are represented by multigene families and are involved in efficient transmission of specific stimuli and also involved in the regulation of the antioxidant defense system in response to stress signaling. In the current review we summarize and investigate the participation of MAPKs as possible mediators of various abiotic stresses in plants. PMID:21512321

  17. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    Agarwala Usha; Blaydes Jeremy P; Maurer Richard I; Essex Jon W; Kilburn Jeremy D; Warenius Hilmar M; Seabra Laurence A

    2011-01-01

    Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the c...

  18. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    Agarwala Usha

    2011-06-01

    Full Text Available Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6 are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP. An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with

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

    Irving, Helen R.

    2012-02-01

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

  20. 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

    Kazuhiro Kikuta; Atsushi Masamune; Masahiro Satoh; Noriaki Suzuki; Tooru Shimosegawa

    2004-01-01

    AIM: Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis,where oxidative stress is thought to play a key role. 4-hydroxy2,3-nonenal (HNE) is generated endogenously during the process of lipid peroxidation, and has been accepted as a mediator of oxidative stress. The aim of this study was to clarify the effects of HNE on the activation of signal transduction pathways and cellular functions in PSCs.METHODS: PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. PSCs were treated with physiologically relevant and non-cytotoxic concentrations (up to 5 μmol/L)of HNE. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay.Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Production of type Ⅰ collagen and monocyte chemoattractant protein-1was determined by enzyme-linked immunosorbent assay.The effect of HNE on the transformation of freshly isolated PSCs in culture was also assessed.RESULTS: HNE activated activator protein-1, but not nuclear factor κB. In addition, HNE activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. HNE increased type Ⅰ collagen production through the activation of p38 MAP kinase and c-Jun N-terminal kinase. HNE did not alter the proliferation,or monocyte chemoattractant protein-1 production. HNE did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype.CONCLUSION: Specific activation of these signal transduction pathways and altered cell functions such as collagen production by HNE may play a role in the pathogenesis of pancreatic

  1. Blunted Activation of Rho-Kinase in Yak Pulmonary Circulation

    Takeshi Ishizaki

    2015-01-01

    Full Text Available Yaks have adapted to high altitude and they do not develop hypoxic pulmonary hypertension. Although we previously identified the important role of augmented nitric oxide synthase activity in the yak pulmonary circulatory system, evidence of the direct involvement of Rho-kinase as a basal vascular tone regulator is lacking. Four domesticated male pure-bred yaks and four bulls that were born and raised at an altitude of 3000 m in the Tien-Shan mountains were studied at an altitude of 3,100 m. Mean pulmonary artery pressure (mPAP was measured before and after fasudil (60 mg in 20 mL of saline was intravenously administered using a Swan-Ganz catheter at a rate of 3.3 mL/min for 30 min. Fasudil decreased mPAP in bulls from 67.8±14.9 to 32.3±5.3 mmHg (P<0.05 after 15 min and the level was maintained for 30 min, but it merely blunted mPAP in yaks from 28.2±4.5 to 25.1±11.1 and 23.2±2.7 mmHg after 5 and 30 min, respectively. These findings comprise the first evidence of a modest role of Rho-kinase in the maintenance of pulmonary artery pressure in the yak.

  2. Blunted activation of Rho-kinase in yak pulmonary circulation.

    Ishizaki, Takeshi; Mizuno, Shiro; Sakai, Akio; Matsukawa, Shigeru; Kojonazarov, Baktybek; Zamirbek, Baiserkeev; Umeda, Yukihiro; Morikawa, Miwa; Anzai, Masaki; Ishizuka, Tamotsu; Aldashev, Almaz

    2015-01-01

    Yaks have adapted to high altitude and they do not develop hypoxic pulmonary hypertension. Although we previously identified the important role of augmented nitric oxide synthase activity in the yak pulmonary circulatory system, evidence of the direct involvement of Rho-kinase as a basal vascular tone regulator is lacking. Four domesticated male pure-bred yaks and four bulls that were born and raised at an altitude of 3000 m in the Tien-Shan mountains were studied at an altitude of 3,100 m. Mean pulmonary artery pressure (mPAP) was measured before and after fasudil (60 mg in 20 mL of saline) was intravenously administered using a Swan-Ganz catheter at a rate of 3.3 mL/min for 30 min. Fasudil decreased mPAP in bulls from 67.8±14.9 to 32.3±5.3 mmHg (P < 0.05) after 15 min and the level was maintained for 30 min, but it merely blunted mPAP in yaks from 28.2±4.5 to 25.1±11.1 and 23.2±2.7 mmHg after 5 and 30 min, respectively. These findings comprise the first evidence of a modest role of Rho-kinase in the maintenance of pulmonary artery pressure in the yak. PMID:25654121

  3. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  4. Horse chestnut extract induces contraction force generation in fibroblasts through activation of Rho/Rho kinase.

    Fujimura, Tsutomu; Moriwaki, Shigeru; Hotta, Mitsuyuki; Kitahara, Takashi; Takema, Yoshinori

    2006-06-01

    Contraction forces generated by non-muscle cells such as fibroblasts play important roles in determining cell morphology, vasoconstriction, and/or wound healing. However, few factors that induce cell contraction forces are known, such as lysophosphatidic acid and thrombin. Our study analyzed various plant extracts for ingredients that induce generation of cell contraction forces in fibroblasts populating collagen gels. We found that an extract of Horse chestnut (Aesculus hippocastanum) is able to induce such contraction forces in fibroblasts. The involvement of actin polymerization and stress fiber formation in the force generation was suggested by inhibition of this effect by cytochalasin D and by Rhodamine phalloidin. Rho kinase inhibitors (Y27632 and HA1077) and a Rho inhibitor (exoenzyme C3) significantly inhibited the force generation induced by the Horse chestnut extract. H7, which inhibits Rho kinase as well as other protein kinases, also significantly inhibited induction of force generation. However, inhibitors of other protein kinases such as myosin light chain kinase (ML-9), protein kinase C (Calphostin), protein kinase A (KT5720), and tyrosine kinase (Genistein, Herbimycin A) had no effect on force generation induced by Horse chestnut extract. These results suggest that the Horse chestnut extract induces generation of contraction forces in fibroblasts through stress fiber formation followed by activation of Rho protein and Rho kinase but not myosin light chain kinase or other protein kinases. PMID:16754996

  5. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    Ichiro N. Maruyama

    2014-04-01

    Full Text Available Receptor tyrosine kinases (RTKs play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

  6. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

    Glaser, P; Elmaoglou-Lazaridou, A; Krin, E.; Ladant, D.; Bârzu, O; Danchin, A

    1989-01-01

    In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture sup...

  7. Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

    Miao Xu; Yuanyuan Xiao; Jun Yin; Wolin Hou; Xueying Yu; Li Shen; Fang Liu; Li Wei; Weiping Jia

    2014-01-01

    Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation wer...

  8. Involvement of Hypothalamic AMP-Activated Protein Kinase in Leptin-Induced Sympathetic Nerve Activation

    Mamoru Tanida; Naoki Yamamoto; Toshishige Shibamoto; Kamal Rahmouni

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effec...

  9. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2013-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in...

  10. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

    Procházka, Radek; Blaha, Milan

    2015-01-01

    Roč. 61, č. 6 (2015), s. 495-502. ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.515, year: 2014

  11. Redox regulation of the AMP-activated protein kinase.

    Yingying Han

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  12. The role of AMP-activated protein kinase in regulation of skeletal muscle metabolism

    Anna Dziewulska; Paweł Dobrzyń; Agnieszka Dobrzyń

    2010-01-01

    AMP-activated protein kinase (AMPK) is a conserved, ubiquitously expressed eukaryotic enzyme that is activated in response to increasing AMP level. Regulation of AMPK activity in skeletal muscle is coordinated by contraction and phosphorylation by upstream kinases and a growing number of hormones and cytokines. Once activated, AMPK turns on catabolic, ATP-generating pathways, and turns off ATP-consuming metabolic processes such as biosynthesis and proliferation. Activation of AMPK promotes gl...

  13. Expression and Purification of PI3 Kinase {alpha} and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

    Boldyreff, Brigitte; Rasmussen, Tine L; Jensen, Hans H; Cloutier, Alexandre; Beaudet, Lucille; Roby, Philippe; Issinger, Olaf-Georg

    2008-01-01

    Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal....... In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. (Journal of Biomolecular Screening XXXX:xx-xx)....

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

    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...

  15. Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells. PMID:15363631

  16. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  17. A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1

    Frödin, Morten; Jensen, Claus J.; Merienne, Karine; Gammeltoft, Steen

    2000-01-01

    The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation...

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

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

    2013-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases 1,2 . 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 lesions 3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for dead...

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

    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.

  20. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  1. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

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

    Matsuura, Daizou; Maeda, Teruo

    2008-01-01

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

  3. Protein kinase A binds and activates heat shock factor 1.

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  4. Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

    Mahajan, Nupam P.; Liu, Yuanbo; Majumder, Samarpan; Warren, Maria R.; Parker, Carol E.; Mohler, James L.; Earp, H. Shelton; Whang, Young E.

    2007-01-01

    Activation of the androgen receptor (AR) may play a role in androgen-independent progression of prostate cancer. Multiple mechanisms of AR activation, including stimulation by tyrosine kinases, have been postulated. We and others have recently shown involvement of activated Cdc42-associated tyrosine kinase Ack1 in advanced human prostate cancer. Here we provide the molecular basis for interplay between Ack1 and AR in prostate cancer cells. Activated Ack1 promoted androgen-independent growth o...

  5. Histidine kinase activity in nuclei of Physarum polycephalum

    Nuclei of the true slime mold Physarum polycephalum, contain a kinase that specifically phosphorylates the 1-nitrogen of histidine-75 of histone H4, in vitro. Phosphohistidine is alkali stable and acid labile. Similar alkali stable phosphorylation has been observed with beef heart extracts and S-100 extracts from S. cerevisiae. The activity may be similar to that previously reported by R.A. Smith and his colleagues in several mammalian tissues. They have begun a search for nuclear proteins that contain phosphohistidine. Cultures of Physarum were grown in the presence of 32P-phosphate using several different labeling protocols. Labeled nuclear proteins were fractionated on a Superose-12 column. Alkali stable phosphate label eluted close to the position of histone H1, although it was not on H1 itself. No alkali stable phosphate eluted at the position of histone H4, which was obtained in high yield by this procedure. The absence of alkali-stable phosphorylation of histone H4 was confirmed by gel electrophoresis of the crude nuclear proteins. The fraction containing alkali-stable phosphate was shown to contain phosphohistidine by amino acid analysis of a total alkaline hydrolysate. They conclude that Physarum nuclei possess at least one protein that contains phosphohistidine in vivo and that histone H4 does not contain phosphohistidine in this system

  6. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  7. HCLK2 is required for activity of the DNA damage response kinase ATR

    Rendtlew Danielsen, Jannie M; Larsen, Dorthe Helena; Schou, Kenneth Bødtker;

    2008-01-01

    ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation...... of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with...

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

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

    2010-09-21

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

  9. Phosphorylation and inhibition of. gamma. -glutamyl transferase activity by cAMP-dependent protein kinase

    Kolesnichenko, L.S.; Chernov, N.N.

    1986-10-20

    It was shown that preparations of bovine kidney ..gamma..-glutamyl transferase of differing degrees of purity are phosphorylated by cAMP-dependent protein kinase. This is accompanied by a decrease in both the transferase and hydrolase activities of the enzyme. Consequently, ..gamma..-glutamyl transferase may serve as the substrate and target of the regulation of cAMP-dependent protein kinase.

  10. Skeletal muscle Ca(2+)-independent kinase activity increases during either hypertrophy or running

    Fluck, M.; Waxham, M. N.; Hamilton, M. T.; Booth, F. W.

    2000-01-01

    Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

  11. Regulation of Lymphoid Enhancer Factor 1/T-Cell Factor by Mitogen-Activated Protein Kinase-Related Nemo-Like Kinase-Dependent Phosphorylation in Wnt/β-Catenin Signaling

    Ishitani, Tohru; Ninomiya-Tsuji, Jun; Matsumoto, Kunihiro

    2003-01-01

    The Wnt/β-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic β-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) s...

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

    Yun-Hee Choi

    2015-11-01

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

  13. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  14. Identification and characterization of a mitogen-activated S6 kinase.

    Jenö, P; Ballou, L M; Novak-Hofer, I; Thomas, G.

    1988-01-01

    Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appear...

  15. Stimulation of casein kinase II by epidermal growth factor: Relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit

    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [32P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [32P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [32P]phosphate was found in the β subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyronsine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [32P]phosphate content (i.e., hyperphosphorylation) of casein kinase II β subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state

  16. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  17. Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit.

    Ackerman, P; Glover, C V; Osheroff, N

    1990-01-01

    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [32P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [32P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled ...

  18. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase

    Mendoza-Mendoza, Artemio; Pozo, María J.; Grzegorski, Darlene; Martínez, Pedro; García, Juan M.; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-01-01

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant ...

  19. INHIBITION OF IL-6-INDUCED STAT3 ACTIVATION IN MYELOMA CELLS BY PROTEIN KINASE A

    宋伦; 黎燕; 沈倍奋

    2001-01-01

    To investigate the regulation effect of protein kinase A on IL-6-induced STAT3 activation in myeloma cells. Methods: Two human myeloma cell lines-Sko-007 and U266 were pretreated with Forskolin, a protein kinase A antagonist, and then stimulated by IL-6. The activation state of STAT3 in these two cells were examined by electrophoretic mobility shift assay (EMSA). Results: Although PKA pathway itself doesn't participate in IL-6 signal transduction in Sko-007 and U266 cells, activation of protein kinase A can inhibit IL-6-induced STAT3 activation in these two cell lines. Conclusion: There exists an inhibitory effect of protein kinase A on STAT3 activation in human myeloma cells treated by IL-6.

  20. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

    Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

  1. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

    Ohannessian Arthur

    2004-05-01

    Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

  2. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass

    Shah, M; Kola, B; Bataveljic, A.; Arnett, T. R.; Viollet, B.; Saxon, L.; Korbonits, M.; C. Chenu

    2010-01-01

    Adenosine 5′-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cult...

  3. Characterization of the endogenous protein kinase activity of the hepatitis B virus.

    Kann, M; Thomssen, R; Köchel, H G; Gerlich, W H

    1993-01-01

    During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles. PMID:8260877

  4. Detection of protein kinase activity by renaturation in sodium dodecyl sulfate-polyacrylamide gels

    The authors have developed a procedure for identifying protein kinase activity in protein samples following electrophoresis on SDS-polyacrylamide gels. Proteins are allowed to renature directly in the gel by removal of detergent. The gel is then incubated with [γ-32P]ATP to allow renatured protein kinases to autophosphorylate or to phosphorylate various substrates which can be incorporated into the gel. The positions of the radiolabeled proteins can then be detected by autoradiography. With this technique, using purified catalytic subunit of cAMP-dependent protein kinase, enzyme concentrations as low as 0.01 μg can be detected on gels containing 1.0 mg/ml casein. The procedure is also applicable for the determination of active subunits of multisubunit protein kinases. For example, when the two subunits of casein kinase II are separated by SDS-polyacrylamide gel electrophoresis and allowed to renature, only the larger α subunit shows activity. This procedure can also be used to detect and distinguish kinases present in heterogeneous mixtures. Starting with a particulate fraction from LSTRA, a murine T cell lymphoma, several distinct enzymes were detected, including a 30,000 Dalton protein with protein-tyrosine kinase activity. This same enzyme has also been detected in T lymphocytes and other T lymphoid cell lines

  5. [Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

    Ishchuk, O P; Iatsyshyn, V Iu; Dmytruk, K V; Voronovs'kyĭ, A Ia; Fedorovych, D V; Sybirnyĭ, A A

    2006-01-01

    The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers. PMID:17290783

  6. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  7. Kinase Activity Profiling of Gram-Negative Pneumonia

    Hoogendijk, Arie J.; Diks, Sander H.; Peppelenbosch, Maikel P.; van der Poll, Tom; Wieland, Catharina W.

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential t

  8. Kinase activity profiling of gram-negative pneumonia

    A.J. Hoogendijk (Arie); S.H. Diks (Sander); M.P. Peppelenbosch (Maikel); T. van der Poll (Tom); C.W. Wieland (Catharina )

    2011-01-01

    textabstractPneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processe

  9. Kinase activity profiling of gram-negative pneumonia

    A.J. Hoogendijk; S.H. Diks; M.P. Peppelenbosch; T. van der Poll; C.W. Wieland

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential

  10. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  11. A placental polypeptide activator of a membranous protein kinase and its relation to histone 1.

    Abdel-Ghany, M; Riegler, C; Racker, E

    1984-01-01

    Crude transforming growth factor preparations of placenta contain a polypeptide that is required for the activity of a protein kinase that has been purified from plasma membrane preparations of Ehrlich ascites tumor cells. The kinase activator has been separated from transforming growth factor beta by reversed-phase HPLC and affinity chromatography. Like the transforming growth factor, it is heat stable and trypsin labile, but it is not inactivated by dithiothreitol. In sodium dodecyl sulfate...

  12. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  13. Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors.

    Burger, Matthew T; Han, Wooseok; Lan, Jiong; Nishiguchi, Gisele; Bellamacina, Cornelia; Lindval, Mika; Atallah, Gordana; Ding, Yu; Mathur, Michelle; McBride, Chris; Beans, Elizabeth L; Muller, Kristine; Tamez, Victoriano; Zhang, Yanchen; Huh, Kay; Feucht, Paul; Zavorotinskaya, Tatiana; Dai, Yumin; Holash, Jocelyn; Castillo, Joseph; Langowski, John; Wang, Yingyun; Chen, Min Y; Garcia, Pablo D

    2013-12-12

    Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K is < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described. PMID:24900629

  14. Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson's disease?

    Rudenko Iakov N

    2012-02-01

    Full Text Available Abstract Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are a common cause of familial Parkinson's disease (PD. Variation around the LRRK2 locus also contributes to the risk of sporadic PD. The LRRK2 protein contains a central catalytic region, and pathogenic mutations cluster in the Ras of complex protein C terminus of Ras of complex protein (mutations N1437H, R1441G/C and Y1699C and kinase (G2019S and I2020T domains. Much attention has been focused on the kinase domain, because kinase-dead versions of mutant LRRK2 are less toxic than kinase-active versions of the same proteins. Furthermore, kinase inhibitors may be able to mimic this effect in mouse models, although the currently tested inhibitors are not completely specific. In this review, we discuss the recent progress in the development of specific LRRK2 kinase inhibitors. We also discuss non-kinase-based therapeutic strategies for LRRK2-associated PD as it is possible that different approaches may be needed for different mutations.

  15. Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

    Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

    2015-10-01

    TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. PMID:26055819

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

    Debora Bogani

    2009-09-01

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

  17. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  18. Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

    AN Wei-wei; WANG Min-wei; Tashiro Shin-ichi; Onodera Satoshi; Ikejima Takashi

    2005-01-01

    Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression. Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.

  19. Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

    Rikke Bøgebo

    Full Text Available Recent progress in the understanding of seven-transmembrane receptor (7TMR signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.

  20. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (Pmechanism of fibrosis. PMID:27385084

  1. Distinct Mechanisms of Receptor and Nonreceptor Tyrosine Kinase Activation by Reactive Oxygen Species in Vascular Smooth Muscle Cells: Role of Metalloprotease and Protein Kinase C-δ

    Frank, Gerald D.; Mifune, Mizuo; Inagami, Tadashi; Ohba, Motoi; Sasaki, Terukatsu; Higashiyama, Shigeki; Dempsey, Peter J; Eguchi, Satoru

    2003-01-01

    Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor...

  2. Induction and phosphorylation of protein kinase C-α and mitogen-activated protein kinase by hypoxia and by radiation in Chinese hamster V79 cells

    Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase are protein-serine/threonine kinases which are important regulators of diverse cellular processes including metabolism, proliferation and differentiation. This study shows that both hypoxia and X irradiation of serum-deprived Chinese hamster V79 cells cause the induction and phosphorylation of the PKC-α isoform. The increased induction and phosphorylation of PKC occur mainly in the nuclear fraction. Unlike the PKC activator TPA, neither hypoxic nor radiation stress causes translocation of PKC-α from the cytosol to the membrane. The induction of PKC-α by hypoxia is accompanied by an increased expression of MAP kinase but, in contrast, this does not occur when PKC-α is induced by radiation. Radiation, like TPA, causes a complete redistribution of MAP kinase from the cytosol to the nucleus. 28 refs., 7 figs

  3. Activation of c-Jun N-terminal Kinases by Ribotoxic Stresses

    Dong-Yun Ouyang; Yuan-Yuan Wang; Yong-Tang Zheng

    2005-01-01

    The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response.

  4. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  5. Stretch activates myosin light chain kinase in arterial smooth muscle

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively

  6. Stretch activates myosin light chain kinase in arterial smooth muscle

    Barany, K.; Rokolya, A.; Barany, M. (Univ. of Illinois, Chicago (USA))

    1990-11-30

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol (32P)phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the (32P)phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in (32P)phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

  7. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  8. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. PMID:26851072

  9. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  10. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  11. Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

    Wei Dai; Weidong Li; Jun Lu; Yingge A; Ya Tu

    2010-01-01

    Recent studies have shown that vaned stress stimuli activate c-Jun N-terminal kinase(JNK),protein kinase B(Akt),and p38 mitogen-activated protein kinase(p38)signal transduction pathway,and also regulate various apoptotic cascades.JNK and p38 promote apoptosis,but Akt protects against apoptosis,in hippocampal neurons.However,changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood.Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group(P 0.05).These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

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

    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

  13. Association between mitogen-activated protein kinase kinase kinase 1 polymorphisms and breast cancer susceptibility: a meta-analysis of 20 case-control studies.

    Qiaoli Zheng

    Full Text Available BACKGROUND: The genome-wide single-nucleotide polymorphisms (SNPs profiles can be used as diagnostic markers for human cancers. The associations between mitogen-activated protein kinase kinase kinase 1 (MAP3K1 SNPs rs889312 A>C, rs16886165 T>G and breast cancer risk have been widely evaluated, but the results were inconsistent. To derive a conclusive assessment of the associations, we performed a meta-analysis by combining data from all eligible case-control studies up to date. METHODS: By searching PubMed, ISI web of knowledge, Embase and Cochrane databases, we identified all eligible studies published before September 2013. Odds ratios (ORs with 95% confidence intervals (CIs were used to assess the strength of associations in fixed-effect or random-effect model. False-positive report probability (FPRP was calculated to confirm the significance of the results. RESULTS: A total of 59670 cases in 20 case-control studies were included in this meta-analysis. Significant associations with breast cancer risk were observed for SNPs rs889312 and rs16886165 polymorphisms with a per-allele OR of 1.11 (95% CI: 1.09-1.13 and 1.14 (95% CI: 1.09-1.20 respectively. For rs889312, in subgroup analysis by ethnicity, significant associations were identified in Europeans and Asians, but not in Africans. When stratified by estrogen receptor (ER expression status, rs889312 was associated with both ER-positive and ER-negative breast cancers. Results from the FPRP analyses were consistent with and supportive to the above results. CONCLUSIONS: The present meta-analysis suggests that rs889312-C allele and rs16886165-G allele might be risk factors for breast cancer, especially in Europeans and Asians.

  14. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with NG-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ► Puerarin induced the phosphorylation of eNOS and the production of NO. ► Puerarin activated eNOS through ER-dependent PI3-kinase and Ca2+-dependent AMPK. ► Puerarin-induced NO was involved in the inhibition of NF-kB activation. ► Puerarin may help for prevention of vascular dysfunction and diabetes.

  15. Involvement of mitogen-activated protein kinase pathways in N-methyl-D-aspartate-induced excitotoxicity

    Xiaorong Yang; Ping Sun; Huaping Qin; Rui Wang; Ye Wang; Ruihong Shi; Xin Zhao; Ce Zhang

    2011-01-01

    Previous studies have shown that mitogen-activated protein kinase (MAPK) signaling pathways are involved in N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. However, a systematic observation or analysis of the role of these various MAPK pathways in excitotoxicity processes does not exist. The present study further evaluated the role and contribution of three MAPK pathways extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK in an NMDA-mediated excitotoxicity model using MAPK-specific inhibitor. Results demonstrated that c-Jun N-terminal kinase inhibitor SP600125 and/or p38 MAPK inhibitor SB203580 inhibited NMDA-induced reduction in cell viability, as well as reduced NMDA-induced lactate dehydrogenase leakage and reactive oxygen species production. However, PD98059, an inhibitor of extracellular signal-regulated kinase, did not influence this model. Results demonstrated an involvement of c-Jun N-terminal kinase and p38 MAPK, but not extracellular signal-regulated kinase, in NMDA-mediated excitotoxicity in cortical neurons.

  16. MEK kinase 1 activity is required for definitive erythropoiesis in the mouse fetal liver

    Bonnesen, Barbara; Ørskov, Cathrine; Rasmussen, Susanne;

    2005-01-01

    Mitogen-activated protein kinase/extracellular signal to regulated kinase (MEK) kinase 1 (MEKK1) is a c-Jun N-terminal kinase (JNK) activating kinase known to be implicated in proinflammatory responses and cell motility. Using mice deficient for MEKK1 kinase activity (Mekk1(DeltaKD)) we show a role...... for MEKK1 in definitive mouse erythropoiesis. Although Mekk1(DeltaKD) mice are alive and fertile on a 129 x C57/BL6 background, the frequency of Mekk1(DeltaKD) embryos that develop past embryonic day (E) 14.5 is dramatically reduced when backcrossed into the C57/BL6 background. At E13.5, Mekk1(Delta......KD) embryos have normal morphology but are anemic due to failure of definitive erythropoiesis. When Mekk1(DeltaKD) fetal liver cells were transferred to lethally irradiated wild-type hosts, mature red blood cells were generated from the mutant cells, suggesting that MEKK1 functions in a non...

  17. Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

    Châtel, A; Hamer, B; Talarmin, H; Dorange, G; Schröder, H C; Müller, W E G

    2010-03-01

    Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution. PMID:19948362

  18. Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

    Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

    2016-04-01

    Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

  19. Mitogen-Activated Protein Kinase Cascade Required for Regulation of Development and Secondary Metabolism in Neurospora crassa▿

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A.

    2008-01-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hy...

  20. Dependence of the hormonal stimulation of adenylate cyclase on the fraction of the plasma membrane accessible for lateral displacement of proteins of the adenylate cyclase complex

    Hormonal activation of the adenylate cyclase complex is associated with lateral displacement in the membrane of the proteins that constitute this complex. In this work an experimental investigation was made of the changes in the interaction of the proteins of the adenylate cyclase complex with the changing fraction of fluid lipids in the cell membrane. A decrease in the fraction of fluid lipids of rat reticulocyte membranes led to a decrease (all the way down to a total suppression) of the interaction of the β-adrenoreceptors with the regulatory N-proteins. The interaction of the N-proteins with the catalytic proteins was also suppressed. On the other hand, an increase in the fraction of fluid lipids led to more effective interaction. It was shown that in this case the functional intactness of the interacting proteins is unimpaired. An analysis of the results obtained, performed on the basis of the percolation theory, suggests the conclusion that the hormonal stimulation of adenylate cyclase depends on the fraction of fluid lipids in the membrane, and the proteins are displaced during interaction over distances comparable with the size of the membrane itself. It was also shown that characteristic activity of the β-agonist 1-isoproterenol varies from 1.0 to 0, depending on the fraction of fluid lipids in the membrane. The data obtained suggest that in the absence of guanylic nucleotides in the membrane in vitro there are no preexisting complexes with a high affinity for the agonist

  1. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  2. Action of radioprotectors - venoms of Central Asian snakes and radiation on the adenylate cyclase system

    Action of venoms of Central Asian snakes (Maja oxiana and Vipera labertina turahica) as radioprotectors on 3'-5'-AMP content and activity of adenylate cyclase and phosphodiesterase in homogenates of liver and spleen of rats 1 and 24 hours after irradiation (800 R) has been studied. c-AMP content and adenylate cyclase activity have been shown to decrease drastically in the organs under study after the action of ionizing radiation. Preventive administration of venoms of cobra (150 μ g/kg) and (700 μ g/kg) one hour before irradiation restores the activity of the enzyme and c - AMP content of the spleen up to 53% and of the liver, to 30%. Phosphodiesterase activity increased markedly after irradiation being practically unaffected by the protector

  3. Azorella compacta methanolic extract induces apoptosis via activation of mitogen-activated protein kinase.

    Sung, Min Hee; Kwon, Ok-Kyoung; Oh, Sei-Ryang; Lee, Joongku; Park, Sang-Hong; Han, Sang Bae; Ahn, Kyung-Seop

    2015-11-01

    Azorella compacta Phil. (AC) is an alpine medicinal plant used traditionally for antibacterial treatment. Recent studies have revealed that this plant also has anti‑diabetic effects, but that it is toxic. The present study investigated the underlying mechanisms of action of AC extract against human leukemia HL60 cells. Apoptosis induction was measured by MTT assay, fluorescence microscopy, DNA fragmentation assay, flow cytometric analysis, reverse transcription quantitative polymerase chain reaction and western blot analyses. It was found that AC extract inhibited the growth of HL60 and other cancer cell lines in a dose‑dependent manner. The cytotoxic effects of AC extract on HL60 cells were associated with apoptosis characterized by DNA fragmentation and dose‑dependent increases in Annexin V‑positive cells, as determined by flow cytometric analysis. AC‑extract‑induced apoptosis was accompanied by activated/cleaved caspase‑3, caspase‑9 and poly(adenosine diphosphate‑ribose) polymerase (PARP). The increases in apoptosis were also associated with decreases of the apoptosis-inhibitor B-cell lymphoma 2 (Bcl‑2), upregulation of pro‑apoptotic Bcl-2-associated X (Bax) protein and downregulation of anti‑apoptotic Bcl extra large protein. Furthermore, western blot analysis of mitogen-activated protein kinase (MAPK)-associated proteins indicated that treatment with AC extract increased the levels of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38. In addition, the expression of Bax and cleaved PARP was blocked when AC treatment was performed in the presence of MAPK inhibitors. It was therefore concluded that AC induced apoptosis in human leukemia HL60 cells via an intrinsic pathway controlled through MAPK-associated signaling. PMID:26397193

  4. Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity

    Layton Meredith J; Saad Mirette; Church Nicole L; Pearson Richard B; Mitchell Christina A; Phillips Wayne A

    2012-01-01

    Abstract Background The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. Results We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activi...

  5. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  6. Resveratrol inhibits Trypanosoma cruzi arginine kinase and exerts a trypanocidal activity.

    Valera Vera, Edward A; Sayé, Melisa; Reigada, Chantal; Damasceno, Flávia S; Silber, Ariel M; Miranda, Mariana R; Pereira, Claudio A

    2016-06-01

    Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease. PMID:26976067

  7. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H. [de Duve Institute, Universite catholique de Louvain, Avenue Hippocrate, B-1200 Brussels (Belgium); Horman, Sandrine, E-mail: sandrine.horman@uclouvain.be [Institute of Experimental and Clinical Research - Pole of Cardiovascular Research, Universite catholique de Louvain, Avenue Hippocrate, B-1200 Brussels (Belgium)

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  8. Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

    Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+)

  9. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    Massip, L; Garand, C; Labbé, A;

    2010-01-01

    show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1...... contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.......Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we...

  10. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  11. A Placental Polypeptide Activator of a Membranous Protein Kinase and Its Relation to Histone 1

    Abdel-Ghany, M.; Riegler, C.; Racker, E.

    1984-12-01

    Crude transforming growth factor preparations of placenta contain a polypeptide that is required for the activity of a protein kinase that has been purified from plasma membrane preparations of Ehrlich ascites tumor cells. The kinase activator has been separated from transforming growth factor β by reversed-phase HPLC and affinity chromatography. Like the transforming growth factor, it is heat stable and trypsin labile, but it is not inactivated by dithiothreitol. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis the purified preparation shows a major double band at about 31,000 daltons. Comparisons of electrophoretic mobility, protein kinase stimulatory activity, and cross-reactivity with an antibody against histone 1 suggest that the placental activator is identical with histone 1.

  12. Corticosterone activates Erk1/2 mitogen-activated protein kinase in primary hippocampal cells through rapid nongenomic mechanism

    QI Aiqun; QIU Jian; XIAO Lin; CHEN Yizhang

    2005-01-01

    Nongenomic effects of glucocorticoids (GC) in various cell types have been well documented, but it still remains unknown whether the mechanism also works in hippocampus which is a crucial target of glucocorticoids in neural system during physiological and/or pathophysiological processes. We present here that corticosterone (B) could rapidly activate Erk1/2 mitogen-activated protein kinase (MAPK) in primarily cultured hippocampal cells within minutes, with a bell-shaped time dependent curve which peaked at 15min and then went down to normal level in 30 min. This activation was blocked by protein kinase C (PKC) inhibitor (Go6976), G protein inhibitor (GDPβs), and MEK(MAPK/extracellular signal-regulated kinase kinase) inhibitor(PD98059), but not by protein kinase A (PKA) inbibitor (H89), tyrosine kinase inhibitor (genistein), and glucocorticoid receptor ( GR ) antagonist (RU38486). Thus, the rapid activation of Erk1/2 MAPK in primary hippocampal cells induced by B was likely mediated by a G protein coupled receptor (GPCR) pathway with involvement of PKC, which belonged to the nongenomic rather than genomic mechanism of GC' s effects.

  13. AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

    King, Tanya S; Russe, Otto Quintus; Möser, Christine V; Ferreirós, Nerea; Kynast, Katharina L; Knothe, Claudia; Olbrich, Katrin; Geisslinger, Gerd; Niederberger, Ellen

    2015-09-01

    AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties. PMID:26049010

  14. Identification of a protein kinase activity in purified foot- and-mouth disease virus.

    Grubman, M J; Baxt, B; La Torre, J L; Bachrach, H L

    1981-01-01

    Purified preparations of foot-and-mouth disease virus types A, O, and C contain a protein kinase activity which can transfer the gamma phosphate of [32P]ATP to virion structural proteins VP2 and VP3 and exogenous acceptor proteins. Utilizing protamine sulfate as an acceptor, the kinase activity can be demonstrated in disrupted virus but not in intact virus. The enzyme is heat labile with optimal activity at pH 7 or greater. Serine residues of protamine sulfate were identified as the amino aci...

  15. Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori.

    Li, Sheng; Zhang, Qi-Rui; Xu, Wei-Hua; Schooley, David A

    2005-11-01

    Juvenile hormone (JH) diol kinase (JHDK) is an important enzyme involved in the JH degradation pathway. Bombyx mori (Bommo)-JHDK cDNA (637bp) contains an open reading frame encoding a 183-amino acid protein, which reveals a high degree of identity to the two previously reported JHDKs. JHDK is similar to GTP-binding proteins with three conserved sequence elements involved in purine nucleotide binding, contains eight alpha-helices and three EF-hand motifs, and resembles the three-dimensional model of 2SCP and some other calcium-binding proteins. The Bommo-JHDK gene has only a single copy in the silkworm haploid genome, contains only one exon, and its 5'-upstream sequence does not have a JH response element. Although Bommo-JHDK is highly expressed in the gut of the silkworm, its mRNA expression remains at a constant level during larval development suggesting this enzyme is constitutive and not regulated by JH, at least at the transcriptional level. Recombinant Bommo-JHDK catalyzed the conversion of 10S-JH diol into JH diol phosphate, confirming its enzymatic function. Recombinant enzyme formed a dimer and had biochemical characteristics similar to other JHDKs. Bommo-JHDK, a calcium-binding protein with kinase activity, provides unique insights on how JH levels are regulated in the silkworm. PMID:16203205

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

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

    2015-12-15

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

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

    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 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.

  18. Locating a nucleotide-binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences.

    Gentry, G A

    1985-01-01

    Computer techniques were used to locate related segments of amino acid sequences in the thymidine kinases of vaccinia virus and of herpes simplex virus type 1 and in porcine adenylate kinase. As determined by a procedure that evaluates triply aligned sequences, the probability that the similarities among the segments described here arose by chance was no greater than 0.001. Because the sequence in porcine adenylate kinase is a nucleotide phosphate-binding site it is concluded that the segment...

  19. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  20. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

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

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan;

    2012-01-01

    Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, b...

  2. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.

    Clarke, Christopher J; Ohanian, Vasken; Ohanian, Jacqueline

    2007-05-01

    The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction. PMID:17208990

  3. Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

    Vitart, V.; Christodoulou, J.; Huang, J. F.; Chazin, W. J.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

  4. Cytosolic adenylate changes during exercise in prawn muscle

    31P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

  5. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  6. Diacylglycerol kinase theta and zeta isoforms : regulation of activity, protein binding partners and physiological functions

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblasto

  7. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis;

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested or....... In recovery, glucose feeding increased plasma glucose and insulin concentrations whereas ghrelin and PYY decreased to (ghrelin) or below (PPY) resting levels. It is concluded that 1 h of strenuous exercise in rats does not elicit significant changes in hypothalamic AMPK activity despite an increase...... ran for 30 or 60 min on a treadmill (22 m/min, 10% slope) were sacrificed immediately after exercise or after 60 min recovery either in the fasted state or after oral gavage with glucose (3 g/kg body weight). Exercise decreased muscle and liver glycogen substantially. Hypothalamic total or a2...

  8. In vitro and in vivo assays of protein kinase CK2 activity.

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  9. Activation of multifunctional calcium/calmodullin dependent protein kinase and phosphorylation of MAP-2 in GH3 cells

    Jefferson, A.B.

    1990-01-01

    The author utilized the pituitary-derived cell line, GH3, as a model system for studying the in situ regulation of multifunctional Ca{sup 2+}/calmodulin-dependent protein kinase (CaM kinase). The author partially purified a Ca{sup 2+}/ calmodulin-dependent protein kinase from GH3 cells and demonstrated that it is similar in biochemical properties to neuronal CaM kinase. Autophosphorylation at the autonomy site converts the kinase into a Ca{sup 2+}-independent enzyme. Regulation of CaM kinase in situ was examined by high K{sup +} depolarization of ({sup 32}P)Pi-labeled H3 cells followed by immunoprecipitation and trypic phosphopeptide mapping. The enzyme displayed a Ca{sup 2+} dependent increase in phosphorylation of the autonomy site. Accordingly, this led to a considerable increase in the Ca{sup 2+}-independent or autonomous activity of the enzyme. Thus, activation of CaM kinase by Ca{sup 2}/calmodulin and the subsequent formation of a Ca{sup 2+}-independent species, previously established in vitro, occur after Ca{sup 2+} influx in situ. In a parallel study the author tested whether microtubule-associated protein-2 (MAP-2), an in vitro substrate of CaM kinase, is phosphorylated by CaM kinase in GH3 cells. MAP-2 phosphorylation is enhanced by depolarization with high K{sup +} at sites characteristic of those recognized by CaM kinase and distinct from those phosphorylated by cAMP kinase or protein kinase C. Thyrotropin releasing hormone (TRH) increased Ca{sup 2+} via the phosphatidyl inositol signaling pathway but neither stimulated autophosphorylation of CaM kinase nor increased phosphorylation of the CaM kinase array of sites on MAP-2. TRH does increase MAP-2 phosphorylation but at sites which closely match those stimulated by phorbol esters that activate protein kinase C.

  10. Activation of multifunctional calcium/calmodullin dependent protein kinase and phosphorylation of MAP-2 in GH3 cells

    The author utilized the pituitary-derived cell line, GH3, as a model system for studying the in situ regulation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). The author partially purified a Ca2+/ calmodulin-dependent protein kinase from GH3 cells and demonstrated that it is similar in biochemical properties to neuronal CaM kinase. Autophosphorylation at the autonomy site converts the kinase into a Ca2+-independent enzyme. Regulation of CaM kinase in situ was examined by high K+ depolarization of [32P]Pi-labeled H3 cells followed by immunoprecipitation and trypic phosphopeptide mapping. The enzyme displayed a Ca2+ dependent increase in phosphorylation of the autonomy site. Accordingly, this led to a considerable increase in the Ca2+-independent or autonomous activity of the enzyme. Thus, activation of CaM kinase by Ca2/calmodulin and the subsequent formation of a Ca2+-independent species, previously established in vitro, occur after Ca2+ influx in situ. In a parallel study the author tested whether microtubule-associated protein-2 (MAP-2), an in vitro substrate of CaM kinase, is phosphorylated by CaM kinase in GH3 cells. MAP-2 phosphorylation is enhanced by depolarization with high K+ at sites characteristic of those recognized by CaM kinase and distinct from those phosphorylated by cAMP kinase or protein kinase C. Thyrotropin releasing hormone (TRH) increased Ca2+ via the phosphatidyl inositol signaling pathway but neither stimulated autophosphorylation of CaM kinase nor increased phosphorylation of the CaM kinase array of sites on MAP-2. TRH does increase MAP-2 phosphorylation but at sites which closely match those stimulated by phorbol esters that activate protein kinase C

  11. Mitogen-activated protein kinase-activated protein kinase 2 mediates resistance to Hydrogen peroxide-induced oxidative stress in Human hepatobiliary Cancer cells

    Nguyen Ho-Bouldoires, Thang Huong; Clapéron, Audrey; Mergey, Martine; Wendum, Dominique; Desbois-Mouthon, Christèle; Tahraoui, Sylvana; Fartoux, Laetitia; Chettouh, Hamza; Merabtene, Fatiha; Scatton, Olivier; Gaestel, Matthias; Praz, Françoise; Housset, Chantal; Fouassier, Laura

    2015-01-01

    The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/Heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. Besides to ...

  12. Long-acting β2-agonists increase fluticasone propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1 in airway smooth muscle cells.

    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.

  13. Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity

    Layton Meredith J

    2012-12-01

    Full Text Available Abstract Background The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. Results We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα. Conclusions Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.

  14. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

    Shah, M; Kola, B; Bataveljic, A; Arnett, T R; Viollet, B; Saxon, L; Korbonits, M; Chenu, C

    2010-08-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation

  15. Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

    Zhen, Yong-Zhan; Lin, Ya-Jun; Shang, Bo-Yang; Zhen, Yong-Su

    2009-07-01

    In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM. PMID:19468799

  16. Pharmacokinetics, Safety, and Tolerability of GLPG0259, a Mitogen-Activated Protein Kinase-Activated Protein Kinase 5 (MAPKAPK5) Inhibitor, Given as Single and Multiple Doses to Healthy Male Subjects

    Namour, Florence; Vanhoutte, Frédéric P.; Beetens, Johan; Blockhuys, Stan; Weer, Marc De; Wigerinck, Piet

    2012-01-01

    Background and Objectives GLPG0259 is a small-molecule inhibitor of mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), a kinase enzyme that plays a role in important inflammatory pathways. The main objectives of the phase I clinical studies in early development were to characterize the pharmacokinetics, tolerability, and safety of GLPG0259 in healthy subjects, including the development of a solid dosage form (free-base pellets and fumarate salt capsules) and the potential...

  17. ArhGAP9, a novel MAP kinase docking protein, inhibits Erk and p38 activation through WW domain binding

    2007-01-01

    We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 and p38α through complementarily charged residues in the WW domain of ArhGAP9 and the CD domains of Erk2 and p38α. This interaction sequesters the MAP kinases in their inactive states through displacement of MAP kinase kinases targeting the same sites. While over-expression of wild type ArhGAP9 caused MAP kinase activation by the epidermal growth factor receptor (EGFR) to be suppressed and preserve...

  18. Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity

    Argetsinger, Lawrence S.; Stuckey, Jeanne A.; Robertson, Scott A.; Koleva, Rositsa I.; Cline, Joel M.; Marto, Jarrod A.; Myers, Martin G.; Carter-Su, Christin

    2010-01-01

    Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry a...

  19. Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth*

    Takemura, Miyohiko; Mishima, Toshiaki; Wang, Yan; Kasahara, Jiro; Fukunaga, Kohji; Ohashi, Kazumasa; Mizuno, Kensaku

    2009-01-01

    Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppres...

  20. Mechanism of Regulation of Casein Kinase I Activity by Group I Metabotropic Glutamate Receptors

    Liu, Feng; Virshup, David M.; Nairn, Angus C.; Greengard, Paul

    2002-01-01

    Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on p...

  1. Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase

    1995-01-01

    EGF-receptor (EGF-R) tyrosine kinase is required for the down- regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of ...

  2. Mitogen-activated Protein Kinase Phosphatase (Mkp)-1 Protects Mice against Acetaminophen-induced Hepatic Injury

    Wancket, Lyn M.; Meng, Xiaomei; Rogers, Lynette K.; Liu, Yusen

    2012-01-01

    c-Jun N-terminal kinase (JNK) activation promotes hepatocyte death during acetaminophen overdose, a common cause of drug-induced liver failure. While mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1 is a critical negative regulator of JNK MAPK, little is known about the role of Mkp-1 during hepatotoxicity. In this study, we evaluated the role of Mkp-1 during acute acetaminophen toxicity. Mkp-1+/+ and Mkp-1−/− mice were dosed ip with vehicle or acetaminophen at 300 mg/kg (for mechan...

  3. Regulation of AMP-activated protein kinase by natural and synthetic activators.

    Grahame Hardie, David

    2016-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function. PMID:26904394

  4. Regulation of AMP-activated protein kinase by natural and synthetic activators

    David Grahame Hardie

    2016-01-01

    Full Text Available The AMP-activated protein kinase (AMPK is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.

  5. A stimulated S6 kinase from rat liver: identity with the mitogen activated S6 kinase of 3T3 cells.

    Kozma, S C; Lane, H A; Ferrari, S; Luther, H; Siegmann, M; Thomas, G.

    1989-01-01

    A number of approaches were tested for their ability to induce S6 phosphorylation and S6 kinase activation in rat liver, including i.p. injection of insulin, sodium orthovanadate or cycloheximide, as well as refeeding starved animals. All treatments led to increased S6 phosphorylation and activation of the apparent same enzyme. The most potent activator of the S6 kinase in liver extracts was cycloheximide. Maximum activation was achieved in 20 min at 1 mg cycloheximide/100 g body weight, with...

  6. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

    Riballo, Enriqueta; Woodbine, Lisa; Stiff, Thomas; Walker, Sarah A; Goodarzi, Aaron A; Jeggo, Penny A

    2009-02-01

    XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex. PMID:19056826

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

    Wong, Aloysius Tze

    2014-06-01

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

  8. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. C...

  9. Protein kinase and phosphatase activities of thylakoid membranes

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  10. Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

    Vincent, Olivier; Kuchin, Sergei; Hong, Seung-Pyo; Townley, Robert; Vyas, Valmik K; Carlson, Marian

    2001-01-01

    Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptio...

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

    Ines eLassowskat

    2014-10-01

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

  12. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  13. Unique catalytic activities and scaffolding of p21 activated kinase-1 in cardiovascular signaling

    YunboKe

    2013-09-01

    Full Text Available P21 activated kinase-1 has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from in vitro studies,emerging evidence has indicated that Pak1 may function as a signaling molecule through a unique molecular mechanism—scaffolding. By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s. Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.

  14. Role of calmodulin (δ-subunit) in activation of phosphorylase kinase from rabbit skeletal muscles

    The structure of the inactivated and activated forms of phospholyase kinase was compared. The enzyme was activated by incubation in an alkaline medium (pH 8.5), phosphorylation of the catalytic subunit of cAMP-dependent protein kinase, and limited proteolysis. Hydrophobic chromatography on phenyl-Sepharose and electrophoresis in a polyacrylamide gel density gradient were employed for a comparison of these forms of the enzyme. Activation of the enzyme was accompanied by the separation of a low-molecular-weight component (M/sub r/ about 17,000). The low-molecular-weight protein was obtained in a homogeneous state by chromatography on phenyl-Sepharose. It was established that its properties are similar to those of calmodulin. The presence of calmodulin in preparations of phosphorylase kinase was judged by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in much the same way as bovine brain calmodulin. The results obtained suggest that the δ-subunit is a protein inhibitor of the enzyme

  15. Oral glucose ingestion attenuates exercise-induced activation of 5'-AMP-activated protein kinase in human skeletal muscle

    Åkerström, Thorbjörn; Birk, Jesper Bratz; Klein, Ditte Kjærsgaard;

    2006-01-01

    5'-AMP-activated protein kinase (AMPK) has been suggested to be a 'metabolic master switch' regulating various aspects of muscle glucose and fat metabolism. In isolated rat skeletal muscle, glucose suppresses the activity of AMPK and in human muscle glycogen loading decreases exercise-induced AMPK...

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

    Robert-Gangneux F.

    2000-06-01

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

  17. [Effect of macro-creatine kinase in serum on dry chemistry methods results for total creatine kinase activity].

    Tozawa, T; Hashimoto, M

    1999-02-01

    Most enzymes in serum that are measured in clinical laboratories can occur in macro-molecular forms in a significantly number of patients. Within dry chemistry (DC) multilayer film, physical barriers may prevent contact macro-molecular enzyme forms with the active reagent ingredients. Here, serum samples with macro-creatine kinase (macro-CK) type 1: CK-immunoglobulin complex or type 2: oligomer mitochondrial CK (CKm) were analyzed for total CK activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM SP4410 and a classic wet chemistry (WC) analyzer: HITACHI 7350. Macro-CKs were detected and identified by electrophoresis on cellulose acetate. Serum with high amounts of oligomer CKm gave CK values by all of DC methods significantly lower than that by the WC method (p < 0.05). Oligomer CKm gradually converts into monomer forms in serum after storage. With increase in day after storage at 4 degrees C, there was a gradual shift in which percent of total CK activity for oligomer CKm decreased while the ratio of total CK activity, DC method/WC method increased. The principle of analytical method for CK activity determination is commonly to all of the DC methods, the WC method and the electrophoretic analysis. These suggest that oligomer CKm is sieved by DC multilayer film elements. In contrast, each of DC method produced highly corrected CK activities for sample containing CK-immunoglobulin complex. This difference in the effects of macro-CKs may depend upon physicochemical characteristics of analytical DC elements. PMID:10097631

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

    Muleya, Victor

    2014-09-23

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

  19. Age-related changes in AMP-activated protein kinase after stroke

    Liu, Fudong; Benashski, Sharon E; Persky, Rebecca; Xu, Yan; Li, Jun; McCullough, Louise D.

    2011-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor sensitive to changes in cellular AMP/ATP ratio which is activated by phosphorylation (pAMPK). pAMPK levels decrease in peripheral tissues with age, but whether this also occurs in the aged brain, and how this contributes to the ability of the aged brain to cope with ischemic stress is unknown. This study investigated the activation of AMPK and the response to AMPK inhibition after induced stroke...

  20. AMP-Activated Protein Kinase alpha1 Dependent Signaling in Renal Tissue Fibrosis

    Mia, Sobuj

    2016-01-01

    Tubulointerstitial fibrosis is a common hallmark of chronic kidney disease caused by diabetes, hypertension, ischemia, renal injury and obstructive uropathy. The hetereotrimeric AMP-activated protein kinase (AMPK) consists of three subunits (α, β, γ) and is a master sensor of cellular energy status. Activation of AMPK contributes to monocyte-fibroblast transition and production of matrix protein even though accumulating evidence suggests that activated AMPK inhibits tissue fibrosis, which may...

  1. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity

    The Homo sapiens and Arabidopsis thaliana genomes are believed to encode >500 and >1,000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Mass spectrometry (MS)-based approaches have been integral to the large-scale mapp...

  2. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D’Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  3. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan; Jensen, Lars Juhl; Berthelsen, Jens

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...

  4. Role of 5'AMP-activated protein kinase in skeletal muscle

    Treebak, Jonas Thue; Wojtaszewski, Jørgen F. P.

    2008-01-01

    5'AMP-activated protein kinase (AMPK) is recognized as an important intracellular energy sensor, shutting down energy-consuming processes and turning on energy-generating processes. Discovery of target proteins of AMPK has dramatically increased in the past 10 years. Historically, AMPK was first...

  5. Biochemical methods for quantifying sphingolipids: ceramide, sphingosine, sphingosine kinase-1 activity, and sphingosine-1-phosphate.

    Brizuela, Leyre; Cuvillier, Olivier

    2012-01-01

    Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabeled molecules ((3)H, (32)P) for their mass determination. Quantitation of sphingosine kinase-1 activity, the most studied isoform, is also included. PMID:22528435

  6. Creatine Kinase Activity Weakly Correlates to Volume Completed Following Upper Body Resistance Exercise

    Machado, Marco; Willardson, Jeffrey M.; Silva, Dailson P.; Frigulha, Italo C.; Koch, Alexander J.; Souza, Sergio C.

    2012-01-01

    In the current study, we examined the relationship between serum creatine kinase (CK) activity following upper body resistance exercise with a 1- or 3-min rest between sets. Twenty men performed two sessions, each consisting of four sets with a 10-repetition maximum load. The results demonstrated significantly greater volume for the 3-min…

  7. AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B

    van Gorp, A. G. M.; van der Vos, K. E.; Brenkman, A. B.; Bremer, A.; van den Broek, N.; Zwartkruis, F.; Hershey, J. W.; Burgering, B. M. T.; Calkhoven, C. F.; Coffer, P. J.

    2009-01-01

    Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using a

  8. An emerging role for p21-activated kinases (Paks) in viral infections

    Van den Broeke, Celine; Radu, Maria; Chernoff, Jonathan;

    2010-01-01

    p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and...... motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in...

  9. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    Walkiewicz, Katarzyna W.

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  10. Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

    Gesslein, Bodil; Håkansson, Gisela; Carpio, Ronald;

    2010-01-01

    The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion.......The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion....

  11. Inhibition of elongation factor-2 kinase augments the antitumor activity of Temozolomide against glioma.

    Xiao-Yuan Liu

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM, the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and in vitro and in vivo glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ. CONCLUSIONS/SIGNIFICANCE: These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.

  12. EFFECT OF PHORBOL ESTER ON cAMP-DEPENDENT PROTEIN KINASE ACTIVITY IN CARDIOMYOCYTES

    周文华; 肖殿模; 郑超强; 王小鲁; 张俊保

    1995-01-01

    Cardiomyocytes isolated from neonatal rats were treated with phorbol-12-myristate-13-acetate(PMA) ranging from 10-11 to 10-7mol/L for 20 min,causing cytosol protein kinase A (PKA) activity to decrease while particulate PKA activity increase in a concentration-dependent manner.The change of PKA activity induced by PMA was abolished completely by pretreatment of polymyxin B or depletion of protein kinase C (PKC).Type Ⅱ PKA activity in particulate fraction was enhanced remarkably,while that of type I PKA was not altered when the cells were treated with 100 nmol/L PMA.The results suggested that subcellular distribution and activity of PKA in cardiomyocytes may be regulated by PKC.

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

    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...... proteoglycan with heparin-binding moieties. This correlates with protein kinase C (PKC) activation, and PKCalpha can become localized to focal adhesions in normal, but not transformed, cells. PKC activation has been thought to be downstream of initial receptor-ligand interactions. We now show, however, that...... syndecan-4 transmembrane heparan sulfate proteoglycan and PKC co-immunoprecipitate and co-patch in vivo. The core protein of syndecan-4 can directly bind the catalytic domain of PKCalpha and potentiate its activation by phospholipid mediators. It can also directly activate PKCalpha in the absence of other...

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

    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

  15. DNA-dependent protein kinase participates in the radiation activation of NF-kB

    The NF-kB transcription factor is maintained in an inactive state by binding to the lkBa inhibitory protein. Activation requires phosphorylation and degradation of lkBa, releasing active NF-kB. NF-kB can be activated by cytokines, antigens, free radicals and X-ray irradiation. The protein kinase responsible for phosphorylation of lkBa in vivo has not been fully characterized. Here, we have examined the role of the DNA-dependent protein kinases (DNA-PK) in the radiation-activation of NF-kB. Wortmannin is an inhibitor of DNA-PK and related kinases. Exposure of SW480 cells to wortmannin inhibited the radioactivation of NF-kB DNA-binding. Analysis of lkBa levels by western blotting indicated that wortmannin blocked the radiation induced degradation of lkBa. In in vitro experiments, purified DNA-PK was able to efficiently phosphorylate lkBa, and this phosphorylation was inhibited by wortmannin. In contrast, the induction of NF-kB activity by TNFa was unaffected by wortmannin. The results suggest that DNA-PK may phosphorylate lkBa following irradiation, leading to degradation of lkBa and the release of active NF-kB. The inability of wortmannin to block TNFa activation of NF-kB indicates there may be more than one pathway for the activation of NF-kB

  16. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl2-induced VEGF secretion in mast cells occurs by a Ca2+-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl2) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl2-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl2-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

  17. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  18. Creatine supplementation: effects on blood creatine kinase activity responses to resistance exercise and creatine kinase activity measurement

    Marco Machado

    2009-12-01

    Full Text Available The purpose of this study was to determine the effects of creatine supplementation and exercise on the integrity of muscle fiber, as well as the effect of the supplementation on the creatine kinase (CK assay measurement. Forty-nine sedentary individuals participated in a double-blind study and were divided into two groups: C (n=26 received 4x5-day packages of 0.6 g.kg-1 of body weight contained 50% of creatine + 50% of dextrose, and P (n=23 received packages containing only dextrose. On the first day the groups performed a 1RM test for bench press, seated row, leg extension, leg curl and leg press. On D7 they received the supplements. On the fourteenth day, they performed a training session of five exercises, each in three sets of ten repetitions at 75% of 1RM. Blood was collected before (D14 and after the exercise session (D15. Differing levels of blood creatine were tested to determine the influence on the assay measurements of CK. ANOVA and Tukey's post-hoc tests were used to compare groups and different times of study protocol (PO objetivo do presente estudo foi determinar o efeito da suplementação de creatina e do exercício na integridade das fibras musculares e, também, o efeito da suplementação na técnica de mensuração da atividade da creatina kinase (CK. Quarenta e nove sedentários participaram de um estudo duplo-cego e foram divididos em dois grupos: C (n=26 que receberam 4x5 dias embalagens com 0,6 g.kg-1 de massa corporal com 50% de creatina + 50% de dextrose, e P (n=23 que receberam embalagens contendo apenas dextrose. No primeiro dia, eles realizaram o teste de 1RM para os exercícios supino reto, remada sentada, cadeira extensora, mesa flexora, e leg press. No D7 receberam os suplementos. No décimo quarto dia eles realizaram uma sessão de treinos com os cinco exercícios, cada um com 3x10 repetições a 75% de 1RM. Sangue foi coletado antes (D14 e depois da sessão de exercícios (D15. Diferentes concentrações de

  19. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects. Treatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs. These results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis

  20. Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells.

    Lee, Wei-Hwa; Lin, Ren-Jye; Lin, Shyr-Yi; Chen, Yu-Chien; Lin, Hsiu-Ming; Liang, Yu-Chih

    2011-12-28

    AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes. PMID:22098542

  1. Protein kinaseactivates NF-κB in response to camptothecin-induced DNA damage

    Highlights: → Protein kinase C-eta (PKCη) is an upstream regulator of the NF-κB signaling pathway. → PKCη activates NF-κB in non-stressed conditions and in response to DNA damage. → PKCη regulates NF-κB by activating IκB kinase (IKK) and inducing IκB degradation. -- Abstract: The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.

  2. Pim-1 kinase phosphorylates RUNX family transcription factors and enhances their activity

    Peltola Katriina J

    2006-05-01

    Full Text Available Abstract Background The pim family genes encode oncogenic serine/threonine kinases which in hematopoietic cells have been implicated in cytokine-dependent signaling as well as in lymphomagenesis, especially in cooperation with other oncogenes such as myc, bcl-2 or Runx family genes. The Runx genes encode α-subunits of heterodimeric transcription factors which regulate cell proliferation and differentiation in various tissues during development and which can become leukemogenic upon aberrant expression. Results Here we have identified novel protein-protein interactions between the Pim-1 kinase and the RUNX family transcription factors. Using the yeast two-hybrid system, we were able to show that the C-terminal part of human RUNX3 associates with Pim-1. This result was confirmed in cell culture, where full-length murine Runx1 and Runx3 both coprecipitated and colocalized with Pim-1. Furthermore, catalytically active Pim-1 kinase was able to phosphorylate Runx1 and Runx3 proteins and enhance the transactivation activity of Runx1 in a dose-dependent fashion. Conclusion Altogether, our results suggest that mammalian RUNX family transcription factors are novel binding partners and substrates for the Pim-1 kinase, which may be able to regulate their activities during normal hematopoiesis as well as in leukemogenesis.

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

    McEneaney, Victoria

    2010-01-01

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

  4. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  5. Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

    The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector

  6. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase [A-kinase], from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from 32P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the 32P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase

  7. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

    1987-05-01

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase (A-kinase), from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from /sup 32/P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the /sup 32/P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase.

  8. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  9. Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

    Kim, Jong Hyun; Park, Ji-Man; Yea, Kyungmoo; Kim, Hyun Wook; Suh, Pann-Ghill; Ryu, Sung Ho

    2010-01-01

    Background Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of gluc...

  10. Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

    Disatnik, M H; Hernandez-Sotomayor, S M; G. Jones; Carpenter, G.; Mochly-Rosen, D

    1994-01-01

    Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We ...

  11. Activation of transfer RNA-guanine ribosyltransferase by protein kinase C.

    Morris, R C; Brooks, B. J.; Eriotou, P; Kelly, D F; Sagar, S.; Hart, K L; Elliott, M.S.

    1995-01-01

    Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TGRase activity. TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70 degrees C. Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test. The addition of micromolar ...

  12. Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase

    Nguyen, Aaron Ngocky; Lee, Albert; Place, Warren; Shiozaki, Kazuhiro

    2000-01-01

    In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe. The fission yeast mpr1+ gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative ...

  13. Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated ...

  14. Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

    Jong Hyun Kim; Ji-Man Park; Kyungmoo Yea; Hyun Wook Kim; Pann-Ghill Suh; Sung Ho Ryu

    2010-01-01

    BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glu...

  15. Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle.

    Aronson, D; Violan, M A; Dufresne, S D; Zangen, D; FIELDING, R.A.; Goodyear, L J

    1997-01-01

    Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pat...

  16. Metabolic Basis for Thyroid Hormone Liver Preconditioning: Upregulation of AMP-Activated Protein Kinase Signaling

    Videla, Luis A; Virginia Fernández; Pamela Cornejo; Romina Vargas

    2012-01-01

    The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK) constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reve...

  17. Protein Kinase C-Independent Activation of the Epstein-Barr Virus Lytic Cycle

    Gradoville, Lyndle; Kwa, David; El-Guindy, Ayman; Miller, George

    2002-01-01

    The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activa...

  18. Adiponectin Increases Skeletal Muscle Mitochondrial Biogenesis by Suppressing Mitogen-Activated Protein Kinase Phosphatase-1

    Qiao, Liping; Kinney, Brice; Yoo, Hyung sun; Lee, Bonggi; Schaack, Jerome; Shao, Jianhua

    2012-01-01

    Adiponectin enhances mitochondrial biogenesis and oxidative metabolism in skeletal muscle. This study aimed to investigate the underlying mechanisms through which adiponectin induces mitochondrial biogenesis in skeletal muscle. Mitochondrial contents, expression, and activation status of p38 mitogen-activated protein kinase (MAPK) and PPARγ coactivator 1α (PGC-1α) were compared between skeletal muscle samples from adiponectin gene knockout, adiponectin-reconstituted, and control mice. Adenovi...

  19. LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

    John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette;

    2010-01-01

    The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified......-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search...

  20. Resveratrol improves non-alcoholic fatty liver disease by activating AMP-activated protein kinase

    Jing SHANG; Lu-lu CHEN; Eang-xi XIAO; Hui SUN; Hong-cheng DING; Hu XIAO

    2008-01-01

    Aim: To investigate whether resveratrol (RSV) can improve non-alcoholic fatty liver disease (NAFLD) and to find the possible mechanism. Methods: Rats fed a high-fat diet were treated with RSV. The liver histology was observed. Hyperinsulinemic euglycemic clamp was performed to assess insulin sensitivity. Fat accumulation was induced in HepG2 cells, and the cells were treated with RSV. AMP-activated protein kinase (AMPK) phosphorylation levels were de-termined both in the animal study and cell study. Results: Rats fed a high-fat diet developed abdominal obesity, NAFLD, and insulin resistance (IR), which were markedly improved by 10 weeks of RSV administration. RSV treatment prevented triacylglycerol (TG) accumulation in HepG2 cells that were incubated with high concentration of glucose and insulin. Both in vivo and in vitro studies showed that RSV treatment could promote the phosphorylation of AMPK, which in this study, suppressed 2 lipogenesis gene expressions, contributing to the improvement of NAFLD and IR. Conclusion: The results indicated that by re-ducing TG accumulation and improving IR, RSV could protect the liver from NAFLD. The activation of AMPK was involved in the mechanism. RSV has the therapeutic potential for preventing or treating NAFLD and IR-related metabolic disorders.

  1. Activation of mitogen activated protein kinases via complement receptor type 2

    LUO Min-hua 罗敏华; CHEN Ming-liang 陈明亮; Heribert Stoiber; Manfred P Dierich

    2004-01-01

    Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS.Results FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp160. In HOS-CD4CR2 cells, MAPK activation was induced by HIV-gp160, 10% NHS, and HIV-gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV-gp160+NHS, and lasted for 1 hour. The cell proliferation results showed that HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells (P<0.01) and that NHS enhanced the effect of HIV-gp160 (P<0.01).Conclusions The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation.

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

    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. Structural Basis for a Reciprocating Mechanism of Negative Cooperativity in Dimeric Phosphagen Kinase Activity

    Wu, X.; Ye, S; Guo, S; Yan, W; Bartlam, M; Rao, Z

    2010-01-01

    Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 {angstrom}, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widely than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase. - Wu, X., Ye, S., Guo, S., Yan, W., Bartlam, M., Rao, Z. Structural basis for a reciprocating mechanism of negative cooperativity in dimeric phosphagen kinase activity.

  4. Structure and ubiquitination-dependent activation of TANK-binding kinase 1.

    Tu, Daqi; Zhu, Zehua; Zhou, Alicia Y; Yun, Cai-hong; Lee, Kyung-Eun; Toms, Angela V; Li, Yiqun; Dunn, Gavin P; Chan, Edmond; Thai, Tran; Yang, Shenghong; Ficarro, Scott B; Marto, Jarrod A; Jeon, Hyesung; Hahn, William C; Barbie, David A; Eck, Michael J

    2013-03-28

    Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents. PMID:23453972

  5. Structure and Ubiquitination-Dependent Activation of TANK-Binding Kinase 1

    Daqi Tu

    2013-03-01

    Full Text Available Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1 induces type I interferon expression and modulates nuclear factor κB (NF-κB signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ∊ (IKK∊ but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

  6. Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

    Silvia Volpe

    Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

  7. Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation.

    Mamoru Tanida

    Full Text Available In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb. We investigated the potential of AMPKα2 in the sympathetic effects of leptin using in vivo siRNA injection to knockdown AMPKα2 in rats, to produce reduced hypothalamic AMPKα2 expression. Leptin effects on body weight, food intake, and blood FFA levels were eliminated in AMPKα2 siRNA-treated rats. Leptin-evoked enhancements of the sympathetic nerve outflows to the kidney, brown and white adipose tissues were attenuated in AMPKα2 siRNA-treated rats. To check whether AMPKα2 was specific to sympathetic changes induced by leptin, we examined the effects of injecting MT-II, a melanocortin-3 and -4 receptor agonist, on the sympathetic nerve outflows to the kidney and adipose tissue. MT-II-induced sympatho-excitation in the kidney was unchanged in AMPKα2 siRNA-treated rats. However, responses of neural activities involving adipose tissue to MT-II were attenuated in AMPKα2 siRNA-treated rats. These results suggest that hypothalamic AMPKα2 is involved not only in appetite and body weight regulation but also in the regulation of sympathetic nerve discharges to the kidney and adipose tissue. Thus, AMPK might function not only as an energy sensor, but as a key molecule in the cardiovascular, thermogenic, and lipolytic effects of leptin through the sympathetic nervous system.

  8. Scutellarein Reduces Inflammatory Responses by Inhibiting Src Kinase Activity

    Sung, Nak Yoon; Kim, Mi-Yeon; Cho, Jae Youl

    2015-01-01

    Flavonoids are plant pigments that have been demonstrated to exert various pharmacological effects including anti-cancer, anti-diabetic, anti-atherosclerotic, anti-bacterial, and anti-inflammatory activities. However, the molecular mechanisms in terms of exact target proteins of flavonoids are not fully elucidated yet. In this study, we aimed to evaluate the anti-inflammatory mechanism of scutellarein (SCT), a flavonoid isolated from Erigeron breviscapus, Clerodendrum phlomidis and Oroxylum i...

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

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

    2015-01-01

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

  10. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.;

    2007-01-01

    bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and....... The tested Gram-negative bacteria were susceptible to 3"-azido-3"-deoxythymidine (AZT) in the concentration range 0.032-31.6 µM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine...... deoxyadenosine kinase had a Km for gemcitabine of 33.5 µM and kcat/Km of 5.1 × 10^3 s-1 M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Bacterial dNKs efficiently activate nucleoside analogues in a species...

  11. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  12. Carboxyl terminal of rhodopsin kinase is required for the phosphorylation of photo—activated rhodopsin

    YUQINGMING; LANMA; 等

    1998-01-01

    Human rhodopsin kinase (RK) and a carboxyl terminus-truncated mutant RK lacking the last 59 amino acids (RKC) were expressed in human embryonic kidney 293 cells to investigate the role of the carboxyl terminus of RK in recognition and phosphorylation of rhodopsin.RKC,like the wild-type RK,was detected in both plasma membranes and cytosolic fractions.The Cterminal truncated rhodopsin kinase was unable to phosphorylate photo-activated rhodopsin,but possesses kinase activity similar to the wild-type RK in phosphorylation of small peptide substrate.It suggests that the truncation did not disturb the gross structures of RK catalytic domain.Our results also show that RKC failed to translocate to photo-activated rod out segments.Taken together,our study demonstrate the carboxyl terminus of RK is required for phosphorylation of photo-activated rhodopsin and strongly indicate that carboxyl-terminus of RK may be involved in interaction with photo-activated rhodopsin.

  13. Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation

    Giuseppe Vassalli

    2012-01-01

    Full Text Available In solid organ transplantation, ischemia/reperfusion (IR injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK, extracellular signal- regulated kinase-1/2 (ERK1/2, p38 MAPK, and big MAPK-1 (BMK1/ERK5. Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion.

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

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

    2016-01-01

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

  15. Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

    Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression

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

    Nasar Virk

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

  17. Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase.

    Mendoza-Mendoza, Artemio; Pozo, María J; Grzegorski, Darlene; Martínez, Pedro; García, Juan M; Olmedo-Monfil, Vianey; Cortés, Carlos; Kenerley, Charles; Herrera-Estrella, Alfredo

    2003-12-23

    The production of lytic enzymes in Trichoderma is considered determinant in its parasitic response against fungal species. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed a clear increase in the level of the expression of mycoparasitism-related genes under simulated mycoparasitism and during direct confrontation with the plant pathogen Rhizoctonia solani. The null mutants displayed an increased protein secretion phenotype as measured by the production of lytic enzymes in culture supernatant compared to the wild type. Consistently, biocontrol assays demonstrated that the null mutants were considerably more effective in disease control than the wild-type strain or a chemical fungicide. In addition, tvk1 gene disruptant strains sporulated abundantly in submerged cultures, a condition that is not conducive to sporulation in the wild type. These data suggest that Tvk1 acts as a negative modulator during host sensing and sporulation in T. virens. PMID:14673101

  18. Sequential Activation and Inactivation of Dishevelled in the Wnt/β-Catenin Pathway by Casein Kinases

    Bernatik, Ondrej; Ganji, Ranjani Sri; Dijksterhuis, Jacomijn P.; Konik, Peter; Cervenka, Igor; Polonio, Tilman; Krejci, Pavel; Schulte, Gunnar; Bryja, Vitezslav

    2011-01-01

    Dishevelled (Dvl) is a key component in the Wnt/β-catenin signaling pathway. Dvl can multimerize to form dynamic protein aggregates, which are required for the activation of downstream signaling. Upon pathway activation by Wnts, Dvl becomes phosphorylated to yield phosphorylated and shifted (PS) Dvl. Both activation of Dvl in Wnt/β-catenin signaling and Wnt-induced PS-Dvl formation are dependent on casein kinase 1 (CK1) δ/ϵ activity. However, the overexpression of CK1 was shown to dissolve Dv...

  19. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    Brandauer, Josef; Vienberg, Sara Gry; Andersen, Marianne Agerholm;

    2013-01-01

    -activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for...... increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein...

  20. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

    Inositol 1,4,5-triphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, they propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, and intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [γ-32P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg2+ > > Mn2+ > Ca2+) for the 3-kinase

  1. Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

    Kampen, G T; Stafford, S; Adachi, T;

    2000-01-01

    Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3 and...... the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to...... assess the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay...

  2. Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells.

    Edsall, L C; Cuvillier, O; Twitty, S; Spiegel, S; Milstien, S

    2001-03-01

    Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic

  3. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-01

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. PMID:26912723

  4. Regulation of skeletal muscle sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) by metabolic stress and diabetes.

    Rune, A.; Osler, M. E.; Fritz, T.; Zierath, J. R.

    2010-01-01

    Aims/hypothesis Sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) is involved in cellular stress responses linked to obesity and type 2 diabetes. We determined the role of SNARK in response to metabolic stress and insulin action on glucose and lipid metabolism in skeletal muscle. Methods Vastus lateralis skeletal muscle biopsies were obtained from normal glucose tolerant (n = 35) and type 2 diabetic (n = 31) men and women for SNARK expression studies. Primary myotu...

  5. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  6. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  7. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity

  8. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  9. Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases.

    Dayalan Naidu, Sharadha; Sutherland, Calum; Zhang, Ying; Risco, Ana; de la Vega, Laureano; Caunt, Christopher J; Hastie, C James; Lamont, Douglas J; Torrente, Laura; Chowdhry, Sudhir; Benjamin, Ivor J; Keyse, Stephen M; Cuenda, Ana; Dinkova-Kostova, Albena T

    2016-09-15

    Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response. PMID:27354066

  10. Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

    Stefely, Jonathan A; Licitra, Floriana; Laredj, Leila; Reidenbach, Andrew G; Kemmerer, Zachary A; Grangeray, Anais; Jaeg-Ehret, Tiphaine; Minogue, Catherine E; Ulbrich, Arne; Hutchins, Paul D; Wilkerson, Emily M; Ruan, Zheng; Aydin, Deniz; Hebert, Alexander S; Guo, Xiao; Freiberger, Elyse C; Reutenauer, Laurence; Jochem, Adam; Chergova, Maya; Johnson, Isabel E; Lohman, Danielle C; Rush, Matthew J P; Kwiecien, Nicholas W; Singh, Pankaj K; Schlagowski, Anna I; Floyd, Brendan J; Forsman, Ulrika; Sindelar, Pavel J; Westphall, Michael S; Pierrel, Fabien; Zoll, Joffrey; Dal Peraro, Matteo; Kannan, Natarajan; Bingman, Craig A; Coon, Joshua J; Isope, Philippe; Puccio, Hélène; Pagliarini, David J

    2016-08-18

    The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease. PMID:27499294

  11. Adipocyte lipolysis-stimulated interleukin-6 production requires sphingosine kinase 1 activity.

    Zhang, Wenliang; Mottillo, Emilio P; Zhao, Jiawei; Gartung, Allison; VanHecke, Garrett C; Lee, Jen-Fu; Maddipati, Krishna R; Xu, Haiyan; Ahn, Young-Hoon; Proia, Richard L; Granneman, James G; Lee, Menq-Jer

    2014-11-14

    Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of β3-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes. PMID:25253697

  12. DMPD: Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcascades during intracellular Toxoplasma gondii infection. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 15361242 Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-sig...mmunol Rev. 2004 Oct;201:191-205. (.png) (.svg) (.html) (.csml) Show Manipulation of mitogen-activated protein kinase/nuclear... gondii infection. PubmedID 15361242 Title Manipulation of mitogen-activated protein kinase/nuclear factor-k

  13. Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death

    Yixia Ye

    2009-01-01

    Full Text Available Cyclin-dependent kinase 5 (Cdk5 is similar to other Cdks but is activated during cell differentiation and cell death rather than cell division. Since activation of Cdk5 has been reported in many situations leading to cell death, we attempted to determine if it was required for any form of cell death. We found that Cdk5 is activated during apoptotic deaths and that the activation can be detected even when the cells continue to secondary necrosis. This activation can occur in the absence of Bim, calpain, or neutral cathepsins. The kinase is typically activated by p25, derived from p35 by calpain-mediated cleavage, but inhibition of calpain does not affect cell death or the activation of Cdk5. Likewise, RNAi-forced suppression of the synthesis of Cdk5 does not affect the incidence or kinetics of cell death. We conclude that Cdk5 is activated as a consequence of metabolic changes that are common to many forms of cell death. Thus its activation suggests processes during cell death that will be interesting or important to understand, but activation of Cdk5 is not necessary for cells to die.

  14. Activation of Mitogen-Activated Protein Kinase Is Required for Migration and Invasion of Placental Site Trophoblastic Tumor

    Köbel, Martin; Pohl, Gudrun; Schmitt, Wolfgang D.; Hauptmann, Steffen; Wang, Tian-Li; Shih, Ie-Ming

    2005-01-01

    Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAP...

  15. Activation of resting human T cells requires prolonged stimulation of protein kinase C

    Berry, N.; Ase, K.; Kishimoto, A.; Nishizuka, Y. (Kobe Univ. School of Medicine (Japan))

    1990-03-01

    Purified resting human T cells can be induced to express the {alpha} subunit of the interleukin 2 receptor and to proliferate by treatment with 12-0-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-0-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the {alpha} subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.

  16. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver. PMID:26876787

  17. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    Sung, C.; Okabayashi, Y.; Williams, J.

    1987-05-01

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.

  18. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact 32P-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the Ca2+ ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in β-glycerophosphate buffer and assayed for the kinase using γ-32P ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither Ca2+ or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase

  19. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide. PMID:26535916

  20. Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

    Higginson, James; Wackerhage, Henning; Woods, Niall;

    2002-01-01

    and mitogen-activated protein kinase kinase (MEK1/2) blockade with U0126 upon myosin heavy chain (MHC) isoform mRNA levels and activities of metabolic enzymes after 1 day, 3 days and 7 days of treatment in primary cultures of spontaneously twitching rat skeletal muscle. U0126 treatment significantly......Activation of either the calcineurin or the extracellular signal-regulated kinase (ERK1/2) pathway increases the percentage of slow fibres in vivo suggesting that both pathways can regulate fibre phenotypes in skeletal muscle. We investigated the effect of calcineurin blockade with cyclosporin A...

  1. Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

    Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise;

    2006-01-01

    The mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, play a major role in the regulation of pivotal cellular processes such as cell death/survival balance, cell cycle progression, and cell migration. MAPK...... activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many of...

  2. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

    Nielsen-Preiss, Sheila M; Allen, Melissa P; Xu, Mei; Linseman, Daniel A; Pawlowski, John E; Bouchard, R J; Varnum, Brian C; Heidenreich, Kim A; Wierman, Margaret E

    2007-06-01

    GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration. PMID:17332061

  3. Protein kinase C activity is associated with prefrontal cortical decline in aging

    Brennan, Avis R.; Yuan, Peixiong; Dickstein, Dara L; Rocher, Anne B.; Hof, Patrick R.; Manji, Husseini; Arnsten, Amy F.T.

    2007-01-01

    Aging is associated with deficiencies in the prefrontal cortex, including working memory impairment, and compromised integrity of neuronal dendrites. Although protein kinase C (PKC) is implicated in structural plasticity, and overactivation of PKC results in working memory impairments in young animals, the role of PKC in prefrontal cortical impairments in the aged has not been examined. This study provides the first evidence that PKC activity is associated with prefrontal cortical dysfunction...

  4. Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

    Prigent, C.; Lasko, D D; Kodama, K.; Woodgett, J R; Lindahl, T

    1992-01-01

    Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation...

  5. Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

    Reszka, A. A.; Seger, R.; Diltz, C D; Krebs, E G; Fischer, E H

    1995-01-01

    Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detect...

  6. Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.

    Pettit, F H; Humphreys, J; Reed, L J

    1982-01-01

    Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffe...

  7. Skeletal muscle metabolic flexibility : The roles of AMP-activated protein kinase and calcineurin

    Long, Yun Chau

    2007-01-01

    Skeletal muscle fibers differ considerably in their metabolic and physiological properties. The metabolic properties of skeletal muscle display a high degree of flexibility which adapts to various physiological demands by shifting energy substrate metabolism. Studies were conducted to evaluate the roles of AMP-activated protein kinase (AMPK) and calcineurin in the regulation of skeletal muscle metabolism. Fasting elicited a coordinated expression of genes involved in lipid ...

  8. Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein.

    Shuba, L. M.; Asai, T.; Pelzer, S.; McDonald, T. F.

    1996-01-01

    1. Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP-dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, were applied to guinea-pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl- channels. 2. Myocytes in the standard whole-cell configuration were pulsed to various pot...

  9. Transgenic Inhibition of Neuronal Protein Kinase A Activity Facilitates Fear Extinction

    Isiegas, Carolina; Park, Alice; Kandel, Eric R.; Abel, Ted; Lattal, K. Matthew

    2006-01-01

    Much is known about the neurobiology of memory storage for learned fear. In contrast, the molecular mechanisms underlying extinction of fear memory are just beginning to be delineated. Here, we investigate the role of protein kinase A (PKA) in extinction of memory for contextual fear by using conventional and temporally regulated transgenic approaches that allow us to inhibit PKA activity in neurons within brain regions thought to be involved in extinction. Strikingly, reduction of PKA activi...

  10. Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

    Smith, A Ian; Lew, Rebecca A.; Thomas, Walter G; Tochon-Danguy, Nathalie

    2006-01-01

    The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC)....

  11. Modular Total Synthesis of Protein Kinase C Activator (-)-Indolactam V.

    Haynes-Smith, Jeremy; Diaz, Italia; Billingsley, Kelvin L

    2016-05-01

    A concise, eight-step total synthesis of (-)-indolactam V, a nanomolar agonist of protein kinase C, is reported. The synthesis relies upon an efficient copper-catalyzed amino acid arylation to establish the indole C4-nitrogen bond. This cross-coupling method is applicable to a range of hydrophobic amino acids, providing a platform for further diversification of indolactam alkaloid scaffolds and studies on their potent biological activity. PMID:27074538

  12. Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

    Pai, J K; Dobek, E A; Bishop, W R

    1991-01-01

    Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of pho...

  13. Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells

    Webb, Jerry G.; Yang, Xiaofeng; Crosson, Craig E.

    2011-01-01

    Bradykinin stimulation of B2 kinin receptors has been shown to promote matrix metalloproteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cell...

  14. Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

    Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna

    2012-01-01

    Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tole...

  15. Mitogen-Activated Protein Kinase Signaling in Male Germ Cell Apoptosis in the Rat1

    Jia, Yue; Castellanos, Jesse; Wang, Christina; Sinha-Hikim, Indrani; Lue, YanHe; Swerdloff, Ronald S.; Sinha-Hikim, Amiya P.

    2008-01-01

    Programmed germ cell death is critical for functional spermatogenesis. Increased germ cell apoptosis can be triggered by various regulatory stimuli, including testicular hyperthermia or deprivation of gonadotropins and intratesticular testosterone. We have previously shown the involvement of the mitogen-activated protein kinase (MAPK) 14 in apoptotic signaling of male germ cells across species after hormone deprivation. This study investigates the role of MAPK14 in germ cell apoptosis in rats...

  16. Crosstalk between mitogen-activated protein kinases and mitochondria in cardiac diseases: therapeutic perspectives

    Javadov, Sabzali; Jang, Sehwan; Agostini, Bryan

    2014-01-01

    Cardiovascular diseases cause more mortality and morbidity worldwide than any other diseases. Although many intracellular signaling pathways influence cardiac physiology and pathology, the mitogen-activated protein kinase (MAPK) family has garnered significant attention because of its vast implications in signaling and cross-talk with other signaling networks. The extensively studied MAPKs ERK1/2, p38, JNK, and ERK5, demonstrate unique intracellular signaling mechanisms, responding to a myria...

  17. The Receptor for Activated C Kinase in Plant Signaling: Tale of a Promiscuous Little Molecule

    Islas-Flores, Tania; Rahman, Ahasanur; Ullah, Hemayet; Villanueva, Marco A.

    2015-01-01

    Two decades after the first report of the plant homolog of the Receptor for Activated C Kinase 1 (RACK1) in cultured tobacco BY2 cells, a significant advancement has been made in the elucidation of its cellular and molecular role. The protein is now implicated in many biological functions including protein translation, multiple hormonal responses, developmental processes, pathogen infection resistance, environmental stress responses, and miRNA production. Such multiple functional roles are co...

  18. Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

    Grimsey, Neil J.; Aguilar, Berenice; Smith, Thomas H.; Le, Phillip; Soohoo, Amanda L.; Puthenveedu, Manojkumar A.; Nizet, Victor

    2015-01-01

    Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption. PMID:26391660

  19. Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials.

    Fiore, Mario; Forli, Stefano; Manetti, Fabrizio

    2016-04-28

    The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases to prevent tumor metastasis and to increase tumor sensitivity to chemotherapeutics. PMID:26502061

  20. Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration

    Polyphosphate kinase partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity. The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor. Chromatographic analyses of the product mixture produced from ADP and [32P]polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase, adenosine triphosphatase, and adenylate kinase. Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C. It may be stored indefinitely as a lyophilized powder at -10 degrees C. Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme. A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium. The ease of purification and immobilization of E. coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration

  1. Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

    Feucht, B U; Saier, M H

    1980-01-01

    Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cel...

  2. IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

    Tai Sang Lee; Hyun Ju Song; Ji Hoon Jeong; Young Sil Min; Chang Yell Shin; Uy Dong Sohn

    2006-01-01

    AIM: To examine the pathway related to the IL-1β-induced activation of mitogen-activated protein (MAP)kinases in cat esophageal smooth muscle cells.METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1β. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases.RESULTS: In the primary cultured cells, although IL-1βfailed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP klnase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively.The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level.Phosphokinase C (PKC) was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity.In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds.CONCLUSION: Based on these results, IL-1β-Induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1β.

  3. The CK2 kinase stabilizes CLOCK and represses its activity in the Drosophila circadian oscillator.

    Aron Szabó

    Full Text Available Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER and TIMELESS (TIM proteins, which inhibit CLOCK (CLK transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory β subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.

  4. Protein kinase a activity is increased in rat heart during late hypodynamic phase of sepsis.

    Yang, S L; Hsu, C; Lue, S I; Hsu, H K; Liu, M S

    1997-07-01

    Changes in the activities of protein kinase A (PKA, or cAMP-dependent protein kinase) in rat heart during different cardiodynamic phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals killed at 9 and 18 h, respectively, after cecal ligation and puncture. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two peaks of PKA, type I (eluted at low ionic strength) and type II (eluted at high ionic strength), were collected and their activities were determined based on the rate of incorporation of [gamma-32P]ATP into histone. Results obtained show that during early sepsis, both type I and type II PKA activities were unaffected. During late sepsis, type I PKA activities were stimulated by 66.7-97.7%, while type II PKA activities remained constant. Kinetic analysis of the data on type I PKA during late sepsis reveals that the Vmax values for ATP, cAMP, and histone were increased by 84.7, 66.7, and 97.7%, respectively; while the Km values for ATP, cAMP, and histone were unaltered. These data indicate that type I PKA is activated in rat heart during late hypodynamic phase of sepsis. Since kinase-mediated phosphorylation plays an important role in regulating myocardial function and metabolism, an activation of type I PKA during late sepsis may contribute to the development of altered myocardial function during hypodynamic phase of sepsis. PMID:9249915

  5. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  6. Suppression of Platelet Aggregation by Bordetella pertussis Adenylate Cyclase Toxin

    Iwaki, Masaaki; Kamachi, Kazunari; Heveker, Nikolaus; Konda, Toshifumi

    1999-01-01

    The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 μM)-induced aggregation of rabbit platelets at 3 μg/ml and strongly suppressed thrombin (0.2 U/ml)-induced aggregation at 10 μg/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive p...

  7. Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites.

    Simon, Bertrand; Huart, Anne-Sophie; Temmerman, Koen; Vahokoski, Juha; Mertens, Haydyn D T; Komadina, Dana; Hoffmann, Jan-Erik; Yumerefendi, Hayretin; Svergun, Dmitri I; Kursula, Petri; Schultz, Carsten; McCarthy, Andrew A; Hart, Darren J; Wilmanns, Matthias

    2016-06-01

    The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation. PMID:27133022

  8. Analysis of Somatic Mutations in Cancer: Molecular Mechanisms of Activation in the ErbB Family of Receptor Tyrosine Kinases

    The ErbB/EGFR/HER family of kinases consists of four homologous receptor tyrosine kinases which are important regulatory elements in many cellular processes, including cell proliferation, differentiation, and migration. Somatic mutations in, or over-expression of, the ErbB family is found in many cancers and is correlated with a poor prognosis; particularly, clinically identified mutations found in non-small-cell lung cancer (NSCLC) of ErbB1 have been shown to increase its basal kinase activity and patients carrying these mutations respond remarkably to the small tyrosine kinase inhibitor gefitinib. Here, we analyze the potential effects of the currently catalogued clinically identified mutations in the ErbB family kinase domains on the molecular mechanisms of kinase activation. Recently, we identified conserved networks of hydrophilic and hydrophobic interactions characteristic to the active and inactive conformation, respectively. Here, we show that the clinically identified mutants influence the kinase activity in distinctive fashion by affecting the characteristic interaction networks

  9. Sestrins Inhibit mTORC1 Kinase Activation through the GATOR Complex

    Anita Parmigiani

    2014-11-01

    Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition.

  10. The Mitogen-Activated Protein Kinase (MAPK) Pathway: Role in Immune Evasion by Trypanosomatids.

    Soares-Silva, Mercedes; Diniz, Flavia F; Gomes, Gabriela N; Bahia, Diana

    2016-01-01

    Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host's MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. PMID:26941717

  11. A highly sensitive square wave voltammetry based biosensor for kinase activity measurements.

    Snir, Elza; Amit, Einav; Friedler, Assaf; Yitzchaik, Shlomo

    2015-09-01

    An electrochemical biosensor has been developed for ultrasensitive, label-free determination of protein kinase activity. The sensor is composed of a unique peptide monolayer on a gold electrode. It identifies the order change in the monolayer upon phosphorylation, via square wave voltametry (SWV) measurements. Disorder caused by the introduction of the phosphate groups onto the middle of the peptide sequence results in pinhole formation and therefore an increase in the electrochemical signal. The measured sensitivity was 100 nM of kinase and the dynamic range was 100 nM up to 11 μM. Sensitivity was an order of magnitude higher, and the dynamic range wider by two orders of magnitude, as compared to our previously reported impedimetric method, in which the sensitivity was 1 μM, and the dynamic range was 1-20 μM. PMID:25851749

  12. Activation of sphingosine kinase-1 in cancer: implications for therapeutic targeting.

    Cuvillier, Olivier; Ader, Isabelle; Bouquerel, Pierre; Brizuela, Leyre; Malavaud, Bernard; Mazerolles, Catherine; Rischmann, Pascal

    2010-06-01

    Sphingolipid metabolites are critical to the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation, cell survival and angiogenesis. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because its produces the pro-survival and pro-angiogenic sphingosine 1-phosphate and decreases the amount of both ceramide and sphingosine, the pro-apoptotic sphingolipids. Moreover, its gene is oncogenic, its mRNA is overproduced in several solid tumors, its overexpression protects cells from apoptosis, and its activity is down-regulated by anti-cancer treatments. Therefore, the sphingosine kinase-1/sphingosine 1-phosphate signaling pathway appears to be a target of interest for therapeutic manipulation. PMID:20302564

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

    Fuglsang, AT; Kristensen, A; Cuin, TA;

    2014-01-01

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

  14. Jiawei Wendan decoction affects mitogen-activated protein kinase signal pathway in the hippocampus of depression rats

    Liping Zhang; Man Zhang; Li Wu; Meng Xia; Guangbin Li

    2011-01-01

    A previous study from our group showed that Jiawei Wendan decoction inhibits protein expression of interleukin-1β, 2, and 6, as well as plasma neuropeptide Y, P substance and somatostatin in the hippocampus of depression rat models. The present study analyzed the influence of Jiawei Wendan decoction on the mitogen-activated protein kinase signal transduction pathway in the hippocampus. Results demonstrated that Jiawei Wendan decoction effectively upregulated expression of small molecular G proteins, extracellular regulated kinase 1/2, and activated ribosomal S6 kinase protein in the rat hippocampus. In addition, Jiawei Wendan decoction exhibits antidepressant effects similar to fluoxetine. The underlying mechanisms were shown to be dependent on increased mitogen-activated protein kinase signal transduction pathway activity.

  15. Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing.

    Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding

    2016-01-21

    Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody-antigen interaction in the presence of casein kinase II (CK2) and adenosine 5'-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis. PMID:26724763

  16. The Role of MEK Kinase 1 in Bcr-Abl-Induced Self-Renewal Activity of Embryonic Stem Cells

    NAKAMURA, Yukinori; Yujiri, Toshiaki; Tanizawa, Yukio

    2006-01-01

    BCR-ABL oncogene, the moecular hallmark of chronic mylogenous leukemia, arises in a promitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to sctivate signal transducers and activators of transcription 3 (STAT3) and to promote self-renewal in embryonic stem (ES)cells, even in the absencs of leukemia in hibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase ...

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

    Bernard, Laura P.; Zhang, Huaye

    2015-01-01

    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 hippoc...

  18. Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase.

    Mustelin, T; Coggeshall, K M; Altman, A

    1989-01-01

    T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endo...

  19. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  20. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K;

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  1. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity

    Harada, Hisashi; Quearry, Bonnie; Ruiz-Vela, Antonio; Korsmeyer, Stanley J.

    2004-01-01

    The “BH3-only” proapoptotic BCL-2 family members initiate the intrinsic apoptotic pathway. A small interfering RNA knockdown of BIM confirms this BH3-only member is important for the cytokine-mediated homeostasis of hematopoietic cells. We show here that the phosphorylation status of BIM controls its proapoptotic activity. IL-3, a hematopoietic survival factor, induces extracellular signal-regulated kinase/mitogen-activated protein kinase-mediated phosphorylation of BIM on three serine sites ...

  2. Differential activation of protein kinase A in various regions of myocardium during sepsis.

    Hsu, C; Yang, S L; Hsu, S P; Hsu, H K; Liu, M S

    1997-08-01

    Changes in the activities of protein kinase A (PKA) (cAMP-dependent protein kinase) in various regions of rat myocardium during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 hr, respectively, after CLP. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two types of PKA, Type I (eluted at low ionic strength) and Type II (eluted at high ionic strength), were collected, and their activities were determined based on the rate of incorporation of [gamma-32P]ATP into histone. Under physiological conditions, Type I PKA activities were unevenly distributed (left atrium > right atrium > pacemaker region > left ventricle > right ventricle > ventricular septum) while Type II PKA activities were evenly distributed among different regions of myocardium. During early sepsis, Type I PKA activities remained unchanged while Type II PKA activities were activated by 32 and 70% in right atrium and pacemaker regions, respectively. During late sepsis, Type I PKA activities were stimulated by 228% in ventricular septum while Type II PKA activities were not affected. These data demonstrate that different PKA activities exist in various regions of the myocardium and that PKA activities were preferentially activated in certain areas during the progression of sepsis. Since PKA plays an important role in the regulation of myocardial function and metabolism, the activation of PKA in different regions of myocardial during different stages of sepsis may contribute to the altered cardiac function during the progression of sepsis. PMID:9299285

  3. The effect of midazolam on neutrophil mitogen-activated protein kinase.

    Ghori, Kamran

    2010-06-01

    Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b\\/CD18.

  4. Mitogen-Activated Protein Kinase Pathway: Genetic Analysis of 95 Adrenocortical Tumors.

    Rubin, Beatrice; Monticelli, Halenya; Redaelli, Marco; Mucignat, Carla; Barollo, Susi; Bertazza, Loris; Mian, Caterina; Betterle, Corrado; Iacobone, Maurizio; Fassina, Ambrogio; Boscaro, Marco; Pezzani, Raffaele; Mantero, Franco

    2015-01-01

    Mitogen-activated protein kinase (MAPK) pathway is often deregulated in adrenocortical tumors (ACT) but with no concrete data confirming alteration rate. The objective of this study was to evaluate genetic alterations in key components of MAPK pathway. We found one BRAF mutation (p.V600E) and four HRAS silent mutations. No alteration was found in NRAS, KRAS, EGFR genes. The patient carrying BRAF mutation was further characterized by investigating his biomolecular and clinico-pathological findings. Therefore, even if MAPK signaling is activated in ACT, our results suggest that genetic alterations do not seem to represent a frequent mechanism of ACT tumorigenesis. PMID:26536286

  5. Modulation of IκB kinase autophosphorylation and activity following brain ischemia

    SHENWan-Hua; ZHANGHun-Yi; 等

    2003-01-01

    ATIM:To investigate the effects of different antagonists on the alteration of IκB kinase(IKK)activity in rat hipoocampus folluwing global brain ischemia,METHODS:Using 4-vessel occlusion(4-VO)as brain ischemia model,IKK protein expression was examined by immunoblotting and immunoprecipitation,and IKK activity was assayed by in vitro kinase assay.RESULTS:There was no alteration of IKK protein expression following ischemia or ischemia/reperfusion different time points,but IKK activity reached its peak level at ischemia 30min.Pretreatment with N-methyl-D-aspartate(NMDA)receptor antagonist ketamine,non-NMDA receptor antagonist DNQX,or NFκB inhibitor PDTC decreased the IKK activity following brain ischemia 30min.The increase in substrate myelin basic protein(MBP)phosphorylation by IKK is associated with an increase in autophosphorylation of IKK,which can also be antagonized by ketamine,DNQX,and PDTC,CONCLUSION:NMDA receptor and non-NMDA receptor mediate the increase of IKK activity following global brain ischemia in rat hippocampus,which contributes to the alterations of expression and activity of downstream factor NF-κB.

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

    Dennis J Templeton

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

  7. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex.

    Cirnaru, Maria D; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

    2014-01-01

    Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

  8. High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.

    Demian, Douglas J; Clugston, Susan L; Foster, Meta M; Rameh, Lucia; Sarkes, Deborah; Townson, Sharon A; Yang, Lily; Zhang, Melvin; Charlton, Maura E

    2009-08-01

    Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. PMID:19641220

  9. Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren's disease fibroblasts

    Krause Carola

    2011-06-01

    Full Text Available Abstract Background Dupuytren's disease is a fibroproliferative disorder of the palmar fascia. The treatment used to date has mostly been surgery, but there is a high recurrence rate. Transforming growth factor β (TGF-β has been implicated as a key stimulator of myofibroblast activity and fascial contraction in Dupuytren's disease. Results We studied Dupuytren's fibroblasts in tissues ex vivo and in cells cultured in vitro and found increased TGF-β expression compared to control fibroblasts. This correlated not only with elevated expression and activation of downstream Smad effectors but also with overactive extracellular signal-regulated kinase 1/2 (ERK1/2/mitogen-activated protein (MAP kinase signalling. Treatment with the TGF-β type I receptor kinase inhibitor SB-431542 and bone morphogenetic protein 6 (BMP6 led to inhibition of elevated Smad and ERK1/2/MAP kinase signalling as well as to inhibition of the increased contractility of Dupuytren's fibroblasts. BMP6 attenuated TGF-β expression in Dupuytren's fibroblasts, but not in control fibroblasts. Platelet-derived growth factor (PDGF expression was strongly promoted by TGF-β in Dupuytren's fibroblasts and was curbed by SB-431542 or BMP6 treatment. High basal expression of phosphorylated ERK1/2 MAP kinase and fibroproliferative markers was attenuated in Dupuytren's fibroblasts by a selective PDGF receptor kinase inhibitor. Cotreatment of Dupuytren's fibroblasts with SB-431542 and the mitogen-activated protein kinase kinase 1 inhibitor PD98059 was sufficient to abrogate proliferation and contraction of Dupuytren's fibroblasts. Conclusions Both TGF-β and ERK1/2 MAP kinase pathways cooperated in mediating the enhanced proliferation and high spontaneous contraction of Dupuytren's fibroblasts. Our data indicate that both signalling pathways are prime targets for the development of nonsurgical intervention strategies to treat Dupuytren's disease.

  10. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of...... tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of...

  11. Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

    Patel, Priyank; Asbach, Benedikt; Shteyn, Elina; Gomez, Cindy; Coltoff, Alexander; Bhuyan, Sadia; Tyner, Angela L; Wagner, Ralf; Blain, Stacy W

    2015-05-01

    Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity. PMID:25733683

  12. Antibacterial and EGFR-Tyrosine Kinase Inhibitory Activities of Polyhydroxylated Xanthones from Garcinia succifolia

    Susawat Duangsrisai

    2014-11-01

    Full Text Available Chemical investigation of the methanol extract of the wood of Garcinia succifolia Kurz (Clusiaceae led to the isolation of 1,5-dihydroxyxanthone (1, 1,7-dihydroxyxanthone (2, 1,3,7-trihydroxyxanthone (3, 1,5,6-trihydroxyxanthone (4, 1,6,7-trihydroxyxanthone (5, and 1,3,6,7-tetrahydroxyxanthone (6. All of the isolated xanthones were evaluated for their antibacterial activity against bacterial reference strains, two Gram-positive (Staphylococcus aureus ATTC 25923, Bacillus subtillis ATCC 6633 and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and environmental drug-resistant isolates (S. aureus B1, Enteroccoccus faecalis W1, and E. coli G1, as well as for their epidermal growth factor receptor (EGFR of tyrosine kinase inhibitory activity. Only 1,5,6-trihydroxy-(4, 1,6,7-trihydroxy-(5, and 1,3,6,7-tetrahydroxyxanthones (6 exhibited antibacterial activity against Gram-positive bacteria, however none was active against vancomycin-resistant E. faecalis. Additionally, 1,7-dihydroxyxanthone (2 showed synergism with oxacillin, but not with ampicillin. On the other hand, only 1,5-dihydroxyxanthone (1 and 1,7-dihydroxyxanthone (2 were found to exhibit the EGFR-tyrosine kinase inhibitory activity, with IC50 values of 90.34 and 223 nM, respectively.

  13. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  14. Atheroprotective effects of antioxidants through inhibition of mitogen-activated protein kinases

    Moe KYAW; Masanori YOSHIZUMI; Koichiro TSUCHIYA; Yuki IZAWA; Yasuhisa KANEMATSU; Toshiaki TAMAKI

    2004-01-01

    Reactive oxygen species (ROS) have been known to play an important role in the pathogenesis of atherosclerosis and several other cardiovascular diseases. It is now apparent that ROS induce endothelial cell damage and vascular smooth muscle cell (VSMC) growth and cardiac remodeling, which are associated with hypertension,atherosclerosis, heart failure, and restenosis. Several lines of evidence have indicated that ROS and mitogenactivated protein (MAP) kinases were involved in vascular remodeling under various pathological conditions. Recenfiy,it was also reported that MAP kinases were sensitive to oxidative stress. MAP kinases play an important role in cell differentiation, growth, apoptosis, and the regulation of a variety of transcription factors and gene expressions.Bioflavonoids and polyphenolic compounds are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases. One of the most widely distributed bioflavonoids, 3,3',4',5,7-pentahydroxyflavone (quercetin) and its metabolite quercetin 3-O-β-D-glucuronide (Q3GA) inhibited Angiotensin Ⅱstimulated JNK activation and resultant hypertrophy of VSMC. Several studies have suggested that various antioxidants including probucol, N-acetyl-L-cysteine, diphenylene iodonium, Trolox C (vitamin E analogue), and vitamin C inhibit VSMC growth, which is associated with pathogenesis of cardiovascular diseases. Therefore, inhibition of MAP kinases by antioxidant treatment may prove to be a therapeutic strategy for cardiovascular diseases. In contrast, some clinical studies have reported that antioxidant vitamins did not show beneficial effects in coronary artery disease or in a number of high-risk people. Thus, further studies are needed to clarify why antioxidants showed beneficial effects in vitro, whereas less satisfactory results were obtained in some clinical conditions.

  15. Translocation and activation of protein kinase C by the plasma cell tumor-promoting alkane pristane.

    Janz, S; Gawrisch, K; Lester, D S

    1995-02-01

    Pristane (2,6,10,14-tetramethylpentadecane) is a C19-isoalkane that promotes the development of plasmacytomas in genetically susceptible BALB/c mice. Similarities between the effects of pristane and protein kinase C (PKC)-activating phorbol esters suggested that the tumor promoting activity of pristane might involve the activation of PKC. Here we show that up to 5 mol% of pristane can be homogeneously incorporated into phosphatidylcholine/phosphatidylserine bilayers. Membrane-incorporated pristane partially activated PKC and increased phorbol ester binding to the bilayer by more than 50%. Pristane (50 microM) delivered as an inclusion complex with beta-cyclodextrin to promyelocytic HL-60 leukemia cells induced a partial long-term translocation of PKC to the cell membrane. This was accompanied by differentiation of HL-60 cells into macrophage-like cells. It is concluded that activation of PKC may comprise an important aspect of the tumor promoting potential of pristane. PMID:7834620

  16. Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

    Takahiro Nemoto; Shunichiro Kubota; Hideyuki Ishida; Nobuo Murata; Daijo Hashimoto

    2005-01-01

    AIM: To investigate the expressions of omithine decarboxylase (ODC), MMP-2, and Erk, and their relationship in human colon tumors.METHODS: ODC activity, MMP-2 expression, and mitogenactivated protein (MAP) kinase activity (Erk phosphorylation) were determined in 58 surgically removed human colon tumors and their adjacent normal tissues, using [1-14C]-ornithine as a substrate, ELISA assay, and Western blotting, respectively.RESULTS: ODC activity, MMP-2 expression, and Erk phosphorylation were significantly elevated in colon tumors, compared to those in adjacent normal tissues. A significant correlation was observed between ODC activities and MMP-2 levels.CONCLUSION: This is the first report showing a significant correlation between ODC activities and MMP-2 levels in human colon tumors. As MMP-2 is involved in cancer invasion and metastasis, and colon cancer overexpresses ODC, suppression of ODC expression may be a rational approach to treat colon cancer which overexpresses ODC.

  17. Oxidized LDL impair adipocyte response to insulin by activating serine/threonine kinases.

    Scazzocchio, Beatrice; Varì, Rosaria; D'Archivio, Massimo; Santangelo, Carmela; Filesi, Carmelina; Giovannini, Claudio; Masella, Roberta

    2009-05-01

    Oxidized LDL (oxLDL) increase in patients affected by type-2 diabetes, obesity, and metabolic syndrome. Likewise, insulin resistance, an impaired responsiveness of target tissues to insulin, is associated with those pathological conditions. To investigate a possible causal relationship between oxLDL and the onset of insulin resistance, we evaluated the response to insulin of 3T3-L1 adipocytes treated with oxLDL. We observed that oxLDL inhibited glucose uptake (-40%) through reduced glucose transporter 4 (GLUT4) recruitment to the plasma membrane (-70%), without affecting GLUT4 gene expression. These findings were associated to the impairment of insulin signaling. Specifically, in oxLDL-treated cells insulin receptor (IR) substrate-1 (IRS-1) was highly degraded likely because of the enhanced Ser(307)phosphorylation. This process was largely mediated by the activation of the inhibitor of kappaB-kinase beta (IKKbeta) and the c-Jun NH(2)-terminal kinase (JNK). Moreover, the activation of IKKbeta positively regulated the nuclear content of nuclear factor kappaB (NF-kappaB), by inactivating the inhibitor of NF-kappaB (IkappaBalpha). The activated NF-kappaB further impaired per se GLUT4 functionality. Specific inhibitors of IKKbeta, JNK, and NF-kappaB restored insulin sensitivity in adipocytes treated with oxLDL. These data provide the first evidence that oxLDL, by activating serine/threonine kinases, impaired adipocyte response to insulin affecting pathways involved in the recruitment of GLUT4 to plasma membranes (PM). This suggests that oxLDL might participate in the development of insulin resistance. PMID:19136667

  18. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    Highlights: ► Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. ► BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. ► BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. ► ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl2) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl2, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl2 exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl2-induced ERK5 but not ERK1/2 phosphorylation. The CdCl2-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl2. These findings suggest that ERK5 pathway activation by CdCl2 exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  19. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.

    Mendez, Aaron S; Alfaro, Jennifer; Morales-Soto, Marisol A; Dar, Arvin C; McCullagh, Emma; Gotthardt, Katja; Li, Han; Acosta-Alvear, Diego; Sidrauski, Carmela; Korennykh, Alexei V; Bernales, Sebastian; Shokat, Kevan M; Walter, Peter

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. PMID:25986605

  20. Activation of NMDA receptors leads to phosphorylation of TRPV1 S800 by protein kinase C and A-Kinase anchoring protein 150 in rat trigeminal ganglia

    Lee, Jongseok; Chung, Man-Kyo; Ro, Jin Y.

    2012-01-01

    A-Kinase anchoring protein 150 (AKAP150) is required for the phosphorylation of transient receptor potential cation channel subfamily V member 1 (TRPV1) by PKA or PKC in sensory neurons and, hence, affects TRPV1-dependent hyperalgesia under pathological conditions. Recently, we showed that the activation of N-methyl-d-aspartate (NMDA) receptors sensitizes TRPV1 by enhancing serine phosphorylation through PKC in trigeminal nociceptors. In this study, we extended this observation by investigati...