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Sample records for bacterial-like deoxyribonucleoside kinases

  1. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  3. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  4. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-01

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

  5. Deoxyribonucleoside kinases in mitochondrial DNA depletion.

    Science.gov (United States)

    Saada-Reisch, Ann

    2004-10-01

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

  6. Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Petersen, G.E.; Sandrini, Michael

    2003-01-01

    In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities......, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine...

  7. Animal deoxyribonucleoside kinases: 'forward' and 'retrograde' evolution of their substrate specificity

    DEFF Research Database (Denmark)

    Piskur, Jure; Sandrini, Michael P; Knecht, Wolfgang

    2004-01-01

    Deoxyribonucleoside kinases, which catalyse the phosphorylation of deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate...

  8. Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

    DEFF Research Database (Denmark)

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

    2007-01-01

    Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathog...... alternative for combating pathogenic bacteria.......Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against...... pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside...

  9. Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

    Science.gov (United States)

    Saada, Ann; Ben-Shalom, Efrat; Zyslin, Rivka; Miller, Chaya; Mandel, Hanna; Elpeleg, Orly

    2003-10-24

    Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

  10. Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Rozpedowska, E.; Le Breton, C.

    2007-01-01

    Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution...

  11. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.

    2007-01-01

    To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Several FDA-approved nucleoside analogue drugs were screened for their potential bacteric......-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria....

  12. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang

    2005-01-01

    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3...

  13. Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'-exonuclease activity

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Zanten, Gabriella van; Berenstein, Dvora

    2016-01-01

    We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development......-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated....

  14. Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Christiansen, Louise Slot; Clausen, Anders R.

    2016-01-01

    , among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of d...... substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs...

  15. Nucleoside analog toxicity and nucleoside kinase deficiency : Effects on mitochondrial DNA

    OpenAIRE

    Bjerke, Mia

    2008-01-01

    Nucleoside analogs are modified nucleosides used in treatment of cancer and viral infections. They are dependent on intracellular phosphorylation to be pharmacologically active. Deoxyribonucleoside kinases catalyze the rate-limiting step in the phosphorylation of many clinically used nucleoside analogs. Human cells contain four distinct deoxyribonucleoside kinases that have partially overlapping substrate specificities for both naturally occurring deoxyribonucleosides as wel...

  16. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarcha...

  17. Human biodistribution and radiation dosimetry of novel PET probes targeting the deoxyribonucleoside salvage pathway

    Energy Technology Data Exchange (ETDEWEB)

    Schwarzenberg, Johannes [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Ahmanson Biological Imaging Division, Los Angeles, CA (United States); Medical University of Vienna, Department of Pediatrics, Vienna (Austria); Radu, Caius G.; Tran, Andrew Q.; Phelps, Michael E.; Satyamurthy, Nagichettiar [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, Los Angeles, CA (United States); Benz, Matthias; Fueger, Barbara; Czernin, Johannes; Schiepers, Christiaan [David Geffen School of Medicine, University of California, Department of Molecular and Medical Pharmacology, Ahmanson Biological Imaging Division, Los Angeles, CA (United States); Witte, Owen N. [David Geffen School of Medicine, University of California, Howard Hughes Medical Institute and Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA (United States)

    2011-04-15

    Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway involved in the production and maintenance of a balanced pool of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis. dCK phosphorylates and therefore activates nucleoside analogs such as cytarabine, gemcitabine, decitabine, cladribine, and clofarabine that are used routinely in cancer therapy. Imaging probes that target dCK might allow stratifying patients into likely responders and nonresponders with dCK-dependent prodrugs. Here we present the biodistribution and radiation dosimetry of three fluorinated dCK substrates, {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC, developed for positron emission tomography (PET) imaging of dCK activity in vivo. PET studies were performed in nine healthy human volunteers, three for each probe. After a transmission scan, the radiopharmaceutical was injected intravenously and three sequential emission scans acquired from the base of the skull to mid-thigh. Regions of interest encompassing visible organs were drawn on the first PET scan and copied to the subsequent scans. Activity in target organs was determined and absorbed dose estimated with OLINDA/EXM. The standardized uptake value was calculated for various organs at different times. Renal excretion was common to all three probes. Bone marrow had higher uptake for L-{sup 18}F-FAC and L-{sup 18}F-FMAC than {sup 18}F-FAC. Prominent liver uptake was seen in L-{sup 18}F-FMAC and L-{sup 18}F-FAC, whereas splenic activity was highest for {sup 18}F-FAC. Muscle uptake was also highest for {sup 18}F-FAC. The critical organ was the bladder wall for all three probes. The effective dose was 0.00524, 0.00755, and 0.00910 mSv/MBq for {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC, respectively. The biodistribution of {sup 18}F-FAC, L-{sup 18}F-FAC, and L-{sup 18}F-FMAC in humans reveals similarities and differences. Differences may be explained by different probe

  18. Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts.

    Science.gov (United States)

    Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

    2010-01-01

    Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. Sequence data from the present study have been deposited in the EMBL database/GenBank under accession numbers: AT3G07800.1 (AtTK1a), At5G...

  20. Studies on Transcriptional Incorporation of 5'-N-Triphosphates of 5'-Amino-5'-Deoxyribonucleosides.

    Directory of Open Access Journals (Sweden)

    Weronika Kotkowiak

    Full Text Available In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleosides (5'NH NTPs. The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5'NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5'NH NTPs incorporation. Based on experimental data it is postulated that 5'NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.

  1. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    that phosphorylates all the natural deoxyribonucleosides and like insects, C. elegans only contains a single deoxyribonucleoside kinase-like gene. In contrast to the insects, however, the protein encoded by the elegans gene is 46 % identical to human TK1 (HuTK1) and have no homology to the insect kinase. Like HuTK1...... the C. elegans kinase (CeTK1) has thymidine as the preferred substrate, but it also displays activity with deoxyguanosine, though with high Km. A number of point mutations have been introduced in the active site of both the human and elegans TK's in order to change the substrate specificity away from...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site....

  2. Application of 2-cyanoethyl n,n,n′,n′-tetraisopropylphosphorodiamidite for in situ preparation of deoxyribonucleoside phosphoramidites and their use in polymer-supported synthesis of oligodeoxyribonucleotides

    DEFF Research Database (Denmark)

    Nielsen, John; Taagaard, Michael; Marugg, John E.

    1986-01-01

    Deoxyribonucleoside phosphoramidites are prepared in situ from 5′-O,N-protected deoxyribonucleosides and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite with tetrazole as catalyst, and the solutions applied directly on an automatic solid-phase DNA synthesizer. Using LCAA-CPG support and a...

  3. Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

    DEFF Research Database (Denmark)

    Mikkelsen, Niels Egil; Munch-Petersen, Birgitte; Eklund, Hans

    2008-01-01

    -drug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. Here we present crystal structures of dNK complexed with four different nucleoside analogs floxuridine (5FdU), brivudine (BVDU), zidovudine (AZT) and zalcitabine (ddC...

  4. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Egeblad, Louise; Munch-Petersen, Birgitte

    2015-01-01

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been t...

  5. Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts

    OpenAIRE

    Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

    2010-01-01

    Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside...

  6. Convenient synthesis of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-methyl-2'-deoxypseudoisocytidine

    CSIR Research Space (South Africa)

    Wellington, Kevin W

    2009-04-01

    Full Text Available The syntheses of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-methyl-2'- deoxypseudoisocytidine via Heck coupling are described. A survey of the attempts to use the Heck coupling to synthesize N,N'-dibenzyl-2,4-diaminopyrimidine...

  7. DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Brendel, M.; Faeth, W.W.; Toper, R.

    1975-01-01

    Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

  8. The influence of calf thymus DNA and deoxyribonucleosides on the induction of different mutation types in Drosophila

    International Nuclear Information System (INIS)

    Ondrej, M.

    1975-01-01

    The influence of an exogenous DNA on the induction of mutations by X rays was compared with the influence of an equimolar mixture of four deoxyribonucleosides. Pre-treatment and post-treatment with the calf thymus DNA did not influence mutation frequency in the specific loci dp, b, cn and bw as well as Minute mutations induced in the Drosophila sperm by X radiation. Pre-treatment with the equimolar mixture of four deoxyribonucleosides increased the frequency of the Minutes but did not affect mutation frequency in the loci dp, b, cn, bw. The equimolar mixture of nucleosides alone induced a low frequency of Minute mutations in the Drosophila sperm. DNA alone induced a low frequency of recessive lethals. These lethals arose as mosaics of small sectors of the gonads of the F 1 females and were revealed as late as in the F 3 generation. (author)

  9. Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

    Science.gov (United States)

    Hocek, Michal

    2014-11-07

    The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

  10. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  11. Cloning and expression of human deoxycytidine kinase cDNA

    International Nuclear Information System (INIS)

    Chottiner, E.G.; Shewach, D.S.; Datta, N.S.; Ashcraft, E.; Gribbin, D.; Ginsburg, D.; Fox, I.H.; Mitchell, B.S.

    1991-01-01

    Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, the authors have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine

  12. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  13. Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts

    International Nuclear Information System (INIS)

    Franzolin, Elisa; Rampazzo, Chiara; Perez-Perez, Maria-Jesus; Hernandez, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

    2006-01-01

    Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1 - cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(β-D-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1

  14. Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.

    Science.gov (United States)

    Franzolin, Elisa; Rampazzo, Chiara; Pérez-Pérez, María-Jesús; Hernández, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

    2006-05-26

    Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

  15. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

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

  16. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

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

  17. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    The central enzyme on the de novo pathway for synthesis of DNA precursors, the deoxyribonucleoside triphosphates, is ribonucleotide reductase (RNR). Deoxythymidine triphosphate (dTTP) has a key role in control of RNR activity shifting the specificity from pyrimidine to purine nucleotide reduction...

  18. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    International Nuclear Information System (INIS)

    Slot Christiansen, Louise; Egeblad, Louise; Munch-Petersen, Birgitte; Piškur, Jure; Knecht, Wolfgang

    2015-01-01

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy

  19. Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion.

    Science.gov (United States)

    Lesko, Nicole; Naess, Karin; Wibom, Rolf; Solaroli, Nicola; Nennesmo, Inger; von Döbeln, Ulrika; Karlsson, Anna; Larsson, Nils-Göran

    2010-03-01

    Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity. Copyright 2009 Elsevier B.V. All rights reserved.

  20. New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

    Energy Technology Data Exchange (ETDEWEB)

    Slot Christiansen, Louise [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Egeblad, Louise [Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Munch-Petersen, Birgitte [Department of Science, Systems and Models, Roskilde University, Roskilde 4000 (Denmark); Piškur, Jure [Department of Biology, Lund University, Lund 22362 (Sweden); Knecht, Wolfgang, E-mail: Louise.Slot_Christiansen@biol.lu.se [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden)

    2015-06-08

    Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

  1. Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with alpha-acetates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA).

    Science.gov (United States)

    Cheng, Guang; Wang, Mingyao; Upadhyaya, Pramod; Villalta, Peter W; Hecht, Stephen S

    2008-03-01

    The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.

  2. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  3. Pyruvate kinase blood test

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2005-01-01

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

  5. Enterococcus faecalis phosphomevalonate kinase

    Science.gov (United States)

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

    2005-01-01

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

  6. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  7. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

    Science.gov (United States)

    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  8. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

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

    Science.gov (United States)

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

    2016-07-29

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

  10. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  11. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  12. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  13. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  14. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  15. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

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

  16. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  17. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

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

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

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

  20. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

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

  1. Signaling network of the Btk family kinases.

    Science.gov (United States)

    Qiu, Y; Kung, H J

    2000-11-20

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

  2. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

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

  3. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

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

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

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

  6. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

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

  7. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

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

  8. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

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

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

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-01-01

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

  10. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

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

    2017-11-01

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

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

    KAUST Repository

    Diaz Galicia, Miriam Escarlet

    2018-05-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  13. A rice kinase-protein interaction map.

    Science.gov (United States)

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

    2009-03-01

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

  14. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases Summary

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

  15. dependent/calmodulin- stimulated protein kinase from moss

    Indian Academy of Sciences (India)

    Unknown

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

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

    National Research Council Canada - National Science Library

    Lannigan, Deborah

    2006-01-01

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

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

    Science.gov (United States)

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

    2008-09-05

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

  18. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  19. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

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

  20. Protein Kinase A in Cancer

    International Nuclear Information System (INIS)

    Caretta, Antonio; Mucignat-Caretta, Carla

    2011-01-01

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

  1. Protein Kinase A in Cancer

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-02-28

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

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

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

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

  3. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

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

  4. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

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

  5. MAP kinase cascades in Arabidopsis innate immunity

    DEFF Research Database (Denmark)

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

    2012-01-01

    Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

  6. Protein Kinases in Human Breast Carcinoma

    National Research Council Canada - National Science Library

    Cane, William

    1998-01-01

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

  7. Ror receptor tyrosine kinases: orphans no more

    OpenAIRE

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

    2008-01-01

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

  8. Mediator kinase module and human tumorigenesis.

    Science.gov (United States)

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

    2015-01-01

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

  9. Fibronectin phosphorylation by ecto-protein kinase

    International Nuclear Information System (INIS)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

    1988-01-01

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

  10. The PIM kinases in hematological cancers.

    Science.gov (United States)

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

    2012-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

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

  12. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

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

  13. Crystal structure of Cryptosporidium parvum pyruvate kinase.

    Directory of Open Access Journals (Sweden)

    William J Cook

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    African Journals Online (AJOL)

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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ting-Lei Gu

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

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Konrad Kubiński

    2017-02-01

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

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

    Science.gov (United States)

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

    1997-01-01

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

  2. Mechanism of polyphosphate kinase from Propionibacterium shermanii

    International Nuclear Information System (INIS)

    Robinson, N.A.

    1986-01-01

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

  3. Radioimmunoassay of bovine heart protein kinase

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  4. The target landscape of clinical kinase drugs.

    Science.gov (United States)

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

    2017-12-01

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

  5. Janus kinase inhibitors: jackpot or potluck?

    Directory of Open Access Journals (Sweden)

    Pavithran Keechilat

    2012-06-01

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

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

    Science.gov (United States)

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

    2018-05-01

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  8. Protein Kinases in Shaping Plant Architecture.

    Science.gov (United States)

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

    2018-02-13

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

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

    Directory of Open Access Journals (Sweden)

    Julianne H. Grose

    2010-06-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  11. 2-Aminobenzimidazoles as potent Aurora kinase inhibitors.

    Science.gov (United States)

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

    2009-09-01

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

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

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

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

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

    Science.gov (United States)

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

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

  14. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Indian Academy of Sciences (India)

    2013-11-06

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

  17. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

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

    2006-01-01

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

  18. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  19. Plant PA signaling via diacylglycerol kinase

    NARCIS (Netherlands)

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

    2009-01-01

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

  20. Nonorthologous gene displacement of phosphomevalonate kinase

    NARCIS (Netherlands)

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

    2001-01-01

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

  1. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

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

    1992-01-01

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

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

    Indian Academy of Sciences (India)

    2012-01-19

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

  3. Kinase-Centric Computational Drug Development

    NARCIS (Netherlands)

    Kooistra, Albert J.; Volkamer, Andrea

    2017-01-01

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

  4. Kinases involved in Rec8 phosphorylation revealed

    Czech Academy of Sciences Publication Activity Database

    Anger, Martin

    2010-01-01

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

  5. Gene regulation by MAP kinase cascades

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2005-05-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

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

    OpenAIRE

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

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

  10. The Roles of Protein Kinases in Learning and Memory

    Science.gov (United States)

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

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

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

    Science.gov (United States)

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2018-01-01

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

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

    Science.gov (United States)

    Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

    2012-06-01

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

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

    Science.gov (United States)

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

    2015-01-06

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

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2011-02-10

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  18. Src family kinases in chronic kidney disease.

    Science.gov (United States)

    Wang, Jun; Zhuang, Shougang

    2017-09-01

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

  19. Tyrosine kinase signalling in breast cancer

    International Nuclear Information System (INIS)

    Hynes, Nancy E

    2000-01-01

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

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

    International Nuclear Information System (INIS)

    Boura, Evzen; Nencka, Radim

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-01

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

  2. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

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

    2008-11-01

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

  3. Aurora kinase inhibitors: Progress towards the clinic

    Czech Academy of Sciences Publication Activity Database

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

    2012-01-01

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

  4. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

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

    2011-01-01

    The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...... these signaling pathways have been exploited for the development of therapeutics and discuss the current knowledge of potential MAPK inhibitors and their anti-inflammatory effects in clinical trials related to IBD....

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

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Nencka, Radim

    2015-01-01

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

  6. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-08-01

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

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

    Science.gov (United States)

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

    2003-08-01

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

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

    Science.gov (United States)

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

    2000-09-12

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

    Science.gov (United States)

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

    2011-12-01

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

  11. PAK4 crystal structures suggest unusual kinase conformational movements.

    Science.gov (United States)

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

    2018-02-01

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

  12. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    OpenAIRE

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

    2000-01-01

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

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

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

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

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

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

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

  17. SH2 domains: modulators of nonreceptor tyrosine kinase activity

    OpenAIRE

    Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

    2009-01-01

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

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

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

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

    Science.gov (United States)

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

    2017-04-01

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

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

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Inger Lindin

    2014-03-01

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

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

    Science.gov (United States)

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2017-01-01

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

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

    Science.gov (United States)

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

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

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

    Science.gov (United States)

    Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

    2009-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Saadat U Aleem

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

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

    Science.gov (United States)

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

    1997-10-31

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

  12. Radioimmunoassay of creatine kinase BB isoenzyme

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-11-01

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

  13. Targeting the Pim kinases in multiple myeloma.

    LENUS (Irish Health Repository)

    Keane, N A

    2015-07-17

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

  14. Radioimmunoassay of creatine kinase BB isoenzyme

    International Nuclear Information System (INIS)

    Geng Jianguo

    1988-01-01

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

  15. Activation of oocyte phosphatidylinositol kinase by polyamines

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Pieterse Corné MJ

    2007-02-01

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

  17. Creatine kinase isozyme expression in embryonic chicken heart

    NARCIS (Netherlands)

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

    1989-01-01

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

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

    NARCIS (Netherlands)

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

    2018-01-01

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

  19. Oral protein kinase c β inhibition using ruboxistaurin

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  20. Enhanced expression of a calcium-dependent protein kinase

    Indian Academy of Sciences (India)

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

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

    Indian Academy of Sciences (India)

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Indian Academy of Sciences (India)

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

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

    Science.gov (United States)

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

    1996-01-01

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

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

    Science.gov (United States)

    Dragoi, Ana-Maria; Agaisse, Hervé

    2014-01-01

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

  6. 2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

    Science.gov (United States)

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

    2018-04-12

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

  7. Protein kinase substrate identification on functional protein arrays

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    2008-02-01

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

  8. Src protein-tyrosine kinase structure and regulation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2004-01-01

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

  9. Structural studies of Schistosoma mansoni adenylate kinases

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ramaswamy Rakshambikai

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

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

    Science.gov (United States)

    2017-01-01

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

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

    Science.gov (United States)

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

  13. Creatine kinase and creatine kinase subunit-B in coronary sinus blood in pacing-induced angina pectoris

    DEFF Research Database (Denmark)

    Bagger, J P; Ingerslev, J; Heinsvig, E M

    1982-01-01

    In nine out of 10 patients with angiographic documented coronary artery disease, pacing-induced angina pectoris provoked myocardial production of lactate, whereas no significant release of either creatine kinase or creatine kinase subunit-B to coronary sinus and peripheral venous blood could...

  14. Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

    Science.gov (United States)

    Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

    2015-12-01

    Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

    Science.gov (United States)

    Polte, T R; Hanks, S K

    1995-11-07

    The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

  16. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  17. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  18. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    International Nuclear Information System (INIS)

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-01-01

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

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

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

  20. Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

    Science.gov (United States)

    Dalton, George D; Dewey, William L

    2006-02-01

    Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

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

    Directory of Open Access Journals (Sweden)

    Anne Dettmann

    2012-09-01

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

  2. RhoA/Rho-Kinase in the Cardiovascular System.

    Science.gov (United States)

    Shimokawa, Hiroaki; Sunamura, Shinichiro; Satoh, Kimio

    2016-01-22

    Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system. © 2016 American Heart Association, Inc.

  3. Kinase inhibition by the Jamaican ball moss, Tillandsia recurvata L.

    Science.gov (United States)

    Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

    2012-10-01

    This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd(50)s were below 20 μg/ml. Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.

  4. Structure of the intact ATM/Tel1 kinase

    Science.gov (United States)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  5. The 'retro-design' concept for novel kinase inhibitors.

    Science.gov (United States)

    Müller, Gerhard; Sennhenn, Peter C; Woodcock, Timothy; Neumann, Lars

    2010-07-01

    Protein kinases are among the most attractive therapeutic targets for a broad range of diseases. This feature review highlights and classifies the main design principles employed to generate active and selective kinase inhibitors. In particular, emphasis is focused on a fragment-based lead-generation approach, which constitutes a novel design method for developing type II kinase inhibitors with distinct binding kinetic attributes. This 'retro-design' strategy relies on a customized fragment library, and contrasts the traditional approach used in the design of type II inhibitors.

  6. Second-generation inhibitors of Bruton tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Jingjing Wu

    2016-09-01

    Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

  7. Nucleolin (C23), a physiological substrate for casein kinase II

    DEFF Research Database (Denmark)

    Schneider, H R; Issinger, O G

    1988-01-01

    Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin...... phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated...... by purified casein kinase II with about two moles phosphate per one mole of nucleolin....

  8. Structural Studies of Archaealthermophilic Adenylate Kinase; TOPICAL

    International Nuclear Information System (INIS)

    Konisky, J.

    2002-01-01

    Through this DOE-sponsored program Konisky has studied the evolution and molecular biology of microbes that live in extreme environments. The emphasis of this work has been the determination of the structural features of thermophilic enzymes that allow them to function optimally at near 100 C. The laboratory has focused on a comparative study of adenylate kinase (ADK), an enzyme that functions to interconvert adenine nucleotides. Because of the close phylogenetic relatedness of members of the Methanococci, differences in the structure of their ADKs will be dominated by structural features that reflect contributions to their optimal temperature for activity, rather than differences due to phylogenetic divergence. We have cloned, sequenced and modeled the secondary structure for several methanococcal ADKs. Using molecular modeling threading approaches that are based on the solved structure for the porcine ADK, we have also proposed a general low resolution three dimensional structure for each of the methanococcal enzymes. These analyses have allowed us to propose structural features that confer hyperthermoactivity to those enzymes functioning in the hyperthermophilic members of the Methanococci. Using protein engineering methodologies, we have tested our hypotheses by examining the effects of selective structural changes on thermoactivity. Despite possessing between 68-81% sequence identity, the methanococcal AKs had significantly different stability against thermal denaturation, with melting points ranging from 69-103 C. The construction of several chimerical AKs by linking regions of the MVO and MJA AKs demonstrated the importance of cooperative interactions between amino- and carboxyl-terminal regions in influencing thermostability. Addition of MJA terminal fragments to the MVO AK increased thermal stability approximately 20 C while maintaining 88% of the mesophilic sequence. Further analysis using structural models suggested that hydrophobic interactions are

  9. Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

    Science.gov (United States)

    Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

    2009-06-15

    Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

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

    Science.gov (United States)

    Huang, Tsai-Lien; Huang, Hao-Jen

    2008-04-01

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

  11. Potassium sensing histidine kinase in Bacillus subtilis.

    Science.gov (United States)

    López, Daniel; Gontang, Erin A; Kolter, Roberto

    2010-01-01

    The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

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

    Science.gov (United States)

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

    2016-03-21

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

  14. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    Science.gov (United States)

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  15. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

    Science.gov (United States)

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

    Science.gov (United States)

    Jones, Mathew J K; Jallepalli, Prasad V

    2016-01-01

    During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

  18. Localization of two mammalian cyclin dependent kinases during mammalian meiosis

    NARCIS (Netherlands)

    Ashley, T.; Walpita, D.; de rooij, D. G.

    2001-01-01

    Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids)

  19. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. ... have shown that the stress associated with cold temperature ..... vation by cyclic-AMP-dependent protein kinase, studied using.

  20. Profiling bacterial kinase activity using a genetic circuit

    DEFF Research Database (Denmark)

    van der Helm, Eric; Bech, Rasmus; Lehning, Christina Eva

    Phosphorylation is a post-translational modification that regulates the activity of several key proteins in bacteria and eukaryotes. Accordingly, a variety of tools has been developed to measure kinase activity. To couple phosphorylation to an in vivo fluorescent readout we used the Bacillus...... subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously...... reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool....

  1. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Jaouani Mouna

    2015-09-26

    Sep 26, 2015 ... to several mutations at the Pyruvate Kinase gene (PKLR) located on chromosome .... Tunisians (Fig. 2) [21]. The screening of whole PKLR gene revealed the presence of ..... newborns: the pitfalls of diagnosis. J Pediatr 2007 ...

  2. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  3. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family. Jaouani Mouna, Hamdi Nadia, Chaouch Leila, Kalai Miniar, Mellouli Fethi, Darragi Imen, Boudriga Imen, Chaouachi Dorra, Bejaoui Mohamed, Abbes Salem ...

  4. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  5. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2001-01-01

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

  6. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2002-01-01

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

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

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

  8. Janus Associated Kinases Inhibitors in the Pharmacological Thera

    Directory of Open Access Journals (Sweden)

    Daniela Santos1

    2017-01-01

    Full Text Available Janus associated kinases inhibitors are a new strategy for the treatment of different clinical conditions like immunologic, inflammatory and oncology disorders. The aim of this study was to perform a review of all Janus associated kinases inhibitors available in national and international pharmaceutical market, their therapeutic indications and adverse effects, and the potential indications for investigation of those already available in the pharmaceutical market. It was also performed a review of the main new Janus associated kinases inhibitors that are still in clinical research. A literature review was conducted by consulting the summary of product characteristics of Janus associated kinases inhibitors available in the pharmaceutical market and a research in the bibliographic database PubMed using the terms «JAK inhibitors», «Janus associated kinases inhibitors» and «Janus kinases inhibitors». Ninety-five publications were included in the present review, published from January 2014 to January 2015. Drug databases of the European Medicines Agency and United States Food and Drug Administration were also consulted to search for Janus associated kinases inhibitors authorized in clinical practice. Currently, ruxolitinib and tofacitinib are available in the pharmaceutical market and oclatinib is approved as a veterinary medicinal product. Both drugs approved for human use have major adverse effects at hematological and immunological levels, which enhance the importance of the pharmacist’s role in the monitoring of patients involved in these treatments. However, several molecules are in pre-clinical and clinical studies trying to prove its potential in the treatment of several immunologic, inflammatory and oncology disorders. Thus, it is still necessary to deepen the knowledge in this area in order to overcome the risks of therapy with these agents. These risks weighed against the benefits of its clinical use have compromised the progress of

  9. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  10. Structural analysis of the Csk homologous kinase CHK

    International Nuclear Information System (INIS)

    Mulhern, T.; Chong, Y.-P.; Cheng, H.-C.

    2003-01-01

    Full text: CHK (Csk homologous kinase) is an intracellular protein tyrosine kinase, which is highly expressed in the haematopoietic system and the brain. The in vivo role of CHK is to specifically phosphorylate and deactivate the Src family of protein tyrosine kinases. The members of the Src family: Src, Blk, Fyn, Fgr, Hck, Lck, Lyn, Yes and Yrk are major players in numerous cell signalling pathways and exquisitely tuned control of Src family activity is fundamental to many processes in normal cells (reviewed in Lowell and Soriano, 1996). For example, the Src family kinase Fyn is highly expressed in the brain and its activity is vital for memory and learning. In the haematopoietic system, the Src family kinase Hck controls cytoskeletal reorganization, cell motility and immunologic activation. While the Csk family enzymes are closely related to the Src proteins (∼37% identity), the x-ray crystal structures of Src (Xu et al., 1997) and Csk (Ogawa et al., 2002) do display several important differences. Unlike Src, the Csk the SH2 and SH3 domains do not bind intramolecular ligands and they adopt a strikingly different disposition to that observed in Src. Another interesting feature is that the linkers between the SH3 and SH2 domains and between the SH2 and kinase domains, are in intimate contact with the N-lobe of kinase and both appear to play important roles in regulation of the kinase activity. However, the structural and functional basis of how this can be altered is still unclear. We describe the results of biochemical analyses of CHK mediated deactivation of Hck, which suggest that in addition to direct tail-phosphorylation, protein-protein interactions are important. We also describe heteronuclear NMR studies of the structure and ligand binding properties of the CHK SH2 and SH3 domains with a particular emphasis on the transmission of regulatory signals from the ligand binding sites to the interdomain linkers

  11. High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38 alpha

    NARCIS (Netherlands)

    Simard, J.R.; Grutter, C.; Pawar, V.; Aust, B.; Wolf, A.; Rabiller, M.; Wulfert, S.; Robubi, A.; Kluter, S.; Ottmann, C.; Rauh, D.

    2009-01-01

    Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases

  12. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    Science.gov (United States)

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  13. Identification of the protein kinase C phosphorylation site in neuromodulin

    International Nuclear Information System (INIS)

    Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

    1990-01-01

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

  14. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    Science.gov (United States)

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  15. Crystal structure of human protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

  16. Putative tyrosine kinases expressed in K-562 human leukemia cells

    International Nuclear Information System (INIS)

    Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

    1990-01-01

    Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

  17. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    M Kasim Diril

    2016-09-01

    Full Text Available The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

  18. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

    Science.gov (United States)

    Diril, M Kasim; Bisteau, Xavier; Kitagawa, Mayumi; Caldez, Matias J; Wee, Sheena; Gunaratne, Jayantha; Lee, Sang Hyun; Kaldis, Philipp

    2016-09-01

    The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

  19. SH2-dependent autophosphorylation within the Tec family kinase Itk.

    Science.gov (United States)

    Joseph, Raji E; Severin, Andrew; Min, Lie; Fulton, D Bruce; Andreotti, Amy H

    2009-08-07

    The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the betaD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.

  20. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.

    Science.gov (United States)

    Kim, Dennis H; Feinbaum, Rhonda; Alloing, Geneviève; Emerson, Fred E; Garsin, Danielle A; Inoue, Hideki; Tanaka-Hino, Miho; Hisamoto, Naoki; Matsumoto, Kunihiro; Tan, Man-Wah; Ausubel, Frederick M

    2002-07-26

    A genetic screen for Caenorhabditis elegans mutants with enhanced susceptibility to killing by Pseudomonas aeruginosa led to the identification of two genes required for pathogen resistance: sek-1, which encodes a mitogen-activated protein (MAP) kinase kinase, and nsy-1, which encodes a MAP kinase kinase kinase. RNA interference assays and biochemical analysis established that a p38 ortholog, pmk-1, functions as the downstream MAP kinase required for pathogen defense. These data suggest that this MAP kinase signaling cassette represents an ancient feature of innate immune responses in evolutionarily diverse species.

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

    KAUST Repository

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

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

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

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  3. Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A; Kuiper, Raoul V; Curbo, Sophie; Karlsson, Anna

    2013-02-15

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK(+/-) transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK(+/-)TK2(-/-) mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK(+/-)TK2(-/-) mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

  4. Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

    Science.gov (United States)

    Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

    2013-01-01

    A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

  5. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  6. Prodrugs of herpes simplex thymidine kinase inhibitors.

    Science.gov (United States)

    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  7. Resveratrol stimulates AMP kinase activity in neurons.

    Science.gov (United States)

    Dasgupta, Biplab; Milbrandt, Jeffrey

    2007-04-24

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

  8. Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    David E Garcia

    Full Text Available The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2 from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3 and purified on a Ni²⁺ column. The KM of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.

  9. Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

    OpenAIRE

    Thauerer, Bettina; zur Nedden, Stephanie; Baier-Bitterlich, Gabriele

    2010-01-01

    Exposure of pheochromocytoma (PC12) cells to hypoxia (1% O2) favors differentiation at the expense of cell viability. Additional incubation with nerve growth factor (NGF) and guanosine, a purine nucleoside with neurotrophin characteristics, rescued cell viability and further enhanced the extension of neurites. In parallel, an increase in the activity of protein kinase C-related kinase (PRK1), which is known to be involved in regulation of the actin cytoskeleton, was observed in hypoxic cells....

  10. Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

    Science.gov (United States)

    Stavenger, Robert A; Cui, Haifeng; Dowdell, Sarah E; Franz, Robert G; Gaitanopoulos, Dimitri E; Goodman, Krista B; Hilfiker, Mark A; Ivy, Robert L; Leber, Jack D; Marino, Joseph P; Oh, Hye-Ja; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Zhang, Daohua; Zhao, Yongdong; Jolivette, Larry J; Head, Martha S; Semus, Simon F; Elkins, Patricia A; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Doe, Christopher P; Bentley, Ross; Chen, Zunxuan X; Hu, Erding; Lee, Dennis

    2007-01-11

    The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

  11. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  12. Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

    OpenAIRE

    Martos, C; Plana, M; Guasch, M D; Itarte, E

    1985-01-01

    Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partia...

  13. Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

    OpenAIRE

    Elbing, Karin; McCartney, Rhonda R.; Schmidt, Martin C.

    2006-01-01

    Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerpr...

  14. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Science.gov (United States)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  15. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  16. The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

    Science.gov (United States)

    Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

    2017-09-22

    The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    OpenAIRE

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulat...

  18. A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

    Science.gov (United States)

    Yuan, Hushan; Pupo, Monica T.; Blois, Joe; Smith, Adam; Weissleder, Ralph; Clardy, Jon; Josephson, Lee

    2009-01-01

    The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min vs Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC50 of 44 nM, compared to Wm’s IC50 of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. PMID:19523825

  19. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  20. Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

    Science.gov (United States)

    Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya; Liu, Yin; James, Michael; Herrero, Juan; Shaw, Stephen

    2005-03-18

    To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

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

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  2. Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Varley, C L; Royds, J A; Brown, B L; Dobson, P R

    2001-01-01

    We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

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

    Directory of Open Access Journals (Sweden)

    Kenyon Colin P

    2012-08-01

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

  4. Effects of obesity on protein kinase C, brain creatine kinase, transcription, and autophagy in cochlea.

    Science.gov (United States)

    Hwang, Juen-Haur

    2017-06-01

    Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-β (PKC-β), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-β, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.

  5. Kinase inhibitors: a new class of antirheumatic drugs

    Directory of Open Access Journals (Sweden)

    Kyttaris VC

    2012-09-01

    Full Text Available Vasileios C KyttarisDivision of Rheumatology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAAbstract: The outlook for patients with rheumatoid arthritis has improved significantly over the last three decades with the use of disease-modifying antirheumatic drugs. However, despite the use of methotrexate, cytokine inhibitors, and molecules targeting T and B cells, a percentage of patients do not respond or lose their response over time. The autoimmune process in rheumatoid arthritis depends on activation of immune cells, which utilize intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. In the past decade, small molecules targeting several kinases, such as p38 MAPK, Syk, and JAK have been developed. Several p38 MAPK inhibitors proved ineffective in treating rheumatoid arthritis. The Syk inhibitor, fostamatinib, proved superior to placebo in Phase II trials and is currently under Phase III investigation. Tofacitinib, a JAK1/3 inhibitor, was shown to be efficacious in two Phase III trials, while VX-509, a JAK3 inhibitor, showed promising results in a Phase II trial. Fostamatinib and tofacitinib were associated with increased rates of infection, elevation of liver enzymes, and neutropenia. Moreover, fostamatinib caused elevations of blood pressure and diarrhea, while tofacitinib was associated with an increase in creatinine and elevation of lipid levels.Keywords: rheumatoid arthritis, kinase inhibitors, mitogen-activated phosphokinase p38, spleen tyrosine kinase, Janus kinases

  6. Fragment-based approaches to the discovery of kinase inhibitors.

    Science.gov (United States)

    Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

    2014-01-01

    Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

  7. The long and the short of SAD-1 kinase.

    Science.gov (United States)

    Kim, Joanne S M; Hung, Wesley; Zhen, Mei

    2010-05-01

    The Ser/Thr SAD kinases are evolutionarily conserved, critical regulators of neural development. Exciting findings in recent years have significantly advanced our understanding of the mechanism through which SAD kinases regulate neural development. Mammalian SAD-A and SAD-B, activated by a master kinase LKB1, regulate microtubule dynamics and polarize neurons. In C. elegans, the sad-1 gene encodes two isoforms, namely the long and the short, which exhibit overlapping and yet distinct functions in neuronal polarity and synaptic organization. Surprisingly, our most recent findings in C. elegans revealed a SAD-1-independent LKB1 activity in neuronal polarity. We also found that the long SAD-1 isoform directly interacts with a STRADalpha pseudokinase, STRD-1, to regulate neuronal polarity and synaptic organization. We elaborate here a working model of SAD-1 in which the two isoforms dimer/oligomerize to form a functional complex, and STRD-1 clusters and localizes the SAD-1 complex to synapses. While the mechanistic difference between the vertebrate and invertebrate SAD kinases may be puzzling, a recent discovery of the functionally distinct SAD-B isoforms predicts that the difference likely arises from our incomplete understanding of the SAD kinase mechanism and may eventually be reconciled as the revelation continues.

  8. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe

    2018-01-22

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

  9. Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

    Science.gov (United States)

    Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2014-01-01

    WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

  10. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

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

    2018-01-01

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

  11. Functions of Aurora kinase C in meiosis and cancer

    Directory of Open Access Journals (Sweden)

    Suzanne M. Quartuccio

    2015-08-01

    Full Text Available The mammalian genome encodes three Aurora kinase protein family members: A, B, and C. While Aurora kinase A (AURKA and B (AURKB are found in cells throughout the body, significant protein levels of Aurora kinase C (AURKC are limited to cells that undergo meiosis (sperm and oocyte. Despite its discovery nearly 15 years ago, we know little about the function of AURKC compared to that of the other 2 Aurora kinases. This lack of understanding can be attributed to the high sequence homology between AURKB and AURKC preventing the use of standard approaches to understand non-overlapping and meiosis I (MI-specific functions of the two kinases. Recent evidence has revealed distinct functions of AURKC in meiosis and may aid in our understanding of why chromosome segregation during MI often goes awry in oocytes. Many cancers aberrantly express AURKC, but because we do not fully understand AURKC function in its normal cellular context, it is difficult to predict the biological significance of this expression on the disease. Here, we consolidate and update what is known about AURKC signaling in meiotic cells to better understand why it has oncogenic potential.

  12. Purification and characterization of a thylakoid protein kinase

    International Nuclear Information System (INIS)

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs

  13. A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

    Science.gov (United States)

    Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

    2017-10-13

    Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

  14. Two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Willson, V J.C.; Jones, H M; Thompson, R J [Cambridge Univ. (UK). Clinical School

    1981-06-18

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and /sup 125/I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive.

  15. A two-site immunoradiometric assay for the MB isoenzyme of creatine kinase

    International Nuclear Information System (INIS)

    Willson, V.J.C.; Jones, H.M.; Thompson, R.J.

    1981-01-01

    A two-site immunoradiometric assay for myocardial creatine kinase MB isoenzyme is described. The method utilizes immobilized anti-human creatine kinase BB antibodies and 125 I-labelled anti-human creatine kinase MM antibodies and can specifically detect creatine kinase MB in the presence of approximately 1000-fold excess of creatine kinase MM or BB. Native kinase MB prepared from human heart and creatine kinase MB prepared by hybridisation of purified human creatine kinase MM and creatine kinase BB appeared to react identically in the assay. Serum estimations on patients with suspected myocardial infarction correlated with the presence of MB band on electrophoresis but preliminary results suggest that the two-site immunoradiometric assay may be more sensitive. (Auth.)

  16. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

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

    DEFF Research Database (Denmark)

    Boldyreff, Brigitte; Rasmussen, Tine L; Jensen, Hans H

    2008-01-01

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

  18. Complete inhibition of creatine kinase in isolated perfused rat hearts

    International Nuclear Information System (INIS)

    Fossel, E.T.; Hoefeler, H.

    1987-01-01

    Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts are able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure. 31 P-NMR of the heart was carried out

  19. Kinase fusions are frequent in Spitz tumors and spitzoid melanomas

    Science.gov (United States)

    Esteve-Puig, Rosaura; Botton, Thomas; Yeh, Iwei; Lipson, Doron; Otto, Geoff; Brennan, Kristina; Murali, Rajmohan; Garrido, Maria; Miller, Vincent A.; Ross, Jeffrey S; Berger, Michael F.; Sparatta, Alyssa; Palmedo, Gabriele; Cerroni, Lorenzo; Busam, Klaus J.; Kutzner, Heinz; Cronin, Maureen T; Stephens, Philip J; Bastian, Boris C.

    2014-01-01

    Spitzoid neoplasms are a group of melanocytic tumors with distinctive histopathologic features. They include benign tumors (Spitz nevi), malignant tumors (spitzoid melanomas), and tumors with borderline histopathologic features and uncertain clinical outcome (atypical Spitz tumors). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbor kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%), and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signaling pathways, are tumorigenic, and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz nevi, 56% of atypical Spitz tumors, and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signaling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms, and may serve as therapeutic targets for metastatic spitzoid melanomas. PMID:24445538

  20. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

    Science.gov (United States)

    Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

    2017-01-01

    ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  1. Novel Bruton's tyrosine kinase inhibitors currently in development

    Directory of Open Access Journals (Sweden)

    D'Cruz OJ

    2013-03-01

    Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

  2. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    International Nuclear Information System (INIS)

    Sprowles, Amy; Robinson, Dan; Wu Yimi; Kung, H.-J.; Wisdom, Ron

    2005-01-01

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli

  3. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

    Directory of Open Access Journals (Sweden)

    Caroline Lund Dahlberg

    Full Text Available Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  4. Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

    Science.gov (United States)

    Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

    2006-06-01

    This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  5. Semiconductor technology in protein kinase research and drug discovery: sensing a revolution.

    Science.gov (United States)

    Bhalla, Nikhil; Di Lorenzo, Mirella; Estrela, Pedro; Pula, Giordano

    2017-02-01

    Since the discovery of protein kinase activity in 1954, close to 600 kinases have been discovered that have crucial roles in cell physiology. In several pathological conditions, aberrant protein kinase activity leads to abnormal cell and tissue physiology. Therefore, protein kinase inhibitors are investigated as potential treatments for several diseases, including dementia, diabetes, cancer and autoimmune and cardiovascular disease. Modern semiconductor technology has recently been applied to accelerate the discovery of novel protein kinase inhibitors that could become the standard-of-care drugs of tomorrow. Here, we describe current techniques and novel applications of semiconductor technologies in protein kinase inhibitor drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Human adenylate kinases – classification, structure, physiological and pathological importance

    Directory of Open Access Journals (Sweden)

    Magdalena Wujak

    2015-01-01

    Full Text Available Adenylate kinase (AK, EC 2.7.4.3 is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy β – and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.

  7. Characterization of pathogenic germline mutations in human Protein Kinases

    Directory of Open Access Journals (Sweden)

    Orengo Christine A

    2011-07-01

    Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

  8. Structural Changes of Creatine Kinase upon Substrate Binding

    OpenAIRE

    Forstner, Michael; Kriechbaum, Manfred; Laggner, Peter; Wallimann, Theo

    1998-01-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzym...

  9. A structural informatics approach to mine kinase knowledge bases.

    Science.gov (United States)

    Brooijmans, Natasja; Mobilio, Dominick; Walker, Gary; Nilakantan, Ramaswamy; Denny, Rajiah A; Feyfant, Eric; Diller, David; Bikker, Jack; Humblet, Christine

    2010-03-01

    In this paper, we describe a combination of structural informatics approaches developed to mine data extracted from existing structure knowledge bases (Protein Data Bank and the GVK database) with a focus on kinase ATP-binding site data. In contrast to existing systems that retrieve and analyze protein structures, our techniques are centered on a database of ligand-bound geometries in relation to residues lining the binding site and transparent access to ligand-based SAR data. We illustrate the systems in the context of the Abelson kinase and related inhibitor structures. 2009 Elsevier Ltd. All rights reserved.

  10. Expression of casein kinase 2 during mouse embryogenesis

    DEFF Research Database (Denmark)

    Mestres, P; Boldyreff, B; Ebensperger, C

    1994-01-01

    This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohisto......This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level...

  11. Proteinase K processing of rabbit muscle creatine kinase

    DEFF Research Database (Denmark)

    Leydier, C; Andersen, Jens S.; Couthon, F

    1997-01-01

    Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However...

  12. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  13. The Azaindole Framework in the Design of Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Jean-Yves Mérour

    2014-11-01

    Full Text Available This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.

  14. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  15. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    International Nuclear Information System (INIS)

    Holzer, Georg W.; Mayrhofer, Josef; Gritschenberger, Werner; Falkner, Falko G.

    2005-01-01

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes

  16. Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

    Science.gov (United States)

    Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

    2017-01-12

    The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

  17. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.

    Science.gov (United States)

    Mehta, Anil

    2007-11-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

  18. Tyrosine kinase, aurora kinase and leucine aminopeptidase as attractive drug targets in anticancer therapy - characterisation of their inhibitors.

    Science.gov (United States)

    Ziemska, Joanna; Solecka, Jolanta

    Cancers are the leading cause of deaths all over the world. Available anticancer agents used in clinics exhibit low therapeutic index and usually high toxicity. Wide spreading drug resistance of cancer cells induce a demanding need to search for new drug targets. Currently, many on-going studies on novel compounds with potent anticancer activity, high selectivity as well as new modes of action are conducted. In this work, we describe in details three enzyme groups, which are at present of extensive interest to medical researchers and pharmaceutical companies. These include receptor tyrosine kinases (e.g. EGFR enzymes) and non-receptor tyrosine kinases (Src enzymes), type A, B and C Aurora kinases and aminopeptidases, especially leucine aminopeptidase. We discuss classification of these enzymes, biochemistry as well as their role in the cell cycle under normal conditions and during cancerogenesis. Further on, the work describes enzyme inhibitors that are under in vitro, preclinical, clinical studies as well as drugs available on the market. Both, chemical structures of discovered inhibitors and the role of chemical moieties in novel drug design are discussed. Described enzymes play essential role in cell cycle, especially in mitosis (Aurora kinases), cell differentiation, growth and apoptosis (tyrosine kinases) as well as G1/S transition (leucine aminopeptidase). In cancer cells, they are overexpressed and only their inhibition may stop tumor progression. This review presents the clinical outcomes of selected inhibitors and argues the safety of drug usage in human volunteers. Clinical studies of EGFR and Src kinase inhibitors in different tumors clearly show the need for molecular selection of patients (to those with mutations in genes coding EGFR and Src) to achieve positive clinical response. Current data indicates the great necessity for new anticancer treatment and actions to limit off-target activity.

  19. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2016-12-16

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae*

    Science.gov (United States)

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M.

    2016-01-01

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1–77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1–77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. PMID:27834677

  1. Glycogen synthase kinase-3: a key kinase in retinal neuron apoptosis in early diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    Li Zhaohui; Ma Ling; Chen Xiaodong; Li Yonghao; Li Shiyi; Zhang Jinglin; Lu Lin

    2014-01-01

    Background Diabetes-related pathogenic factors can cause retinal ganglion cell (RGC) apoptosis,but the specific mechanism is not very clear.The aim of this study is to investigate the correlation between glycogen synthase kinase-3 (GSK-3) activation and retinal neuron apoptosis.Methods In an in vitro experiment,the number of apoptotic RGC-5 cells differentiated by staurosporine was evaluated via flow cytometry and nuclei staining using Hoechst 33258.GSK-3 phosphorylation and caspase-3 activation in RGC-5 cells after serum deprivation were determined using Western blotting.Mitochondrial membrane potential was detected using the dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetrethyl benzimidalyl carbocyanine iodide,and reactive oxygen species (ROS) levels were measured with dihydroethidium.In an in vivo experiment,the number of apoptotic retinal neurons was evaluated via terminal transferase dUTP nick-end labeling (TUNEL),and GSK-3 phosphorylation was determined using Western blotting,in the retinal nerve epithelial tissue of rats in which diabetes was induced by intravenous tail-vein injection of streptozotocin for 4 weeks.Results The levels of phosphorylated Ser21/9 in GSK-3α/β and p-T308/S473-AKT were lower and the cleaved caspase-3 levels were higher in the serum-deprived model (P <0.05).Lithium chloride treatment was associated with a slower rate of apoptosis,increased mitochondrial membrane potential,and decreased ROS levels in differentiated RGC-5 cells (P <0.05).The level of blood glucose and the number of TUNEL-positive cells in the whole-mounted retinas were higher (P <0.01),and the levels of phosphorylated Ser21/9 in GSK-3α/β and body weight were lower (P <0.05).However,the thickness of the retinal nerve epithelial layer was not significantly less in diabetic rats compared with control group.Lithium chloride intravitreal injection increased the levels of phosphorylated Ser21/9 in GSK-3α/β and decreased TUNEL-positive cells in the whole-mounted retinas

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

    Directory of Open Access Journals (Sweden)

    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

  3. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    OpenAIRE

    Jette, Nicholas; Lees-Miller, Susan P.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemi...

  4. Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

    Science.gov (United States)

    Celada, Lindsay J.; Whalen, Margaret M.

    2013-01-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

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

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

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

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

    Science.gov (United States)

    Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    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. PMID:26703626

  7. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

    Science.gov (United States)

    Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

    2015-01-20

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  8. Creatine kinase response to high-intensity aerobic exercise in adult-onset muscular dystrophy

    DEFF Research Database (Denmark)

    Andersen, Søren P; Sveen, Marie-Louise; Hansen, Regitze S

    2013-01-01

    We investigated the effect of high-intensity exercise on plasma creatine kinase (CK) in patients with muscular dystrophies.......We investigated the effect of high-intensity exercise on plasma creatine kinase (CK) in patients with muscular dystrophies....

  9. Clinical Pharmacology of Kinase Inhibitors in Oncology : Personalized and Optimzed Dosing

    NARCIS (Netherlands)

    Verheijen, Remy B.

    2017-01-01

    Kinase inhibitors are an important category of molecularly targeted therapies used for cancer. Verheijen’s doctoral thesis describes several clinical pharmacological studies to optimize and personalize the treatment of cancer with kinase inhibitors, using pharmacokinetics, molecular imaging and

  10. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

  11. Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpesviruses

    International Nuclear Information System (INIS)

    Tenser, R.B.; Jones, J.C.; Ressel, S.J.; Fralish, F.A.

    1983-01-01

    Plaques formed by herpes simplex virus (HSV), pseudorabies virus, and varicella-zoster virus were studied by plaque autoradiography after [ 14 C]thymidine labeling. Standard thymidine kinase-positive (TK+) viruses and TK- mutants of HSV types 1 and 2 and pseudorabies virus were studied, including cell cultured viruses and viruses isolated from animals. Autoradiography was performed with X-ray film with an exposure time of 5 days. After development of films, TK+ plaques showed dark rims due to isotope incorporation, whereas TK- plaques were minimally labeled. Plaque autoradiography of stock TK- viruses showed reversion frequencies to the TK+ phenotype of less than 10(-3). Autoradiography indicated that TK- virus retained the TK- phenotype after replication in vivo. In addition, it was shown that TK- HSV could be isolated from mouse trigeminal ganglion tissue after corneal inoculation of TK- HSV together with TK+ HSV. The plaque autoradiographic procedure was very useful to evaluate proportions of TK+ and TK- virus present in TK+-TK- virus mixtures

  12. Structures of thymidine kinase 1 of human and mycoplasma origin

    DEFF Research Database (Denmark)

    Welin, Martin; Kosinska, Urszula; Mikkelsen, Nils-Egil

    2004-01-01

    Cytosolic thymidine kinase, TK1, is a well-known cell cycle regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs as AZT. We have now determined the first structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in com...

  13. The Role of Protein Kinase CK2 in Glioblastoma Development

    OpenAIRE

    Ji, Haitao; Lu, Zhimin

    2013-01-01

    Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor in adults, and its response to current therapies is limited. Protein kinase CK2 is overexpressed in GBM and regulates GBM cell survival, proliferation, and migration and brain tumorigenesis. Targeting CK2 for GBM treatment may benefit GBM patients.

  14. VHH Activators and Inhibitors for Protein Kinase C Epsilon

    NARCIS (Netherlands)

    Summanen, M.M.I.

    2012-01-01

    Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are

  15. SH2 dependent autophosphorylation within the Tec family kinase Itk

    Science.gov (United States)

    Joseph, Raji E.; Severin, Andrew; Min, Lie; Fulton, D. Bruce; Andreotti, Amy H.

    2009-01-01

    The Tec family kinase, Itk, undergoes an in cis autophosphorylation on Y180 within its SH3 domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening SH2 domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk, a Tec family kinase linked to the B cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA causing mutations might impair Btk phosphorylation. PMID:19523959

  16. FDA-approved small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

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

    2015-01-01

    Kinases have emerged as one of the most intensivelypursued targets in current pharmacological research,especially for cancer, due to their critical roles in cellularsignaling. To date, the US FDA has approved 28 smallmoleculekinase inhibitors, half of which were approvedin the past 3 years. While...

  17. Over-expression of NAD kinase in Corynebacterium crenatum and ...

    African Journals Online (AJOL)

    in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) ... Results: In HOS condition, NAD+ kinase activity increased by 116 %, while ... (NADPH), an important co-enzyme during ... Polymerase, TaKaRa) using C. crenatum .... were washed with cold 100 mM PBS (pH 7.5) ..... Catalase and.

  18. Hyper-IgD syndrome or mevalonate kinase deficiency.

    NARCIS (Netherlands)

    Stoffels, M.; Simon, A.

    2011-01-01

    PURPOSE OF REVIEW: The hyper-IgD and periodic fever syndrome (HIDS) is one of the classical monogenetic hereditary autoinflammatory disorders, and together with the more severe mevalonic aciduria it is also known as 'mevalonate kinase deficiency' (MKD). In this study, we will give an overview of the

  19. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  20. Protein kinase activity associated with the corticosteroid binder IB

    International Nuclear Information System (INIS)

    Vujicic, M.; Djordjevic-Markovic, R.; Radic, O.; Krstic, M.; Kanazir, D.

    1997-01-01

    The physiological effects elicited by glucocorticoids are mediated via glucocorticoid receptors (GR). Analysis of specific glucocorticoid binding to radioactively labelled [ 3 H] triamcinolone acetonide in rat liver cytosol and analysis by ion exchange chromatography have revealed the presence of two distinct molecular species. The major form, designated as binder II appears to correspond to the well characterized glucocorticoid receptor by virtue of its size, charge, steroid binding characteristics and ability to bind to DNA.The second form, designated as corticosteroid binder IB, is a minor binding component in the liver. The binder IB differs from the binder II receptor by virtue of its lower molecular weight and its elution in the pre gradient of DEAE-Sephadex A-50 column which retains the un activated binder II receptor complexes. We examined the kinase activity of partially purified corticosteroid binder IB. Using (γ 3 2 P) ATP we detected kinase activity associated with the IB fraction from the rat liver. This kinase phosphorylate mixed histones and and dose not phosphorylate IB protein in vitro. The kinase activity is completely inhibited by the addition of Mg 2 + ions and is partially inhibited by the addition of Ca 2 +ions. (author)

  1. Evaluation of Creatine Kinase Activity and Inorganic Phosphate ...

    African Journals Online (AJOL)

    subjects presenting with major VOC. Keywords: Serum creatine kinase activity, Serum inorganic phosphate concentration, Sickle cell disease,. Steady state, Vaso‑occlusive crisis. Original Article. Address for correspondence: Dr. John C Aneke,. Department of Hematology,. Nnamdi Azikiwe University Teaching. Hospital ...

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

    DEFF Research Database (Denmark)

    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...

  3. A mathematical model of human thymidine kinase 2 activity

    DEFF Research Database (Denmark)

    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...

  4. Tomato thymidine kinase is subject to inefficient TTP feedback regulation

    DEFF Research Database (Denmark)

    Larsen, Nicolai Balle; Munch-Petersen, Birgitte; Piskur, Jure

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. Transduction with toTK1 has been found to effi...

  5. Skeletal muscle mitochondrial respiration in AMPKa2 kinase dead mice

    DEFF Research Database (Denmark)

    Larsen, Steen; Kristensen, Jonas Møller; Stride, Nis

    2012-01-01

    AIM: To study if the phenotypical characteristics (exercise intolerance; reduced spontaneous activity) of the AMPKa2 kinase-dead (KD) mice can be explained by a reduced mitochondrial respiratory flux rates (JO(2) ) in skeletal muscle. Secondly, the effect of the maturation process on JO(2...

  6. Novel mutations associated with pyruvate kinase deficiency in Brazil

    Directory of Open Access Journals (Sweden)

    Maria Carolina Costa Melo Svidnicki

    2018-01-01

    Full Text Available Background: Pyruvate kinase deficiency is a hereditary disease that affects the glycolytic pathway of the red blood cell, causing nonspherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait and shows a marked variability in clinical expression. This study reports on the molecular characterization of ten Brazilian pyruvate kinase-deficient patients and the genotype–phenotype correlations. Method: Sanger sequencing and in silico analysis were carried out to identify and characterize the genetic mutations. A non-affected group of Brazilian individuals were also screened for the most commonly reported variants (c.1456C>T and c.1529G>A. Results: Ten different variants were identified in the PKLR gene, of which three are reported here for the first time: p.Leu61Gln, p.Ala137Val and p.Ala428Thr. All the three missense variants involve conserved amino acids, providing a rationale for the observed enzyme deficiency. The allelic frequency of c.1456C>T was 0.1% and the 1529G>A variant was not found. Conclusion: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency from South America. The results allowed us to correlate the severity of the clinical phenotype with the identified variants. Keywords: Red cell disorder, Pyruvate kinase, Mutation, Hemolytic anemia, PKLR gene

  7. Cloning, expression and purification of cold adapted acetate kinase ...

    African Journals Online (AJOL)

    shell) Neobuccinum living in the Antarctic ice-covered sea. An open reading frame of 1203 bp, coding for acetate kinase gene, called ack, was amplified, cloned into the expression vector, pETY-16b, and the enzyme was overproduced by ...

  8. Periodontal status and serum creatine kinase levels among young ...

    African Journals Online (AJOL)

    Objectives: It is hypothesized that soccer players with periodontal disease exhibit raised serum creatine kinase (CK) levels as compared to those without periodontal disease. We assessed the clinical gingival status and serum CK levels among young soccer players. Materials and Methods: Demographic data were ...

  9. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    Background and Objectives: There is the recognition of a pattern of elevations of serum enzymes in hyperthyroid and hypothyroid patients. The aims of this study were to determine the activities of serum creatine kinase (CK) and lactate deydrogenase (LDH) in thyroid disorders, and to evaluate the relationship between CK, ...

  10. Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

    African Journals Online (AJOL)

    Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

  11. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    Science.gov (United States)

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-02-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Effect of triiodothyronine on rat liver chromatin protein kinase

    International Nuclear Information System (INIS)

    Kruh, J.; Tichonicky, L.

    1976-01-01

    1) Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2) Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32 P when incubated with [γ- 32 P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32 P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins. (orig.) [de

  13. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The removal of the auto-inhibitory domain that negatively regulates the kinase activity in M. truncatula results in a constitutively-active form, inducing symbiotic responses in the absence of bacterial signals. In this study, we verified the functionality of a DMI3 variant and its ability to induce spontaneous nodules in M.

  14. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    Science.gov (United States)

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.

  15. Variability in bioavailability of small molecular tyrosine kinase inhibitors

    NARCIS (Netherlands)

    Herbrink, Maikel; Nuijen, Bastiaan; Schellens, Jan H M; Beijnen, Jos H.

    2015-01-01

    Small molecular tyrosine kinase inhibitors (smTKIs) are in the centre of the very quickly expanding area of personalized chemotherapy and oral applicability thereof. The number of drugs in this class is rapidly growing, with twenty current approvals by both the European Medicines Agency (EMA) and

  16. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  17. Involvement of protein kinase C-δ activation in betulininduced ...

    African Journals Online (AJOL)

    Purpose: To investigate the clinical benefits and underlying mechanisms of action of betulin in the treatment of cancer using a neuroblastoma (NB) cell model. Method: Cell viability ... of tumor recurrence. Keywords: Betulin, Neuroblastoma, Apoptosis, protein kinase C-δ, Adjuvant chemotherapy, Tumor recurrence, Caspase ...

  18. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu

    2011-05-01

    Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  19. Assessment of creatine kinase and lactate dehydrogenase activities ...

    African Journals Online (AJOL)

    Ina bid to investigate the influence of menopausal on coronary heart disease, plasma creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes were analysed on a prospective cohort of 100 women attending Irrua Specialist Teaching Hospital (ISTH), Irrua, Edo state-Nigeria. They were divided into two groups; ...

  20. Protein kinase C alpha controls erythropoietin receptor signaling.

    NARCIS (Netherlands)

    M.M. von Lindern (Marieke); M. Parren-Van Amelsvoort (Martine); T.B. van Dijk (Thamar); E. Deiner; B. Löwenberg (Bob); E. van den Akker (Emile); S. van Emst-de Vries (Sjenet); P.J. Willems (Patrick); H. Beug (Hartmut)

    2000-01-01

    textabstractProtein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We

  1. Protein kinase C alpha controls erythropoietin receptor signaling

    NARCIS (Netherlands)

    von Lindern, M.; Parren-van Amelsvoort, M.; van Dijk, T.; Deiner, E.; van den Akker, E.; van Emst-de Vries, S.; Willems, P.; Beug, H.; Löwenberg, B.

    2000-01-01

    Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors

  2. Metazoan-like signaling in a unicellular receptor tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  3. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    ... in thyroid function are common endocrine disorders affecting 5-10% of individuals over ... Key words: Hyperthyroidism, hypothyroidism, lactate dehydrogenase, serum creatine kinase ... individuals depends on age, race, lean body mass and physical activity. ... measured by radioimmunoassay on AXSYM System (Abbott.

  4. Unconventional functions of mitotic kinases in kidney tumourigenesis

    Directory of Open Access Journals (Sweden)

    Pauline eHascoet

    2015-10-01

    Full Text Available Human tumours exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumour types, including breast, colon and renal cell carcinoma. The Renal cell cancer (RCC is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC is the most common subtype and represents 85 % of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel Lindau gene but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell-cell adhesion and apical-basal cell polarity that also may be regulated by the mitotic kinases (Plk1, CK2, DLCK1 and Aurora kinases. In this review, we describe the non mitotic unconventional functions of these kinases in renal tumourigenesis.

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

    Indian Academy of Sciences (India)

    admin

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

  6. Changes in neutrophil count, creatine kinases and muscle soreness ...

    African Journals Online (AJOL)

    Objective. A primary objective was to examine circulating neutrophil count after repeated bouts of downhill running. An additional aim was to determine creatine kinase (CK) levels during the initial 12 hours, after repeated DHRs. Design. Eleven healthy, untrained Caucasian males performed 2 x 60 min bouts of DHR ...

  7. Presenilin dependence of phospholipase C and protein kinase C signaling

    DEFF Research Database (Denmark)

    Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola

    2007-01-01

    -stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...

  8. Periodontal status and serum creatine kinase levels among young ...

    African Journals Online (AJOL)

    2015-12-02

    Dec 2, 2015 ... Key words: Periodontal disease, serum creatine kinase, soccer players ... has also been reported that poor oral health status influences the quality of life of an individual ..... A short‑term longitudinal randomized case‑control study. Clin Oral ... crevicular fluid from chronic periodontitis patients before and after.

  9. Evaluation of Creatine Kinase Activity and Inorganic Phosphate ...

    African Journals Online (AJOL)

    Background: Biochemical parameters vary in subjects with different hemoglobin phenotypes, compared with normal controls. Aim: The aim was to evaluate serum creatine kinase (CK) activity and inorganic phosphate concentrations in Nigerian adults with homozygous and heterozygous hemoglobin phenotypes. Subjects ...

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

    Science.gov (United States)

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

    2011-04-01

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

  11. A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

    Science.gov (United States)

    Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

    2013-06-10

    We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

  12. An open library of human kinase domain constructs for automated bacterial expression

    OpenAIRE

    Rodríguez-Laureano, Lucelenie; Işık, Mehtap; Chodera, John; Seeliger, Markus; Jeans, Chris; Gradia, Scott; Hanson, Sonya; Parton, Daniel; Albanese, Steven; Levinson, Nicholas; Behr, Julie

    2017-01-01

    Kinases play a critical role in many cellular signaling pathways and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Since the FDA approval of imatinib in 2001, therapeutics targeting kinases now account for roughly 50% of current cancer drug discovery efforts. The ability to explore human kinase biochemistry, biophysics, and structural biology in the laboratory is essential to making rapid progress in understanding kinase regulation, designing selec...

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

    Science.gov (United States)

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

    2017-10-01

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

  14. Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis

    Science.gov (United States)

    Marston, Adele L.; Wassmann, Katja

    2017-01-01

    Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid. PMID:29322045

  15. The molecular architecture of human N-acetylgalactosamine kinase.

    Science.gov (United States)

    Thoden, James B; Holden, Hazel M

    2005-09-23

    Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

  16. Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis

    Directory of Open Access Journals (Sweden)

    Adele L. Marston

    2017-12-01

    Full Text Available Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3, and Aurora B and C (Ipl1 will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid.

  17. A grammar inference approach for predicting kinase specific phosphorylation sites.

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  18. A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

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

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

    2013-01-01

    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...

  20. KLIFS : a knowledge-based structural database to navigate kinase-ligand interaction space

    NARCIS (Netherlands)

    van Linden, O.P.J.; Kooistra, A.J.; Leurs, R.; de Esch, I.J.P.; de Graaf, C.

    2013-01-01

    Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal

  1. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Creatine phosphokinase/creatine kinase or... Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test system. (a) Identification. A creatine phosphokinase/creatine kinase or isoenzymes test system is a device...

  2. Exclusion of acute myocardial infarction. The value of measuring creatine kinase slope

    NARCIS (Netherlands)

    Bakker, A. J.; Koelemay, M. J.; van Vlies, B.; Gorgels, J. P.; Smits, R.; Tijssen, J. G.; Haagen, F. D.

    1995-01-01

    For the exclusion (and diagnosis) of acute myocardial infarction, we studied timed sequential (slope) measurements of creatine kinase and creatine kinase-MB catalytic activity concentration, creatine kinase-MB mass concentration, troponin T and myoglobin, using data from 242 patients consecutively

  3. Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases

    NARCIS (Netherlands)

    Miller, Malgorzata; Donat, Stefanie; Rakette, Sonja; Stehle, Thilo; Kouwen, Thijs R. H. M.; Diks, Sander H.; Dreisbach, Annette; Reilman, Ewoud; Gronau, Katrin; Becher, Doerte; Peppelenbosch, Maikel P.; van Dijl, Jan Maarten; Ohlsen, Knut

    2010-01-01

    In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of

  4. ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

  5. Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

    DEFF Research Database (Denmark)

    Stalter, G; Siemer, S; Becht, E

    1994-01-01

    of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

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

    DEFF Research Database (Denmark)

    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...

  7. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  8. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  9. Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes

    Czech Academy of Sciences Publication Activity Database

    Balboula, A. Z.; Nguyen, A. L.; Gentilello, A. S.; Quartuccio, S. M.; Drutovič, Dávid; Šolc, Petr; Schindler, K.

    2016-01-01

    Roč. 129, č. 19 (2016), s. 3648-3660 ISSN 0021-9533 R&D Projects: GA MŠk(CZ) LO1609 Institutional support: RVO:67985904 Keywords : haspin * aurora kinase * spindle * MTOC * oocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.431, year: 2016

  10. A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes

    NARCIS (Netherlands)

    Cha, Hoon-Suk; Boyle, David L.; Inoue, Tomoyuki; Schoot, Reineke; Tak, Paul P.; Pine, Polly; Firestein, Gary S.

    2006-01-01

    Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like

  11. Selective Cyclin-Dependent Kinase Inhibitors Discriminating between Cell Cycle and Transcriptional Kinases Future Reality or Utopia?

    Czech Academy of Sciences Publication Activity Database

    Wesierska-Gadek, J.; Kryštof, Vladimír

    2009-01-01

    Roč. 1171, - (2009), s. 228-241 ISSN 0077-8923 R&D Projects: GA ČR GA204/08/0511 Institutional research plan: CEZ:AV0Z50380511 Keywords : cell cycle * CYC202 * cyclin-dependent kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.670, year: 2009

  12. The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions.

    Science.gov (United States)

    Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

    2014-02-21

    We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

    2014-05-01

    mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

  14. Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cγ at the centrosome

    Directory of Open Access Journals (Sweden)

    Tassin Anne-Marie

    2008-04-01

    Full Text Available Abstract Background The t(6;8 translocation found in rare and agressive myeloproliferative disorders results in a chimeric gene encoding the FOP-FGFR1 fusion protein. This protein comprises the N-terminal region of the centrosomal protein FOP and the tyrosine kinase of the FGFR1 receptor. FOP-FGFR1 is localized at the centrosome where it exerts a constitutive kinase activity. Results We show that FOP-FGFR1 interacts with the large centrosomal protein CAP350 and that CAP350 is necessary for FOP-FGFR1 localisation at centrosome. FOP-FGFR1 activates the phosphoinositide-3 kinase (PI3K pathway. We show that p85 interacts with tyrosine 475 of FOP-FGFR1, which is located in a YXXM consensus binding sequence for an SH2 domain of p85. This interaction is in part responsible for PI3K activation. Ba/F3 cells that express FOP-FGFR1 mutated at tyrosine 475 have reduced proliferative ability. Treatment with PI3K pathway inhibitors induces death of FOP-FGFR1 expressing cells. FOP-FGFR1 also recruits phospholipase Cγ1 (PLCγ1 at the centrosome. We show that this enzyme is recruited by FOP-FGFR1 at the centrosome during interphase. Conclusion These results delineate a particular type of oncogenic mechanism by which an ectopic kinase recruits its substrates at the centrosome whence unappropriate signaling induces continuous cell growth and MPD.

  15. Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

    Science.gov (United States)

    Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

    2018-02-13

    Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

  16. 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

    International Nuclear Information System (INIS)

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

    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 [ 32 P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [ 32 P]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 [ 32 P]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 [ 32 P]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

  17. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-01-01

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

  18. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid.

    Science.gov (United States)

    Xu, You; Villa, Alessandra; Nilsson, Lennart

    2017-06-05

    Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  20. Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Fábry, Milan; Hubálek, Martin; Otwinowski, Z.; Řezáčová, Pavlína

    2014-01-01

    Roč. 281, č. 18 (2014), s. 4280-4292 ISSN 1742-464X R&D Projects: GA MŠk ME08016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : dimeric interface * effector binding * Schiff base * transcription repressor * X-ray crystallography Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J) Impact factor: 4.001, year: 2014

  1. A General Regioselective Approach to 2,4-Disubstituted Pyrimidin-5-yl C-2-Deoxyribonucleosides

    Czech Academy of Sciences Publication Activity Database

    Kubelka, Tomáš; Slavětínská, Lenka; Hocek, Michal

    2012-01-01

    Roč. 44, č. 6 (2012), s. 953-965 ISSN 0039-7881 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * pyrimidines * cross - coupling * Heck reaction Subject RIV: CC - Organic Chemistry Impact factor: 2.500, year: 2012

  2. Synthesis of 6-Substituted 2(1H)-Pyridon-3-yl C-2'-Deoxyribonucleosides

    Czech Academy of Sciences Publication Activity Database

    Chapuis, Hubert Jean; Kubelka, Tomáš; Joubert, Nicolas; Pohl, Radek; Hocek, Michal

    -, č. 9 (2012), s. 1759-1767 ISSN 1434-193X R&D Projects: GA AV ČR IAA400550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleosides * pyridones * cross - coupling * Heck reaction Subject RIV: CC - Organic Chemistry Impact factor: 3.344, year: 2012

  3. DNA Photosensitization by an "Insider": Photophysics and Triplet Energy Transfer of 5-Methyl-2-pyrimidone Deoxyribonucleoside.

    Science.gov (United States)

    Bignon, Emmanuelle; Gattuso, Hugo; Morell, Christophe; Dumont, Elise; Monari, Antonio

    2015-08-03

    The main chromophore of (6-4) photoproducts, namely, 5-methyl-2-pyrimidone (Pyo), is an artificial noncanonical nucleobase. This chromophore has recently been reported as a potential photosensitizer that induces triplet damage in thymine DNA. In this study, we investigate the spectroscopic properties of the Pyo unit embedded in DNA by means of explicit solvent molecular-dynamics simulations coupled to time-dependent DFT and quantum-mechanics/molecular-mechanics techniques. Triplet-state transfer from the Pyo to the thymine unit was monitored in B-DNA by probing the propensity of this photoactive pyrimidine analogue to induce a Dexter-type triplet photosensitization and subsequent DNA damage. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

    Science.gov (United States)

    Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

    2018-04-01

    Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

    Science.gov (United States)

    Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

    2009-01-01

    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 create a novel

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

    Directory of Open Access Journals (Sweden)

    Debora Bogani

    2009-09-01

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

  7. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

    Science.gov (United States)

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry

  8. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs Effects on AMP-Activated Protein Kinase (AMPK Regulation of Chicken Sperm Functions.

    Directory of Open Access Journals (Sweden)

    Thi Mong Diep Nguyen

    Full Text Available Sperm require high levels of energy to ensure motility and acrosome reaction (AR accomplishment. The AMP-activated protein kinase (AMPK has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+/calmodulin-dependent protein kinase kinases (CaMKKs mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+, or of CaMKKs inhibitor (STO-609. Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β, CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+. Our results show for the first time the presence of CaMKKs (α and β and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+ entry in sperm through the Ca(2+/CaM/CaMKKs/CaMKI pathway. The Ca(2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2

  9. Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway.

    Science.gov (United States)

    Cheng, Lihong; Ye, Ying; Xiang, Lan; Osada, Hiroyuki; Qi, Jianhua

    2017-01-15

    Neurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood-brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases. The present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance. Extract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments. The ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal

  10. Calcium-dependent but calmodulin-independent protein kinase from soybean

    International Nuclear Information System (INIS)

    Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

    1987-01-01

    A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

  11. The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

    Science.gov (United States)

    Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

    2007-04-01

    The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

  12. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    Science.gov (United States)

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance.

  13. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  14. Sensitization of TRPA1 by Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Jannis E Meents

    Full Text Available The TRPA1 ion channel is expressed in nociceptive (pain-sensitive somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1.

  15. Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

    Science.gov (United States)

    Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

    2018-04-01

    Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

    1981-08-27

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

  17. K63-Linked Ubiquitination in Kinase Activation and Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guocan [Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Gao, Yuan [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Li, Liren [Department of Genomic Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Jin, Guoxiang; Cai, Zhen [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Chao, Jui-I [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan (China); Lin, Hui-Kuan, E-mail: hklin@mdanderson.org [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States)

    2012-01-31

    Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.

  18. K63-Linked Ubiquitination in Kinase Activation and Cancer

    International Nuclear Information System (INIS)

    Wang, Guocan; Gao, Yuan; Li, Liren; Jin, Guoxiang; Cai, Zhen; Chao, Jui-I; Lin, Hui-Kuan

    2012-01-01

    Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.

  19. Tunable signal processing in synthetic MAP kinase cascades.

    Science.gov (United States)

    O'Shaughnessy, Ellen C; Palani, Santhosh; Collins, James J; Sarkar, Casim A

    2011-01-07

    The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    Science.gov (United States)

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

  1. Investigating the role of RIO protein kinases in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Tasha K Mendes

    Full Text Available RIO protein kinases (RIOKs are a relatively conserved family of enzymes implicated in cell cycle control and ribosomal RNA processing. Despite their functional importance, they remain a poorly understood group of kinases in multicellular organisms. Here, we show that the C. elegans genome contains one member of each of the three RIOK sub-families and that each of the genes coding for them has a unique tissue expression pattern. Our analysis showed that the gene encoding RIOK-1 (riok-1 was broadly and strongly expressed. Interestingly, the intestinal expression of riok-1 was dependent upon two putative binding sites for the oxidative and xenobiotic stress response transcription factor SKN-1. RNA interference (RNAi-mediated knock down of riok-1 resulted in germline defects, including defects in germ line stem cell proliferation, oocyte maturation and the production of endomitotic oocytes. Taken together, our findings indicate new functions for RIOK-1 in post mitotic tissues and in reproduction.

  2. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

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

  3. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    International Nuclear Information System (INIS)

    Burnam, M.H.; Shell, W.E.

    1981-01-01

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

  4. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

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

    Science.gov (United States)

    Xiong, Yan; Sheen, Jen

    2015-12-01

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

  6. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

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

    2015-05-01

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

  7. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Boris Pinchuk

    2016-04-01

    Full Text Available In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

  8. Conserved family of glycerol kinase loci in Drosophila melanogaster

    Science.gov (United States)

    Martinez Agosto, Julian A.; McCabe, Edward R.B.

    2009-01-01

    Glycerol kinase (GK) is an enzyme that catalyzes the formation of glycerol 3-phosphate from ATP and glycerol, the rate-limiting step in glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for glycerol kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders. PMID:16545593

  9. Characterization of a MAPKK-like protein kinase TOPK

    International Nuclear Information System (INIS)

    Matsumoto, Suguru; Abe, Yasuhito; Fujibuchi, Taketsugu; Takeuchi, Takashi; Kito, Katsumi; Ueda, Norifumi; Shigemoto, Kazuhiro; Gyo, Kiyofumi

    2004-01-01

    A MAPKK-like protein kinase TOPK expresses in a wide range of proliferating cells and tissues such as cancer cells and testis. However, details of this kinase are still uncovered. We investigated the intracellular distribution of TOPK and its association with cdk1/cyclin B and microtubules. In interphase cells, TOPK expresses in cytosol and nucleus without any significant association with microtubule networks. During mitosis, TOPK-Thr-9 was phosphorylated by cdk1/cyclin B and TOPK significantly associates with mitotic spindles. When TOPK expression was suppressed, formation of spindle midzone was thinned and dimmed and cytokinesis was disturbed. We propose that TOPK plays a role in the formation of spindle midzone and in cytokinesis

  10. Molecular cloning of the human casein kinase II α subunit

    International Nuclear Information System (INIS)

    Meisner, H.; Heller-Harrison, R.; Buxton, J.; Czech, M.P.

    1989-01-01

    A human cDNA encoding the α subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II α subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the α subunits

  11. Comparing the Folding and Misfolding Energy Landscapes of Phosphoglycerate Kinase

    OpenAIRE

    Agocs, Gergely; Szabo, Bence T.; Koehler, Gottfried; Osvath, Szabolcs

    2012-01-01

    Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the prop...

  12. A High Affinity Adenosine Kinase from Anopheles gambiae

    Science.gov (United States)

    Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

  13. Novel arylazopyrazole inhibitors of cyclin-dependent kinases

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Schütznerová, E.; Cankař, P.; Brychtová, Veronika; Navrátilová, Jana; Kryštof, Vladimír

    2015-01-01

    Roč. 23, č. 9 (2015), s. 1975-1981 ISSN 0968-0896 R&D Projects: GA ČR GAP305/12/0783; GA ČR GA14-19590S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Cyclin-dependent kinases * Inhibitor * Cell cycle Subject RIV: CE - Biochemistry Impact factor: 2.923, year: 2015

  14. Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

    OpenAIRE

    Skórko, R; Osipiuk, J; Stetter, K O

    1989-01-01

    Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing ...

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Cyclin-dependent kinase inhibitors as anticancer drugs

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Uldrijan, S.

    2010-01-01

    Roč. 11, č. 3 (2010), s. 291-302 ISSN 1389-4501 R&D Projects: GA ČR GA204/08/0511; GA ČR GA301/08/1649; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50380511 Keywords : CDK * protein kinase * inhibitor Subject RIV: CE - Biochemistry Impact factor: 3.061, year: 2010

  17. Supplementary data: Variation in the PTEN-induced putative kinase ...

    Indian Academy of Sciences (India)

    Variation in the PTEN-induced putative kinase 1 gene associated with the increase risk of type 2 diabetes in northern Chinese. Yanchun Qu, Liang Sun, Ze Yang and Ruifa Han. J. Genet. 90, 125–128. Table 1. Clinical characteristics of cases and controls. Phenotype. T2DM. Controls. P value. Age (years). 49.5 ± 11.1. 50.4 ± ...

  18. Serum creatine kinase isoenzymes in children with osteogenesis imperfecta.

    Science.gov (United States)

    D'Eufemia, P; Finocchiaro, R; Zambrano, A; Lodato, V; Celli, L; Finocchiaro, S; Persiani, P; Turchetti, A; Celli, M

    2017-01-01

    This study evaluates serum creatine kinase isoenzyme activity in children with osteogenesis imperfecta to determine its usefulness as a biochemical marker during treatment with bisphosphonate. The changes of creatine kinase (CK) isoenzyme activity during and after discontinuation therapy were observed. These results could be useful in addressing over-treatment risk prevention. The brain isoenzyme of creatine kinase (CKbb) is highly expressed in mature osteoclasts during osteoclastogenesis, thus plays an important role in bone resorption. We previously identified high serum CKbb levels in 18 children with osteogenesis imperfect (OI) type 1 treated for 1 year with bisphosphonate (neridronate). In the present study, serum CK isoenzymes were evaluated in the same children with continuous versus discontinued neridronate treatment over a further 2-year follow-up period. This study included 18 children with OI type 1, 12 with continued (group A) and 6 with ceased (group B) neridronate treatment. Auxological data, serum biochemical markers of bone metabolism, bone mineral density z-score, and serum total CK and isoenzyme activities were determined in both groups. Serum CKbb was progressively and significantly increased in group A (p < 0.004) but rapidly decreased to undetectable levels in group B. In both groups, the cardiac muscle creatine kinase isoenzyme (CKmb) showed a marked decrease, while serum C-terminal telopeptide (CTx) levels were almost unchanged. This study provides evidence of the cumulative effect of neridronate administration in increasing serum CKbb levels and the reversible effect after its discontinuation. This approach could be employed for verifying the usefulness of serum CKbb as a biochemical marker in patients receiving prolonged bisphosphonate treatment. Moreover, the decreased serum CKmb levels suggest a systemic effect of these drugs.

  19. Purine analogs as phosphatidylinositol 4-kinase III beta inhibitors

    Czech Academy of Sciences Publication Activity Database

    Šála, Michal; Kögler, Martin; Plačková, Pavla; Mejdrová, Ivana; Hřebabecký, Hubert; Procházková, Eliška; Strunin, Dmytro; Lee, G.; Birkuš, G.; Weber, Jan; Mertlíková-Kaiserová, Helena; Nencka, Radim

    2016-01-01

    Roč. 26, č. 11 (2016), s. 2706-2712 ISSN 0960-894X R&D Projects: GA ČR GA15-09310S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : phosphatidylinositol 4-kinase * purine * PI4K III beta * antiviral agent * hepatitis C virus Subject RIV: CC - Organic Chemistry Impact factor: 2.454, year: 2016

  20. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  1. Structural aspects of protein kinase ASK1 regulation

    Czech Academy of Sciences Publication Activity Database

    Obšil, Tomáš; Obšilová, Veronika

    2017-01-01

    Roč. 66, 1 Dec (2017), s. 31-36 ISSN 2212-4926 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ASK1 kinase * apoptosis * thioredoxin * 14-3-3 protein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  2. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    International Nuclear Information System (INIS)

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

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

    Czech Academy of Sciences Publication Activity Database

    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 - others: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

  4. The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

    OpenAIRE

    Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John; Rhind, Nicholas

    2008-01-01

    Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or ...

  5. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  6. Crystal Structure of the FERM Domain of Focal Adhesion Kinase

    International Nuclear Information System (INIS)

    Ceccarelli, D.; Song, H.; Poy, F.; Schaller, M.; Eck, M.

    2006-01-01

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment

  7. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

    OpenAIRE

    Boris Pinchuk; Rebecca Horbert; Alexander Döbber; Lydia Kuhl; Christian Peifer

    2016-01-01

    In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrug...

  8. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

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

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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

  11. Src kinase conformational activation: thermodynamics, pathways, and mechanisms.

    Directory of Open Access Journals (Sweden)

    Sichun Yang

    2008-03-01

    Full Text Available Tyrosine kinases of the Src-family are large allosteric enzymes that play a key role in cellular signaling. Conversion of the kinase from an inactive to an active state is accompanied by substantial structural changes. Here, we construct a coarse-grained model of the catalytic domain incorporating experimental structures for the two stable states, and simulate the dynamics of conformational transitions in kinase activation. We explore the transition energy landscapes by constructing a structural network among clusters of conformations from the simulations. From the structural network, two major ensembles of pathways for the activation are identified. In the first transition pathway, we find a coordinated switching mechanism of interactions among the alphaC helix, the activation-loop, and the beta strands in the N-lobe of the catalytic domain. In a second pathway, the conformational change is coupled to a partial unfolding of the N-lobe region of the catalytic domain. We also characterize the switching mechanism for the alphaC helix and the activation-loop in detail. Finally, we test the performance of a Markov model and its ability to account for the structural kinetics in the context of Src conformational changes. Taken together, these results provide a broad framework for understanding the main features of the conformational transition taking place upon Src activation.

  12. Cdc7 kinase - a new target for drug development.

    Science.gov (United States)

    Swords, Ronan; Mahalingam, Devalingam; O'Dwyer, Michael; Santocanale, Corrado; Kelly, Kevin; Carew, Jennifer; Giles, Francis

    2010-01-01

    The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.

  13. Glycogen synthase kinase 3: more than a namesake.

    Science.gov (United States)

    Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

    2009-03-01

    Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

  14. RON kinase isoforms demonstrate variable cell motility in normal cells.

    Science.gov (United States)

    Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

    2016-09-01

    Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

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

    International Nuclear Information System (INIS)

    Barany, K.; Rokolya, A.; Barany, M.

    1990-01-01

    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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  17. Improved radioimmunoassay for creatine kinase isoenzymes in plasma

    International Nuclear Information System (INIS)

    Ritter, C.S.; Mumm, S.R.; Roberts, R.

    1981-01-01

    We describe convenient and relatively rapid procedures for purifying creatine kinase isoenzymes MM, BB, and MB, and their use in an improved radioimmunoassay for creatine kinase isoenzymes in plasma. The modifications include use of: (a) BB with a specific activity of 400 kU/G, which can be labeled with a specific radioactivity of 20 Ci/g; (b) albumin-free purified MB as inhibitor; (c) antiserum to MB creatine kinase; and (d) a second-antibody technique that necessitates only a 15-min incubation. The radioimmunoassay for MB has a sensitivity of 0.2 μg/L (80 mU/L) and a CV of <5%. Plasma MB average 22 (SD 12) μg/L in 200 normal subjects; 24 (SD 12) μg/L in 200 patients with chest pain without infarction; and 23 (SD 7) μg/L in 43 patients with renal disease, whether measured before or after dialysis. Peak values for plasma MB averaged 191 (SD 86) μg/L in 325 patients with documented myocardial infarction; BB was negligible. Extensive clinical experience indicates the radioimmunoassay to be suitably rapid, highly sensitive, and reliable as a diagnostic assay for MB on plasma

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-09-29

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

  19. PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina.

    Science.gov (United States)

    Kicka, Sébastien; Silar, Philippe

    2004-03-01

    MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation.

  20. Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

    Science.gov (United States)

    Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

    2010-06-01

    Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

  1. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

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

    Science.gov (United States)

    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. © 2016 Federation of European Biochemical Societies.

  3. Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

    2018-01-01

    Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

  4. LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development.

    Science.gov (United States)

    Sparrow, Alexander J; Sweetman, Dylan; Welham, Simon J M

    2017-10-01

    Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; Plabelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells*

    Science.gov (United States)

    Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

    2012-01-01

    The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. PMID:22669945

  6. Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells.

    Science.gov (United States)

    Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

    2012-07-27

    The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

  7. Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

    NARCIS (Netherlands)

    Koning, W. de; Weusthuis, R.A.; Harder, W.; Dijkhuizen, L.

    Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250 mM) and an additional substrate (0.5%, w/v) to

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

    International Nuclear Information System (INIS)

    Yang, Se-Ran; Cho, Sung-Dae; Ahn, Nam-Shik; Jung, Ji-Won; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sung-Hoon; Lee, Bong-Hee; Kang, Kyung-Sun; Lee, Yong-Soon

    2005-01-01

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

  9. Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

    International Nuclear Information System (INIS)

    Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

    1985-01-01

    A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and]2number 2 PO 4 /mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 34 PO 4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro

  10. Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

    Science.gov (United States)

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-10-01

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

  11. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

    Directory of Open Access Journals (Sweden)

    Boon Siang Nicholas Tan

    Full Text Available Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL cells from mouse embryonic stem (mES cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

  12. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    Science.gov (United States)

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

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

    Directory of Open Access Journals (Sweden)

    Guan Kunliang

    2003-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Eike Hendrik Rademacher

    2012-11-01

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

  15. Structural insight into the mechanism of synergistic autoinhibition of SAD kinases.

    Science.gov (United States)

    Wu, Jing-Xiang; Cheng, Yun-Sheng; Wang, Jue; Chen, Lei; Ding, Mei; Wu, Jia-Wei

    2015-12-02

    The SAD/BRSK kinases participate in various important life processes, including neural development, cell cycle and energy metabolism. Like other members of the AMPK family, SAD contains an N-terminal kinase domain followed by the characteristic UBA and KA1 domains. Here we identify a unique autoinhibitory sequence (AIS) in SAD kinases, which exerts autoregulation in cooperation with UBA. Structural studies of mouse SAD-A revealed that UBA binds to the kinase domain in a distinct mode and, more importantly, AIS nestles specifically into the KD-UBA junction. The cooperative action of AIS and UBA results in an 'αC-out' inactive kinase, which is conserved across species and essential for presynaptic vesicle clustering in C. elegans. In addition, the AIS, along with the KA1 domain, is indispensable for phospholipid binding. Taken together, these data suggest a model for synergistic autoinhibition and membrane activation of SAD kinases.

  16. High quality, small molecule-activity datasets for kinase research [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rajan Sharma

    2016-10-01

    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.

  17. The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum.

    Science.gov (United States)

    Lin, Benjamin C; Harris, Darcy R; Kirkman, Lucy M D; Perez, Astrid M; Qian, Yiwen; Schermerhorn, Janse T; Hong, Min Y; Winston, Dennis S; Xu, Lingyin; Lieber, Alexander M; Hamilton, Matthew; Brandt, Gabriel S

    2017-12-01

    The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC 50 =2μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    KAUST Repository

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

    2012-01-01

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

  19. Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

    DEFF Research Database (Denmark)

    Wang, X; Uhler, M D; Billestrup, N

    1992-01-01

    The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels...... of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present...... in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific....

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

    KAUST Repository

    Irving, Helen R.

    2012-02-01

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

  1. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  3. Bruton tyrosine kinase inhibition in chronic lymphocytic leukemia.

    Science.gov (United States)

    Maddocks, Kami; Jones, Jeffrey A

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and remains incurable outside of the setting of allogeneic stem cell transplant. While the standard therapy for both initial and relapsed CLL has traditionally included monoclonal antibody therapy in combination with chemotherapy, there are patients with high-risk disease features including unmutated IgVH, del(11q22) and del(17p13) that are associated with poor overall responses to these therapies with short time to relapse and shortened overall survival. Additionally, many of these therapies have a high rate of infectious toxicity in a population already at increased risk. Targeting the B-cell receptor (BCR) signaling pathway has emerged as a promising therapeutic advance in a variety of B-cell malignancies, including CLL. Bruton agammaglobulinemia tyrosine kinase (Btk) is a tyrosine kinase in the BCR pathway critical to the survival of both normal and malignant B cells and inhibition of this kinase has shown to block the progression of CLL. Ibrutinib, a first in class oral inhibitor of Btk, has shown promise as a very effective agent in the treatment of CLL-in both relapsed and upfront therapy, alone and in combination with other therapies, and in patients of all-risk disease-which has led to its approval in relapsed CLL and as frontline therapy in patients with the high-risk del(17p13) disease. Several studies are ongoing to evaluate the efficacy and safety of ibrutinib in combination with chemotherapy as frontline treatment for CLL and investigation into newer-generation Btk inhibitors is also underway. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

    Science.gov (United States)

    Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

    2017-11-01

    Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

  5. Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

    OpenAIRE

    Lewis, R E; Czech, M P

    1987-01-01

    Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for ...

  6. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    OpenAIRE

    Fabian V Filipp

    2013-01-01

    Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor acti...

  7. DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

    OpenAIRE

    Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

    2007-01-01

    The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

  8. The Discovery of Aurora Kinase Inhibitor by Multi-Docking-Based Virtual Screening

    Directory of Open Access Journals (Sweden)

    Jun-Tae Kim

    2014-11-01

    Full Text Available We report the discovery of aurora kinase inhibitor using the fragment-based virtual screening by multi-docking strategy. Among a number of fragments collected from eMololecules, we found four fragment molecules showing potent activity (>50% at 100 μM against aurora kinase. Based on the explored fragment scaffold, we selected two compounds in our synthesized library and validated the biological activity against Aurora kinase.

  9. Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

    Science.gov (United States)

    Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

    2016-07-01

    The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

  10. Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-14-1-0251 TITLE: Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase... Tyrosine Kinase Inhibitors 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0251 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kiran Mahajan 5d...ABSTRACT Central to all cycling cells-including prostate cancer stem cells- is the expression of WEE1 tyrosine kinase. WEE1 monitors duplication of

  11. Theoretical Insights Reveal Novel Motions in Csk's SH3 Domain That Control Kinase Activation.

    Directory of Open Access Journals (Sweden)

    Sulyman Barkho

    Full Text Available The Src family of tyrosine kinases (SFKs regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk. Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk's activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk.

  12. Csk Homologous Kinase, a Potential Regulator of CXCR4-mediated Breast Cancer Cell Metastasis

    Science.gov (United States)

    2010-08-31

    SH2 ) and SH3 domains and lacks the consensus tyrosine phosphorylation and myristylation sites found in Src family kinases . CHK has been shown to...0350 TITLE: Csk Homologous Kinase , a Potential Regulator of CXCR4-mediated Breast Cancer Cell Metastasis PRINCIPAL INVESTIGATOR: Byeong-Chel...1 AUG 2009 - 31 JUL 2010 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-09-1-0350 Csk Homologous Kinase , a Potential Regulator

  13. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

    OpenAIRE

    Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

    1994-01-01

    Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the ...

  14. The tyrosyl residues in creatine kinase. Modification by iodine.

    Science.gov (United States)

    Fattoum, A; Kassab, R; Pradel, L A

    1975-10-20

    The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.

  15. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

    OpenAIRE

    Garone, Caterina; Garc??a-D??az, Beatriz; Emmanuele, Valentina; L??pez Garc??a, Luis Carlos; Tadesse, Saba; Akman, Hasan O.; Tanji, Kurenai; Quinzii, Catarina M.; Hirano, Michio

    2014-01-01

    Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 −/− ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-...

  16. Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

    Science.gov (United States)

    Skórko, R; Osipiuk, J; Stetter, K O

    1989-09-01

    Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.

  17. Protein kinase CK2 structure-function relationship

    DEFF Research Database (Denmark)

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

    1994-01-01

    Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations...

  18. Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

    Science.gov (United States)

    Radich, Jerald P; Mauro, Michael J

    2017-08-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Protein Kinase Signalling in the Moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Azevedo de Silva, Raquel

    Adaptation to environmental cues trigger a plethora of intracellular pathways capable of maintaining homeostasis. Receptors in the plasma membrane and in the cytosol recognize extracellular or intracellular signals initiating defense against pathogens or stress-adaptation. MAPK cascade are one...... of the pathways involved in stress signalling, phosphorylating several downstream substrates in order to produce appropriate responses. We report here that P. patens has a receptor-like kinase CERK1 responsible for chitin perception which can rescue Atcerk1 mutant. Activation of PpCERK1 triggers the activation...

  20. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

    Science.gov (United States)

    Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  1. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK Complex.

    Directory of Open Access Journals (Sweden)

    Stefan Frey

    Full Text Available The striatin-interacting phosphatase and kinase (STRIPAK complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP. In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

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

    Science.gov (United States)

    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. © The Author(s) 2015.

  3. Partial contribution of Rho-kinase inhibition to the bioactivity of Ganoderma lingzhi and its isolated compounds: insights on discovery of natural Rho-kinase inhibitors.

    Science.gov (United States)

    Amen, Yhiya; Zhu, Qinchang; Tran, Hai-Bang; Afifi, Mohamed S; Halim, Ahmed F; Ashour, Ahmed; Shimizu, Kuniyoshi

    2017-04-01

    Recent studies identified Rho-kinase enzymes (ROCK-I and ROCK-II) as important targets that are involved in a variety of diseases. Synthetic Rho-kinase inhibitors have emerged as potential therapeutic agents to treat disorders such as hypertension, stroke, cancer, diabetes, glaucoma, etc. Our study is the first to screen the total ethanol extract of the medicinal mushroom Ganoderma lingzhi with thirty-five compounds for Rho-kinase inhibitory activity. Moreover, a molecular binding experiment was designed to investigate the binding affinity of the compounds at the active sites of Rho-kinase enzymes. The structure-activity relationship analysis was investigated. Our results suggest that the traditional uses of G. lingzhi might be in part due to the ROCK-I and ROCK-II inhibitory potential of this mushroom. Structure-activity relationship studies revealed some interesting features of the lanostane triterpenes that potentiate their Rho-kinase inhibition. These findings would be helpful for further studies on the design of Rho-kinase inhibitors from natural sources and open the door for contributions from other researchers for optimizing the development of natural Rho-kinase inhibitors.

  4. Contraction-associated translocation of protein kinase C in rat skeletal muscle

    DEFF Research Database (Denmark)

    Richter, Erik; Cleland, P J; Rattigan, S

    1987-01-01

    Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short...... tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism....

  5. Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase

    International Nuclear Information System (INIS)

    Qian, Kevin C.; Studts, Joey; Wang, Lian; Barringer, Kevin; Kronkaitis, Anthony; Peng, Charline; Baptiste, Alistair; LaFrance, Roger; Mische, Sheenah; Farmer, Bennett

    2004-01-01

    Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P6 5 , with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit

  6. Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

    Science.gov (United States)

    Labbe, Benjamin D; Kristich, Christopher J

    2017-11-01

    Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  8. Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free...... suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP...

  9. Visual Snapshots of Intracellular Kinase Activity At The Onset of Mitosis

    Science.gov (United States)

    Dai, Zhaohua; Dulyaninova, Natalya G.; Kumar, Sanjai; Bresnick, Anne R.; Lawrence, David S.

    2007-01-01

    Summary Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell division cycle. Robust protein kinase activity transpires just prior to, but not immediately following, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC β isoform with subsequent NEB. PMID:18022564

  10. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

    Science.gov (United States)

    Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

    2009-03-24

    ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

  11. Approved and Experimental Small-Molecule Oncology Kinase Inhibitor Drugs: A Mid-2016 Overview.

    Science.gov (United States)

    Fischer, Peter M

    2017-03-01

    Kinase inhibitor research is a comparatively recent branch of medicinal chemistry and pharmacology and the first small-molecule kinase inhibitor, imatinib, was approved for clinical use only 15 years ago. Since then, 33 more kinase inhibitor drugs have received regulatory approval for the treatment of a variety of cancers and the volume of reports on the discovery and development of kinase inhibitors has increased to an extent where it is now difficult-even for those working in the field-easily to keep an overview of the compounds that are being developed, as currently there are 231 such compounds, targeting 38 different protein and lipid kinases (not counting isoforms), in clinical use or under clinical investigation. The purpose of this review is thus to provide an overview of the biomedical rationales for the kinases being targeted on the one hand, and the design principles, as well as chemical, pharmacological, pharmaceutical, and toxicological kinase inhibitor properties, on the other hand. Two issues that are especially important in kinase inhibitor research, target selectivity and drug resistance, as well as the underlying structural concepts, are discussed in general terms and in the context of relevant kinases and their inhibitors. © 2016 Wiley Periodicals, Inc.

  12. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...... and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...

  13. Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    Directory of Open Access Journals (Sweden)

    Ren-You Gan

    2014-09-01

    Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

  14. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States); Busenlehner, Laura [Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 (United States); Marcus, Stevan, E-mail: smarcus@bama.ua.edu [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States)

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  15. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    Science.gov (United States)

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  16. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-01-01

    Research highlights: → Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. → The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. → Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. → PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

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

    Science.gov (United States)

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

    2009-06-03

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

  18. Structural changes of creatine kinase upon substrate binding.

    Science.gov (United States)

    Forstner, M; Kriechbaum, M; Laggner, P; Wallimann, T

    1998-08-01

    Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

  19. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    Science.gov (United States)

    Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

    2004-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

  20. Pyruvate kinase M2: a potential target for regulating inflammation

    Directory of Open Access Journals (Sweden)

    Jose Carlos eAlves-Filho

    2016-04-01

    Full Text Available Pyruvate kinase (PK is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signalling pathways, affecting both the enzymatic activity of PKM2 as a pyruvate kinase and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for PKM2 as a therapeutic target in inflammatory and metabolic disorders.