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Sample records for putative sensor kinase

  1. The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila.

    PubMed

    Tiaden, André; Spirig, Thomas; Sahr, Tobias; Wälti, Martin A; Boucke, Karin; Buchrieser, Carmen; Hilbi, Hubert

    2010-05-01

    The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and Lqs

  2. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  3. Putative modifier genes in mevalonate kinase deficiency.

    PubMed

    Marcuzzi, Annalisa; Vozzi, Diego; Girardelli, Martina; Tricarico, Paola Maura; Knowles, Alessandra; Crovella, Sergio; Vuch, Josef; Tommasini, Alberto; Piscianz, Elisa; Bianco, Anna Monica

    2016-04-01

    Mevalonate kinase deficiency (MKD) is an autosomal recessive auto‑inflammatory disease, caused by impairment of the mevalonate pathway. Although the molecular mechanism remains to be elucidated, there is clinical evidence suggesting that other regulatory genes may be involved in determining the phenotype. The identification of novel target genes may explain non‑homogeneous genotype‑phenotype correlations, and provide evidence in support of the hypothesis that novel regulatory genes predispose or amplify deregulation of the mevalonate pathway in this orphan disease. In the present study, DNA samples were obtained from five patients with MKD, which were then analyzed using whole exome sequencing. A missense variation in the PEX11γ gene was observed in homozygosis in P2, possibly correlating with visual blurring. The UNG rare gene variant was detected in homozygosis in P5, without correlating with a specific clinical phenotype. A number of other variants were found in the five analyzed DNA samples from the MKD patients, however no correlation with the phenotype was established. The results of the presents study suggested that further analysis, using next generation sequencing approaches, is required on a larger sample size of patients with MKD, who share the same MVK mutations and exhibit 'extreme' clinical phenotypes. As MVK mutations may be associated with MKD, the identification of specific modifier genes may assist in providing an earlier diagnosis.

  4. WNK3 is a putative chloride-sensing kinase.

    PubMed

    Pacheco-Alvarez, Diana; Gamba, Gerardo

    2011-01-01

    The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex. PMID:22179001

  5. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  6. A novel putative tyrosine kinase receptor with oncogenic potential.

    PubMed

    Janssen, J W; Schulz, A S; Steenvoorden, A C; Schmidberger, M; Strehl, S; Ambros, P F; Bartram, C R

    1991-11-01

    We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

  7. Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana

    PubMed Central

    Xie, Yuan; Li, Mei; Chang, Wenrui

    2016-01-01

    The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. PMID:27223615

  8. Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.

    PubMed

    Tassan, J P; Jaquenoud, M; Léopold, P; Schultz, S J; Nigg, E A

    1995-09-12

    Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

  9. Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis

    SciTech Connect

    Newman, Joseph A.; Das, Sanjan K.; Sedelnikova, Svetlana E.; Rice, David W.

    2006-10-01

    A putative pyridoxal kinase from B. subtilis has been cloned, overexpressed, purified and crystallized and data have been collected to 2.8 Å resolution. Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B{sub 6} precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5′-phosphates and play an important role in the vitamin B{sub 6} salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ‘HMPP kinase-like’ pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg{sup 2+}. X-ray diffraction data have been collected from crystals to 2.8 Å resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 Å, α = β = γ = 90°) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.

  10. Crystal structures of putative phosphoglycerate kinases from B. anthracis> and C. jejuni

    PubMed Central

    Zheng, Heping; Filippova, Ekaterina V.; Tkaczuk, Karolina L.; Dworzynski, Piotr; Chruszcz, Maksymilian; Porebski, Przemyslaw J.; Wawrzak, Zdzislaw; Onopriyenko, Olena; Kudritska, Marina; Grimshaw, Sarah; Savchenko, Alexei; Anderson, Wayne F.

    2015-01-01

    Phosphoglycerate kinase (PGK) is indispensable during glycolysis for anaerobic glucose degradation and energy generation. Here we present comprehensive structure analysis of two putative PGKs from Bacillus anthracis str. Sterne and Campylobacter jejuni in the context of their structural homologs. They are the first PGKs from pathogenic bacteria reported in the Protein Data Bank. The crystal structure of PGK from Bacillus anthracis str. Sterne (BaPGK) has been determined at 1.68 Å while the structure of PGK from Campylobacter jejuni (CjPGK) has been determined at 2.14 A resolution. The proteins’ monomers are composed of two domains, each containing a Rossmann fold, hinged together by a helix which can be used to adjust the relative position between two domains. It is also shown that apo-forms, of both BaPGK and CjPGK adopt open conformations as compared to the substrate and ATP bound forms of PGK from other species. PMID:22403005

  11. Genome-wide survey of putative Serine/Threonine protein kinases in cyanobacteria

    PubMed Central

    Zhang, Xiaowen; Zhao, Fangqing; Guan, Xiangyu; Yang, Yu; Liang, Chengwei; Qin, Song

    2007-01-01

    Background Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence

  12. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  13. Identification and Characterization of a Putative Arginine Kinase Homolog from Myxococcus xanthus Required for Fruiting Body Formation and Cell Differentiation

    PubMed Central

    Bragg, Jonathan; Rajkovic, Andrei; Anderson, Chance; Curtis, Rachael; Van Houten, Jason; Begres, Brittany; Naples, Colin; Snider, Mark; Fraga, Dean

    2012-01-01

    Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development. PMID:22389486

  14. Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

    PubMed Central

    Velikova, Nadya; Fulle, Simone; Manso, Ana Sousa; Mechkarska, Milena; Finn, Paul; Conlon, J. Michael; Oggioni, Marco Rinaldo; Wells, Jerry M.; Marina, Alberto

    2016-01-01

    Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials. PMID:27173778

  15. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  16. Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit.

    PubMed

    Park, Inshik; Ives, David H

    2002-03-31

    Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.

  17. Myxococcus xanthus mokA Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance

    PubMed Central

    Kimura, Yoshio; Nakano, Hiromi; Terasaka, Hideaki; Takegawa, Kaoru

    2001-01-01

    A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum. PMID:11157925

  18. Preparation and characterization of human ADCK3, a putative atypical kinase.

    PubMed

    Wheeler, Brody; Jia, Zongchao

    2015-04-01

    AarF domain containing kinase 3 (ADCK3) is a mitochondrial protein known to have a role in the electron transport chain. Despite being required for the biosynthesis of coenzyme Q10, a lipid-soluble electron transporter found to be essential for aerobic cellular respiration, the precise biological function of ADCK3 remains unknown. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. After extensive screening for soluble recombinant protein expression, an N-terminal fusion of maltose-binding protein was found to facilitate the overexpression of the human ADCK3 kinase domain in Escherichia coli as a soluble and biologically active entity. For the first time our work reveals Mg(2+)-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of this protein being a functional atypical kinase.

  19. Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2

    PubMed Central

    Beautrait, Alexandre; Michalski, Kevin R.; Lopez, Thomas S.; Mannix, Katelynn M.; McDonald, Devin J.; Cutter, Amber R.; Medina, Christopher B.; Hebert, Aaron M.; Francis, Charnelle J.; Bouvier, Michel; Tesmer, John J. G.; Sterne-Marr, Rachel

    2014-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. PMID:25049229

  20. Signal Transduction by BvgS Sensor Kinase

    PubMed Central

    Dupré, Elian; Lesne, Elodie; Guérin, Jérémy; Lensink, Marc F.; Verger, Alexis; de Ruyck, Jérôme; Brysbaert, Guillaume; Vezin, Hervé; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-01-01

    The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS. PMID:26203186

  1. The putative lipid transporter, Arv1, is required for activating pheromone-induced MAP kinase signaling in Saccharomyces cerevisiae.

    PubMed

    Villasmil, Michelle L; Ansbach, Alison; Nickels, Joseph T

    2011-02-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P(2) and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5(Q59L), which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P(2) polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

  2. The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

    PubMed Central

    Villasmil, Michelle L.; Ansbach, Alison; Nickels, Joseph T.

    2011-01-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking. PMID:21098723

  3. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  4. Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

    PubMed Central

    Miyoshi, J; Higashi, T; Mukai, H; Ohuchi, T; Kakunaga, T

    1991-01-01

    A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor. Images PMID:2072910

  5. Sensor Domain of Histidine Kinase KinB of Pseudomonas

    PubMed Central

    Tan, Kemin; Chhor, Gekleng; Binkowski, T. Andrew; Jedrzejczak, Robert P.; Makowska-Grzyska, Magdalena; Joachimiak, Andrzej

    2014-01-01

    The overproduction of polysaccharide alginate is responsible for the formation of mucus in the lungs of cystic fibrosis patients. Histidine kinase KinB of the KinB-AlgB two-component system in Pseudomonas aeruginosa acts as a negative regulator of alginate biosynthesis. The modular architecture of KinB is similar to other histidine kinases. However, its periplasmic signal sensor domain is unique and is found only in the Pseudomonas genus. Here, we present the first crystal structures of the KinB sensor domain. The domain is a dimer in solution, and in the crystal it shows an atypical dimer of a helix-swapped four-helix bundle. A positively charged cavity is formed on the dimer interface and involves several strictly conserved residues, including Arg-60. A phosphate anion is bound asymmetrically in one of the structures. In silico docking identified several monophosphorylated sugars, including β-d-fructose 6-phosphate and β-d-mannose 6-phosphate, a precursor and an intermediate of alginate synthesis, respectively, as potential KinB ligands. Ligand binding was confirmed experimentally. Conformational transition from a symmetric to an asymmetric structure and decreasing dimer stability caused by ligand binding may be a part of the signal transduction mechanism of the KinB-AlgB two-component system. PMID:24573685

  6. Phosphatase and tensin homolog-induced putative kinase 1 and Parkin in diabetic heart: Role of mitophagy.

    PubMed

    Tang, Ying; Liu, Jiankang; Long, Jiangang

    2015-05-01

    Diabetes is an independent risk factor for cardiovascular morbidity and mortality. Diabetes-associated cardiac pathophysiology is recognized to be due to reasons including metabolic consequences on the myocardium. The heart is a highly energy-demanding tissue, with mitochondria supplying over 90% of adenosine triphosphate. The involvement of mitochondrial dysfunction in diabetes-related cardiac pathogenesis has been studied. Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin, initially identified to be associated with the pathogenesis of a familiar form of Parkinson's disease, have recently been recognized to play a critical role in mediating cardiomyocytes' adaption to stresses. Extensive studies have suggested PINK1 and Parkin as key regulators of mitophagy. In the present review article, we will first summarize the new findings on PINK1/Parkin acting in cardioprotection, and then discuss the potential role of PINK1/Parkin in diabetic heart by mediating mitophagy. PMID:25969707

  7. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    PubMed

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  8. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps

    PubMed Central

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A. S.; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-01-01

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K+ uptake system in the Venus flytrap. In search of K+ transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K+-transporter genes into Xenopus oocytes, however, both putative K+ transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K+ transporter 1 (AKT1), we coexpressed the putative K+ transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K+ uptake. DmKT1 was found to be a K+-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around −120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K+, reducing its concentration from millimolar levels down to trace levels. PMID:25997445

  9. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    PubMed

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-01

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels. PMID:25997445

  10. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    PubMed

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-01

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

  11. Identification of salt-inducible peptide with putative kinase activity in halophilic bacterium Virgibacillus halodenitrificans.

    PubMed

    Rafiee, Mahmoud-Reza; Sokhansanj, Ashrafaddin; Yoosefi, Mitra; Naghizadeh, Mohammad-Ali

    2007-09-01

    Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics. PMID:17964480

  12. Sphingosine kinase: a novel putative target for the prevention of infection-triggered preterm birth.

    PubMed

    Vyas, Vibhuti; Ashby, Charles R; Reznik, Sandra E

    2013-01-01

    Preterm birth is defined as any delivery before 37 complete weeks of gestation. It is a universal challenge in the field of obstetrics owing to its high rate of mortality, long-term morbidity, associated human suffering and economic burden. In the United States, about 12.18% deliveries in 2009 were preterm, producing an exorbitant cost of $5.8 billion. Infection-associated premature rupture of membranes (PROM) accounts for 40% of extremely preterm births (<28 weeks of gestation). Major research efforts are directed towards improving the understanding of the pathophysiology of preterm birth and ways to prevent or at least postpone delivery. Endothelin-1 (ET-1) is a potent vasoconstrictor that plays a significant role in infection-triggered preterm birth. Its involvement in a number of pathological mechanisms and its elevation in preterm delivered amniotic fluid samples implicate it in preterm birth. Sphingosine kinase (SphK) is a ubiquitous enzyme responsible for the production of sphingosine-1-phosphate (S1P). S1P acts as second messenger in a number of cell proliferation and survival pathways. SphK is found to play a key role in ET-1 mediated myometrial contraction. This review highlights SphK as a prospective target with great potential to prevent preterm birth. PMID:23818902

  13. Perinatal programming of body weight control by leptin: putative roles of AMP kinase and muscle thermogenesis.

    PubMed

    Pico, Catalina; Jilkova, Zuzana Macek; Kus, Vladimir; Palou, Andreu; Kopecky, Jan

    2011-12-01

    Breastfeeding, compared with infant-formula feeding, confers later protection against obesity. Leptin represents a candidate for the programming of the lean phenotype as suggested by 1) the presence of leptin in breast milk and its absence in infant formula, 2) a human study that showed a negative correlation between leptin concentrations in breast milk and body weights of infants until 2 y of age, and 3) intervention studies in animals. Milk-borne leptin and leptin synthesized in adipose tissue and the stomach may contribute to leptinemia in newborns. Studies in rodents suggested that early leptin treatment may program either a lean or obese phenotype, probably depending on the dose, route of administration, and timing of exposure to high leptin concentrations, whereas these studies also suggested the importance of the physiologic postnatal surge in leptinemia for the programming effect. Leptin oral administration at physiologic doses to neonate rats during the entire lactation period had later positive effects that prevented the animals from overweight and obesity and other metabolic alterations, which were particularly associated with feeding of a high-fat diet. High leptin sensitivity, which is associated with leanness, and leptin resistance in obesity may be programmed by the early life environment. The differential sensitivity to leptin implies a contribution of leptin-inducible energy expenditure to the adult phenotype. Available data have suggested the involvement of nonshivering thermogenesis induced by a leptin-AMP-activated protein kinase axis in oxidative muscles, which is based on lipid metabolism. Additional studies on the programming effects of leptin, mainly in response to the oral intake of leptin, are required.

  14. Crystal Structure of a Histidine Kinase Sensor Domain with Similarity to Periplasmic Binding Proteins

    SciTech Connect

    Cheung, J.; Le-Khac, M; Hendrickson, W

    2009-01-01

    Histidine kinase receptors are elements of the two-component signal transduction systems commonly found in bacteria and lower eukaryotes, where they are crucial for environmental adaption through the coupling of extracellular changes to intracellular responses. The typical two-component system consists of a membrane-spanning histidine kinase sensor and a cytoplasmic response regulator. In the calssic system, extracellular signals such as small molecule ligands and ions are detected by the periplasmic sensor domain of the histidine kinase receptor, which modulates the catalytic activity of the cytoplasmic histidine kinase domain and promotes ATP-dependent autophosphorylation of a conserved histidine residue. G. sulfurreducens genomic DNA was used.

  15. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  16. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito

    PubMed Central

    Hart, Robert J.; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S.; Ben Mamoun, Choukri; Aly, Ahmed S. I.

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  17. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

  18. Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

    PubMed Central

    Zhang, Zhen; Hendrickson, Wayne A.

    2012-01-01

    Histidine kinase receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi and other protists. Typical histidine kinase receptors have an extracellular sensor portion to detect the signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While the kinase domains are highly conserved, sensor domains are diverse. Histidine kinase receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, Family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane histidine kinases. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated- and apo-state structures provide insights into signal transmission. Some HK1-family proteins prove to be sensors for chemotaxis proteins or for diguanylate cyclase receptors, which implies a combinatorial molecular evolution. PMID:20435045

  19. CHARACTERIZATION AND FUNCTIONAL STUDY OF A PUTATIVE JUVENILE HORMONE DIOL KINASE IN THE COLORADO POTATO BEETLE Leptinotarsa decemlineata (Say).

    PubMed

    Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2015-11-01

    Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide-binding elements, and contains three EF-hand motifs (helix-loop-helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double-stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr-h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation. PMID:26280246

  20. CHARACTERIZATION AND FUNCTIONAL STUDY OF A PUTATIVE JUVENILE HORMONE DIOL KINASE IN THE COLORADO POTATO BEETLE Leptinotarsa decemlineata (Say).

    PubMed

    Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2015-11-01

    Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide-binding elements, and contains three EF-hand motifs (helix-loop-helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double-stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr-h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation.

  1. Methylatable Signaling Helix Coordinated Inhibitory Receiver Domain in Sensor Kinase Modulates Environmental Stress Response in Bacillus Cereus

    PubMed Central

    Chen, Jung-Chi; Liu, Jyung-Hurng; Hsu, Duen-Wei; Shu, Jwu-Ching; Chen, Chien-Yen; Chen, Chien-Cheng

    2015-01-01

    σB, an alternative transcription factor, controls the response of the cell to a variety of environmental stresses in Bacillus cereus. Previously, we reported that RsbM negatively regulates σB through the methylation of RsbK, a hybrid sensor kinase, on a signaling helix (S-helix). However, RsbK comprises a C-terminal receiver (REC) domain whose function remains unclear. In this study, deletion of the C-terminal REC domain of RsbK resulted in high constitutive σB expression independent of environmental stimuli. Thus, the REC domain may serve as an inhibitory element. Mutagenic substitution was employed to modify the putative phospho-acceptor residue D827 in the REC domain of RsbK. The expression of RsbKD827N and RsbKD827E exhibited high constitutive σB, indicating that D827, if phosphorylatable, possibly participates in σB regulation. Bacterial two-hybrid analyses demonstrated that RsbK forms a homodimer and the REC domain interacts mainly with the histidine kinase (HK) domain and partly with the S-helix. In particular, co-expression of RsbM strengthens the interaction between the REC domain and the S-helix. Consistently, our structural model predicts a significant interaction between the HK and REC domains of the RsbK intradimer. Here, we demonstrated that coordinated the methylatable S-helix and the REC domain of RsbK is functionally required to modulate σB-mediated stress response in B. cereus and maybe ubiquitous in microorganisms encoded RsbK-type sensor kinases. PMID:26379238

  2. Ligand-Induced Asymmetry in Histidine Sensor Kinase Complex Regulates Quorum Sensing

    SciTech Connect

    Neiditch,M.; Federle, M.; Pompeani, A.; Kelly, R.; Swem, D.; Jeffrey, P.; Bassler, B.; Hughson, F.

    2006-01-01

    Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

  3. Crystal Structures of Apparent Saccharide Sensors from Histidine Kinase Receptors Prevalent in a Human Gut Symbiont

    PubMed Central

    Zhang, Zhen; Liu, Qun; Hendrickson, Wayne A.

    2014-01-01

    The adult human gut presents a complicated ecosystem where host-bacterium symbiosis plays an important role. Bacteroides thetaiotaomicron is a predominant member of the gut microflora, providing the human digestive tract with a large number of glycolytic enzymes. Expression of many of these enzymes appears to be controlled by histidine kinase receptors that are fused into unusual hybrid two-component systems that share homologous periplasmic sensor domains. These sensor domains belong to the third most populated (HK3) family based on a previous bioinformatics analysis of predicted histidine kinase sensors. Here, we present crystal structures of two sensor domains representative of the HK3 family. Each sensor is folded into three domains: two seven-bladed β-propeller domains and one β-sandwich domain. Both sensors form dimers in crystals and one sensor appears to be physiologically relevant. The folding characteristics in the individual domains, the domain organization, and the oligomeric architecture are all unique to the HK3 sensors. The sequence analysis of the HK3 sensors indicates that these sensors are shared among other signaling molecules, implying a combinatorial molecular evolution. PMID:24995510

  4. Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

    SciTech Connect

    Zhang, Z.; Hendrickson, W

    2010-01-01

    Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.

  5. Structural characterization of the predominant family of histidine kinase sensor domains.

    PubMed

    Zhang, Zhen; Hendrickson, Wayne A

    2010-07-16

    Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.

  6. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility

    SciTech Connect

    Not Available

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  7. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

    PubMed Central

    Han, Xue; Adames, Kelly; Sykes, Ellen M. E.; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase. PMID:26168236

  8. How Important Is the Phosphatase Activity of Sensor Kinases?

    PubMed Central

    Kenney, Linda J.

    2010-01-01

    In two-component signaling systems, phosphorylated response regulators (RRs) are often dephosphorylated by their partner kinases in order to control the in vivo concentration of phospho-RR (RR~P). This activity is easily demonstrated in vitro, but these experiments have typically used very high concentrations of the histidine kinase (HK) compared to the RR~P. Many two-component systems exhibit exquisite control over the ratio of HK to RR in vivo. The question thus arises as to whether the phosphatase activity of HKs is significant in vivo. This topic will be explored in the present review. PMID:20223700

  9. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

    PubMed Central

    Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

    1994-01-01

    The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

  10. Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism

    PubMed Central

    Herrou, Julien; Bompard, Coralie; Wintjens, René; Dupré, Elian; Willery, Eve; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2010-01-01

    Two-component sensory transduction systems control important bacterial programs. In Bordetella pertussis, expression of the virulence regulon is controlled by the unorthodox BvgAS two-component system. BvgS is the prototype of a family of sensor-kinases that harbor periplasmic domains homologous to bacterial solute-binding proteins. Although BvgAS is active under laboratory conditions, no activating signal has been identified, only negative modulators. Here we show that the second periplasmic domain of BvgS interacts with modulators and adopts a Venus flytrap (VFT) fold. X-ray crystallography reveals that the two lobes of VFT2 delimitate a ligand-binding cavity enclosing fortuitous ligands. Most substitutions of putative ligand-binding residues in the VFT2 cavity keep BvgS active, and alteration of the cavity's electrostatic potential affects responsiveness to modulation. The crystal structure of this VFT2 variant conferring constitutive kinase activity to BvgS shows a closed cavity with another nonspecific ligand. Thus, VFT2 is closed and active without a specific agonist ligand, in contrast to typical VFTs. Modulators are antagonists of VFT2 that interrupt signaling. BvgAS is active for most of the B. pertussis infectious cycle, consistent with the proposed mechanism. PMID:20855615

  11. Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.

    PubMed

    Taipale, Mikko; Krykbaeva, Irina; Whitesell, Luke; Santagata, Sandro; Zhang, Jianming; Liu, Qingsong; Gray, Nathanael S; Lindquist, Susan

    2013-07-01

    The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

  12. Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

    PubMed Central

    Gauntlett, Jonathan C.; Oldenburg, Darby G.; Cook, Gregory M.; Rainey, Paul B.

    2015-01-01

    ABSTRACT CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [3H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction. IMPORTANCE Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single

  13. Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

    PubMed

    Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

    2016-02-01

    β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

  14. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

    SciTech Connect

    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  15. Characterization of the RcsC sensor kinase from Erwinia amylovora and other Enterobacteria.

    PubMed

    Wang, Dongping; Korban, Schuyler S; Pusey, P Lawrence; Zhao, Youfu

    2011-06-01

    RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain, and a receiver domain. We have previously demonstrated that, although the Erwinia amylovora rcsC mutant produces more amylovoran than the wild-type (WT) strain in vitro, the mutant remains nonpathogenic on both immature pear fruit and apple plants. In this study, we have comparatively characterized the Erwinia RcsC and its homologs from various enterobacteria. Results demonstrate that expression of the Erwinia rcsC gene suppresses amylovoran production in various amylovoran overproducing WT and mutant strains, thus suggesting the presence of a net phosphatase activity of Erwinia RcsC. Findings have also demonstrated that rcsC homologs from other enterobacteria could not rescue amylovoran production of the Erwinia rcsC mutant in vitro. However, virulence of the Erwinia rcsC mutant is partially restored by rcsC homologs from Pantoea stewartii, Yersinia pestis, and Salmonella enterica but not from Escherichia coli on apple shoots. Domain-swapping experiments have indicated that replacement of the E. coli RcsC sensor domain by those of Erwinia and Yersinia spp. partially restores virulence of the Erwinia rcsC mutant, whereas chimeric constructs containing the sensor domain of E. coli RcsC could not rescue virulence of the Erwinia rcsC mutant on apple. Interestingly, only chimeric constructs containing the histidine kinase and receiver domains of Erwinia RcsC are fully capable of rescuing amylovoran production. These results suggest that the sensor domain of RcsC may be important in regulating bacterial virulence, whereas the activity of the histidine kinase and receiver domains of Erwinia RcsC may be essential for amylovoran production in vitro.

  16. The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

    PubMed Central

    Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

    2015-01-01

    Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

  17. Multiple signals modulate the activity of the complex sensor kinase TodS

    PubMed Central

    Silva-Jiménez, Hortencia; Ortega, Álvaro; García-Fontana, Cristina; Ramos, Juan Luis; Krell, Tino

    2015-01-01

    The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules. PMID:24986263

  18. Structural Analysis of Sensor Domains from the TMAO-Responsive Histidine Kinase Receptor TorS.

    SciTech Connect

    Moore, J.; Hendrickson, W

    2009-01-01

    Histidine kinase receptors respond to diverse signals and mediate signal transduction across the plasma membrane in all prokaryotes and certain eukaryotes. Each receptor is part of a two-component system that regulates a particular cellular process. Organisms that use trimethylamine-N-oxide (TMAO) as a terminal electron acceptor typically control their anaerobic respiration through the TMAO reductase (Tor) pathway, which the TorS histidine kinase activates when sensing TMAO in the environment. We have determined crystal structures for the periplasmic sensor domains of TorS receptors from Escherichia coli and Vibrio parahaemolyticus. TorS sensor domains have a novel fold consisting of a membrane-proximal right-handed four-helical bundle and a membrane-distal left-handed four-helical bundle, but conformational dispositions differ significantly in the two structures. Isolated TorS sensor domains dimerize in solution; and from comparisons with dimeric NarX and Tar sensors, we postulate that signaling through TorS dimers involves a piston-type displacement between helices.

  19. The Janus kinase 2 inhibitor fedratinib inhibits thiamine uptake: a putative mechanism for the onset of Wernicke's encephalopathy.

    PubMed

    Zhang, Qiang; Zhang, Yan; Diamond, Sharon; Boer, Jason; Harris, Jennifer J; Li, Yu; Rupar, Mark; Behshad, Elham; Gardiner, Christine; Collier, Paul; Liu, Phillip; Burn, Timothy; Wynn, Richard; Hollis, Gregory; Yeleswaram, Swamy

    2014-10-01

    The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

  20. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt

    PubMed Central

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-01-01

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression. PMID:26446555

  1. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum.

    PubMed

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2015-09-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA(-)) did not exhibit tip dominance. A striking phenotype of pkcA- was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA(-) to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA(-) slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA(-). PMID:26183108

  2. Role of the putative osmosensor Arabidopsis histidine kinase1 in dehydration avoidance and low-water-potential response.

    PubMed

    Kumar, M Nagaraj; Jane, Wann-Neng; Verslues, Paul E

    2013-02-01

    The molecular basis of plant osmosensing remains unknown. Arabidopsis (Arabidopsis thaliana) Histidine Kinase1 (AHK1) can complement the osmosensitivity of yeast (Saccharomyces cerevisiae) osmosensor mutants lacking Synthetic Lethal of N-end rule1 and SH3-containing Osmosensor and has been proposed to act as a plant osmosensor. We found that ahk1 mutants in either the Arabidopsis Nossen-0 or Columbia-0 background had increased stomatal density and stomatal index consistent with greater transpirational water loss. However, the growth of ahk1 mutants was not more sensitive to controlled moderate low water potential (ψ(w)) or to salt stress. Also, ahk1 mutants had increased, rather than reduced, solute accumulation across a range of low ψ(w) severities. ahk1 mutants had reduced low ψ(w) induction of Δ(1)-Pyrroline-5-Carboxylate Synthetase1 (P5CS1) and 9-cis-Epoxycarotenoid Dioxygenase3, which encode rate-limiting enzymes in proline and abscisic acid (ABA) synthesis, respectively. However, neither Pro nor ABA accumulation was reduced in ahk1 mutants at low ψ(w). P5CS1 protein level was not reduced in ahk1 mutants. This indicated that proline accumulation was regulated in part by posttranscriptional control of P5CS1 that was not affected by AHK1. Expression of AHK1 itself was reduced by low ψ(w), in contrast to previous reports. These results define a role of AHK1 in controlling stomatal density and the transcription of stress-responsive genes. These phenotypes may be mediated in part by reduced ABA sensitivity. More rapid transpiration and water depletion can also explain the previously reported sensitivity of ahk1 to uncontrolled soil drying. The unimpaired growth, ABA, proline, and solute accumulation of ahk1 mutants at low ψ(w) suggest that AHK1 may not be the main plant osmosensor required for low ψ(w) tolerance.

  3. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

    PubMed Central

    Marina, Alberto; Waldburger, Carey D; Hendrickson, Wayne A

    2005-01-01

    The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPβN at 1.9 Å resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts. PMID:16319927

  4. Recruitment and activation of the ATM kinase in the absence of DNA damage sensors

    PubMed Central

    Hartlerode, Andrea J.; Morgan, Mary J.; Wu, Yipin; Buis, Jeffrey; Ferguson, David O.

    2015-01-01

    Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11–Rad50–NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70–Ku80). Using Mus musculus cells harboring targeted mutant alleles of Mre11 and/or Ku70, together with pharmacologic kinase inhibition we demonstrate that ATM can in fact be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. Strikingly, in the absence of both MRN and Ku, ATM is recruited to chromatin, phosphorylates H2AX, and triggers the G2/M cell cycle checkpoint, but DNA repair functions of MRN are not restored. This implies that a complex interplay between sensors plays a significant role in ATM control, rather than straightforward recruitment and activation by MRN. PMID:26280532

  5. Role of the PAS Sensor Domains in the Bacillus subtilis Sporulation Kinase KinA

    PubMed Central

    Winnen, Brit; Anderson, Eric; Cole, James L.; King, Glenn F.

    2013-01-01

    Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environmental signals. KinA is the major histidine kinase required for initiation of sporulation upon nutrient deprivation in Bacillus subtilis. KinA has a large N-terminal region (residues 1 to 382) that is uniquely composed of three tandem Per-ARNT-Sim (PAS) domains that have been proposed to constitute a sensor module. To further enhance our understanding of this “sensor” region, we defined the boundaries that give rise to the minimal autonomously folded PAS domains and analyzed their homo- and heteroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PASA self-associates very weakly, while PASC is primarily a monomer. In contrast, PASB forms a stable dimer (Kd [dissociation constant] of <10 nM), and it appears to be the main N-terminal determinant of KinA dimerization. Analysis of KinA mutants deficient for one or more PAS domains revealed a critical role for PASB, but not PASA, in autophosphorylation of KinA. Our findings suggest that dimerization of PASB is important for keeping the catalytic domain of KinA in a functional conformation. We use this information to propose a model for the structure of the N-terminal sensor module of KinA. PMID:23504013

  6. Structure of the Entire Cytoplasmic Portion of a Sensor Histidine-kinase Protein

    SciTech Connect

    Marina,A.; Waldburger, C.; Hendrickson, W.

    2005-01-01

    The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADP{beta}N at 1.9 Angstrom resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.

  7. ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1.

    PubMed

    Lassak, Jürgen; Henche, Anna-Lena; Binnenkade, Lucas; Thormann, Kai M

    2010-05-01

    The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.

  8. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

    SciTech Connect

    Chen, Shang-Der; Lin, Tsu-Kung; Yang, Ding-I.; Lee, Su-Ying; Shaw, Fu-Zen; Liou, Chia-Wei; Chuang, Yao-Chung

    2015-05-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield.

  9. The ArcB Sensor Kinase of Escherichia coli: Genetic Exploration of the Transmembrane Region

    PubMed Central

    Kwon, Ohsuk; Georgellis, Dimitris; Lynch, A. Simon; Boyd, Dana; Lin, E. C. C.

    2000-01-01

    The Arc two-component signal transduction system of Escherichia coli regulates the expression of numerous operons in response to respiratory growth conditions. Cellular redox state or proton motive force (Δμ̄H+) has been proposed to be the signal for the membrane-associated ArcB sensor kinase. This study provided evidence for a short ArcB periplasmic bridge that contains a His47. The dispensability of this amino acid, the only amino acid with a pK in the physiological range, renders the Δμ̄H+ model unlikely. Furthermore, results from substituting membrane segments of ArcB with counterparts of MalF indicate that the region does not play a stereospecific role in signal reception. PMID:10781568

  10. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  11. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont

    PubMed Central

    Norsworthy, Allison N.; Visick, Karen L.

    2015-01-01

    Summary Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two-component signaling (TCS), composed of an environmental sensor (the sensor kinase, SK) and a cognate, intracellular effector (the response regulator, RR). The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild-type SypF functioned as a SK in vitro, this activity was dispensable for colonization. In fact, only a single non-enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS’s own HPt domain and SypF’s enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators. PMID:25586643

  12. Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.

    PubMed

    Yeo, Kwon Joo; Park, Jin-Wan; Kim, Eun-Hee; Jeon, Young Ho; Hwang, Kwang Yeon; Cheong, Hae-Kap

    2016-10-01

    In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE. PMID:27371359

  13. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light

  14. The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G.

    PubMed

    Ulke-Lemée, Annegret; Trinkle-Mulcahy, Laura; Chaulk, Steve; Bernstein, Nina K; Morrice, Nick; Glover, Mark; Lamond, Angus I; Moorhead, Greg B G

    2007-10-01

    The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

  15. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility]. Progress report, January 1993

    SciTech Connect

    Not Available

    1993-06-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK{sub 6} cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys{sup 524} codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with {sup 32}P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified {sup 32}p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  16. Analysis of periplasmic sensor domains from Anaeromyxobacter dehalogenans 2CP-C: Structure of one sensor domain from a histidine kinase and another from a chemotaxis protein

    PubMed Central

    Pokkuluri, P Raj; Dwulit-Smith, Jeff; Duke, Norma E; Wilton, Rosemarie; Mack, Jamey C; Bearden, Jessica; Rakowski, Ella; Babnigg, Gyorgy; Szurmant, Hendrik; Joachimiak, Andrzej; Schiffer, Marianne

    2013-01-01

    Anaeromyxobacter dehalogenans is a δ-proteobacterium found in diverse soils and sediments. It is of interest in bioremediation efforts due to its dechlorination and metal-reducing capabilities. To gain an understanding on A. dehalogenans' abilities to adapt to diverse environments we analyzed its signal transduction proteins. The A. dehalogenans genome codes for a large number of sensor histidine kinases (HK) and methyl-accepting chemotaxis proteins (MCP); among these 23 HK and 11 MCP proteins have a sensor domain in the periplasm. These proteins most likely contribute to adaptation to the organism's surroundings. We predicted their three-dimensional folds and determined the structures of two of the periplasmic sensor domains by X-ray diffraction. Most of the domains are predicted to have either PAS-like or helical bundle structures, with two predicted to have solute-binding protein fold, and another predicted to have a 6-phosphogluconolactonase like fold. Atomic structures of two sensor domains confirmed the respective fold predictions. The Adeh_2942 sensor (HK) was found to have a helical bundle structure, and the Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in a binding site typical for PAS-like domains. Future work is needed to determine whether Adeh_3718 is involved in acetate sensing by A. dehalogenans. PMID:23897711

  17. Reciprocal regulation as a source of ultrasensitivity in two-component systems with a bifunctional sensor kinase.

    PubMed

    Straube, Ronny

    2014-05-01

    Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations--a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme's phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness--consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase's phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two-component systems, but may

  18. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    PubMed Central

    Lesne, E.; Krammer, E.-M.; Dupre, E.; Locht, C.; Lensink, M. F.

    2016-01-01

    ABSTRACT The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. PMID:26933056

  19. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

    PubMed

    Kim, Hye-Sook; Willett, Jonathan W; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-11-01

    In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.

  20. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signaling pathway

    PubMed Central

    Kim, Hye-Sook; Willett, Jonathan W.; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-01-01

    Summary In the intracellular pathogen Brucella abortus, the general stress response (GSR) signaling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signaling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK efficiently and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signaling pathway that regulates B. abortus stress physiology and infection biology. PMID:25257300

  1. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    PubMed

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

  2. Sensor Kinase PA4398 Modulates Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa PA14

    PubMed Central

    Strehmel, Janine; Neidig, Anke; Nusser, Michael; Geffers, Robert; Brenner-Weiss, Gerald

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. A PA4398− mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398− mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398− mutant to the wild-type strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (P < 0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes, we were able to show that not only pvdQ but also pvdR, fptA, pchA, pchD, and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398− mutant in addition to elevated c-di-GMP levels. PMID:25501476

  3. Small protein B upregulates sensor kinase bvgS expression in Aeromonas veronii

    PubMed Central

    Liu, Zhu; Liu, Peng; Liu, Shuanshuan; Song, Haichao; Tang, Hongqian; Hu, Xinwen

    2015-01-01

    Earlier studies reveal that Small protein B (SmpB), a class of well-conserved tmRNA-binding proteins, is essential for the trans-translation process, which functions as a system for translation surveillance and ribosome rescue. Here, we report a previously unrecognized mechanism by which SmpB alone positively regulates the expression of a sensor kinase, BvgS, in Aeromonas veronii. A reporter plasmid was constructed in which the promoter of bvgS was used to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-transformed with a SmpB expression construct into E. coli, the relative fluorescence intensity increased about threefold. Transformation with a truncated form of smpB gene showed that the C-terminus had little effect, while N-terminus unexpectedly increased eGFP production. Next, a series of SmpB mutants were generated by site-directed mutagenesis. When the mutants SmpB (G11S) or SmpB (E32AG) was used in the experiment, eGFP expression dropped significantly compared with that of wild type SmpB. Further, purified SmpB was shown to bind the promoter regions of bvgS in the agarose gel retardation assay. Quantitative RT-PCR analysis showed that eGFP transcript levels increased approximately 25-fold in the presence of SmpB. Likewise, smpB knockout decreased bvgS transcripts significantly in A. veronii, and also displayed a reduced capability in salt tolerance. Collectively, the data presented here will facilitate a deeper understanding of SmpB-mediated regulatory circuits as a transcriptional factor in A. veronii. PMID:26136727

  4. Sensor kinase PA4398 modulates swarming motility and biofilm formation in Pseudomonas aeruginosa PA14.

    PubMed

    Strehmel, Janine; Neidig, Anke; Nusser, Michael; Geffers, Robert; Brenner-Weiss, Gerald; Overhage, Joerg

    2015-02-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. APA4398 mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398 mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398 mutant to the wildtype strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (P<0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes,we were able to show that not only pvdQ but also pvdR, fptA, pchA, pchD, and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398 mutant in addition to elevated c-di-GMP levels. PMID:25501476

  5. Divergent distribution of the sensor kinase CosS in non-thermotolerant campylobacter species and its functional incompatibility with the response regulator CosR of Campylobacter jejuni.

    PubMed

    Hwang, Sunyoung; Miller, William G; Ryu, Sangryeol; Jeon, Byeonghwa

    2014-01-01

    Two-component signal transduction systems are commonly composed of a sensor histidine kinase and a cognate response regulator, modulating gene expression in response to environmental changes through a phosphorylation-dependent process. CosR is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a major foodborne pathogenic species causing human gastroenteritis. Although CosR is a response regulator, its cognate sensor kinase has not been identified in C. jejuni. In this study, DNA sequence analysis of the cosR flanking regions revealed that a gene encoding a putative sensor kinase, which we named cosS, is prevalent in non-thermotolerant Campylobacter spp., but not in thermotolerant campylobacters. Phosphorylation assays indicated that C. fetus CosS rapidly autophosphorylates and then phosphorylates C. fetus CosR, suggesting that the CosRS system constitutes a paired two-component signal transduction system in C. fetus. However, C. fetus CosS does not phosphorylate C. jejuni CosR, suggesting that CosR may have different regulatory cascades between thermotolerant and non-thermotolerant Campylobacter species. Comparison of CosR homolog amino acid sequences showed that the conserved phosphorylation residue (D51), which is present in all non-thermotolerant Campylobacter spp., is absent from the CosR homologs of thermotolerant Campylobacter species. However, C. jejuni CosR was not phosphorylated by C. fetus CosS even after site-directed mutagenesis of N51D, implying that C. jejuni CosR may possibly function phosphorylation-independently. In addition, the results of cosS mutational analysis indicated that CosS is not associated with the temperature dependence of the Campylobacter spp. despite its unique divergent distribution only in non-thermotolerant campylobacters. The findings in this study strongly suggest that thermotolerant and non-thermotolerant Campylobacter spp. have different signal sensing mechanisms associated with the Cos

  6. Cooperation of Secondary Transporters and Sensor Kinases in Transmembrane Signalling: The DctA/DcuS and DcuB/DcuS Sensor Complexes of Escherichia coli.

    PubMed

    Unden, G; Wörner, S; Monzel, C

    2016-01-01

    Many membrane-bound sensor kinases require accessory proteins for function. The review describes functional control of membrane-bound sensors by transporters. The C4-dicarboxylate sensor kinase DcuS requires the aerobic or anaerobic C4-dicarboxylate transporters DctA or DcuB, respectively, for function and forms DctA/DcuS or DcuB/DcuS sensor complexes. Free DcuS is in the permanent (ligand independent) ON state. The DctA/DcuS and DcuB/DcuS complexes, on the other hand, control expression in response to C4-dicarboxylates. In DctA/DcuS, helix 8b of DctA and the PASC domain of DcuS are involved in interaction. The stimulus is perceived by the extracytoplasmic sensor domain (PASP) of DcuS. The signal is transmitted across the membrane by a piston-type movement of TM2 of DcuS which appears to be pulled (by analogy to the homologous citrate sensor CitA) by compaction of PASP after C4-dicarboxylate binding. In the cytoplasm, the signal is perceived by the PASC domain of DcuS. PASC inhibits together with DctA the kinase domain of DcuS which is released after C4-dicarboxylate binding. DcuS exhibits two modes for regulating expression of target genes. At higher C4-dicarboxylate levels, DcuS is part of the DctA/DcuS complex and in the C4-dicarboxylate-responsive form which stimulates expression of target genes in response to the concentration of the C4-dicarboxylates (catabolic use of C4-dicarboxylates, mode I regulation). At limiting C4-dicarboxylate concentrations (≤0.05mM), expression of DctA drops and free DcuS appears. Free DcuS is in the permanent ON state (mode II regulation) and stimulates low level (C4-dicarboxylate independent) DctA synthesis for DctA/DcuS complex formation and anabolic C4-dicarboxylate uptake.

  7. Cooperation of Secondary Transporters and Sensor Kinases in Transmembrane Signalling: The DctA/DcuS and DcuB/DcuS Sensor Complexes of Escherichia coli.

    PubMed

    Unden, G; Wörner, S; Monzel, C

    2016-01-01

    Many membrane-bound sensor kinases require accessory proteins for function. The review describes functional control of membrane-bound sensors by transporters. The C4-dicarboxylate sensor kinase DcuS requires the aerobic or anaerobic C4-dicarboxylate transporters DctA or DcuB, respectively, for function and forms DctA/DcuS or DcuB/DcuS sensor complexes. Free DcuS is in the permanent (ligand independent) ON state. The DctA/DcuS and DcuB/DcuS complexes, on the other hand, control expression in response to C4-dicarboxylates. In DctA/DcuS, helix 8b of DctA and the PASC domain of DcuS are involved in interaction. The stimulus is perceived by the extracytoplasmic sensor domain (PASP) of DcuS. The signal is transmitted across the membrane by a piston-type movement of TM2 of DcuS which appears to be pulled (by analogy to the homologous citrate sensor CitA) by compaction of PASP after C4-dicarboxylate binding. In the cytoplasm, the signal is perceived by the PASC domain of DcuS. PASC inhibits together with DctA the kinase domain of DcuS which is released after C4-dicarboxylate binding. DcuS exhibits two modes for regulating expression of target genes. At higher C4-dicarboxylate levels, DcuS is part of the DctA/DcuS complex and in the C4-dicarboxylate-responsive form which stimulates expression of target genes in response to the concentration of the C4-dicarboxylates (catabolic use of C4-dicarboxylates, mode I regulation). At limiting C4-dicarboxylate concentrations (≤0.05mM), expression of DctA drops and free DcuS appears. Free DcuS is in the permanent ON state (mode II regulation) and stimulates low level (C4-dicarboxylate independent) DctA synthesis for DctA/DcuS complex formation and anabolic C4-dicarboxylate uptake. PMID:27134023

  8. Suppression of growth factor-mediated MAP kinase activation by v-raf in macrophages: a putative role for the MKP-1 phosphatase.

    PubMed

    Krautwald, S; Büscher, D; Dent, P; Ruthenberg, K; Baccarini, M

    1995-03-16

    Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.2F5 macrophages the expression of virally activated RAF does not correlate with constitutive MAPK activation; on the contrary, growth factor-mediated stimulation of MAPK activity is suppressed in these cells. Suppression correlates with v-RAF expression, as MAPK activation is normal in a revertant cell line that stopped expressing v-RAF. Inhibition of MAPK activation is associated with lack of ERK-2 tyrosine phosphorylation, and is not due to the suppression of CSF-1-mediated MEK activation. Pretreatment with vanadate restores growth factor-stimulated activation and tyrosine phosphorylation of MAPK in v-RAF-expressing macrophages, indicating the involvement of a tyrosine phosphatase. Interestingly, v-RAF-expressing macrophages contain low constitutive levels of MKP-1 mRNA, an immediate early gene that encodes a MAPK-specific phosphatase and is induced in the parental cell line by CSF-1 treatment. The restoration of MAPK activation by vanadate pretreatment and the presence of MKP-1 mRNA in v-RAF-expressing macrophages raise the intriguing possibility that in macrophages RAF may be feeding back on the MAPK pathway by participating in the control of MKP-1 expression. PMID:7700643

  9. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-01

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  10. The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

    PubMed

    Sumita, Kazutaka; Lo, Yu-Hua; Takeuchi, Koh; Senda, Miki; Kofuji, Satoshi; Ikeda, Yoshiki; Terakawa, Jumpei; Sasaki, Mika; Yoshino, Hirofumi; Majd, Nazanin; Zheng, Yuxiang; Kahoud, Emily Rose; Yokota, Takehiro; Emerling, Brooke M; Asara, John M; Ishida, Tetsuo; Locasale, Jason W; Daikoku, Takiko; Anastasiou, Dimitrios; Senda, Toshiya; Sasaki, Atsuo T

    2016-01-21

    While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target. PMID:26774281

  11. Combining chemical and genetic approaches for development of responsive FRET-based sensor systems for protein kinases.

    PubMed

    Manoharan, Ganesh Babu; Enkvist, Erki; Uri, Asko

    2016-04-01

    Chemical and genetic approaches were combined for the development of responsive FRET-based sensor systems for protein kinases, using PIM2 as the model kinase. Fusions of PIM2 and a red fluorescent protein, TagRFP were expressed in mammalian cells and small-molecule ARC-Lum photoluminescent probes possessing different phosphorescent and fluorescent properties were constructed. Based on a variety of Förster-type resonant energy transfer (FRET) mechanisms (including intermolecular or intramolecular energy transfer and transfer between singlet-singlet or triplet-singlet electronic states of interacting luminophores) of the probe and that of the fluorescently tagged PIM2, FRET-based sensor systems were constructed. The developed assays can be applied for analysis of PIM2 in biological samples and screening and characterization of PIM2 inhibitors in cell lysates. In screening studies sub-micromolar affinity of a d-arginine-rich peptide, nona(d-arginine) amide [(d-Arg)9-NH2], towards PIM2 was discovered that points to possible specific effect of this widely used transport peptide to cellular protein phosphorylation balance. PMID:26874332

  12. The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

    PubMed Central

    Bae, Taeok

    2015-01-01

    Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases. PMID:25849574

  13. A role for the sensor kinase PlrS in controlling the response of Bordetella bronchiseptica to increased CO2 levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of Bordetella bronchiseptica to colonize the rodent respiratory tract requires the sensor kinase PlrS, which is presumably part of a two-component regulatory system. Microarray analysis revealed that PlrS influences the expression of several genes required for virulence, including those ...

  14. Divergent distribution of the sensor kinase CosS in non-thermophilic Campylobacter species and its functional incompatibility with the Campylobacter jejuni CosR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two-component signal transduction system is commonly composed of a sensor histidine kinase and a response regulator, modulating gene expression in response to environmental changes through a phosphorylation-dependent process. CosR is an OmpR-type response regulator essential for the viability of Cam...

  15. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

    PubMed Central

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

    2016-01-01

    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  16. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

    PubMed Central

    Hussain, Rohanah; Harding, Stephen E.; Hughes, Charlotte S.; Ma, Pikyee; Patching, Simon G.; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J.F.; Phillips-Jones, Mary K.

    2016-01-01

    This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins–synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs. PMID:27284046

  17. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions.

    PubMed

    Hussain, Rohanah; Harding, Stephen E; Hughes, Charlotte S; Ma, Pikyee; Patching, Simon G; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J F; Phillips-Jones, Mary K

    2016-06-15

    This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins-synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs. PMID:27284046

  18. Requirement of the Pseudomonas aeruginosa CbrA Sensor Kinase for Full Virulence in a Murine Acute Lung Infection Model

    PubMed Central

    Yeung, Amy T. Y.; Janot, Laure; Pena, Olga M.; Neidig, Anke; Kukavica-Ibrulj, Irena; Hilchie, Ashley; Levesque, Roger C.; Overhage, Joerg

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of respiratory tract and other nosocomial infections. The sensor kinase CbrA is a central regulator of carbon and nitrogen metabolism and in vitro also regulates virulence-related processes in P. aeruginosa. Here, we investigated the role of CbrA in two murine models of infection. In both peritoneal infections in leukopenic mice and lung infection models, the cbrA mutant was less virulent since substantially larger numbers of cbrA mutant bacteria were required to cause the same level of infection as wild-type or complemented bacteria. In contrast, in the chronic rat lung model the cbrA mutant grew and persisted as well as the wild type, indicating that the decrease of in vivo virulence of the cbrA mutant did not result from growth deficiencies on particular carbon substrates observed in vitro. In addition, a mutant in the cognate response regulator CbrB showed no defect in virulence in the peritoneal infection model, ruling out the involvement of certain alterations of virulence properties in the cbrA mutant including defective swarming motility, increased biofilm formation, and cytotoxicity, since these alterations are controlled through CbrB. Further investigations indicated that the mutant was more susceptible to uptake by phagocytes in vitro, resulting in greater overall bacterial killing. Consistent with the virulence defect, it took a smaller number of Dictyostelium discoideum amoebae to kill the cbrA mutant than to kill the wild type. Transcriptional analysis of the cbrA mutant during D. discoideum infection led to the conclusion that CbrA played an important role in the iron metabolism, protection of P. aeruginosa against oxidative stress, and the regulation of certain virulence factors. PMID:24379284

  19. Requirement of the Pseudomonas aeruginosa CbrA sensor kinase for full virulence in a murine acute lung infection model.

    PubMed

    Yeung, Amy T Y; Janot, Laure; Pena, Olga M; Neidig, Anke; Kukavica-Ibrulj, Irena; Hilchie, Ashley; Levesque, Roger C; Overhage, Joerg; Hancock, Robert E W

    2014-03-01

    Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of respiratory tract and other nosocomial infections. The sensor kinase CbrA is a central regulator of carbon and nitrogen metabolism and in vitro also regulates virulence-related processes in P. aeruginosa. Here, we investigated the role of CbrA in two murine models of infection. In both peritoneal infections in leukopenic mice and lung infection models, the cbrA mutant was less virulent since substantially larger numbers of cbrA mutant bacteria were required to cause the same level of infection as wild-type or complemented bacteria. In contrast, in the chronic rat lung model the cbrA mutant grew and persisted as well as the wild type, indicating that the decrease of in vivo virulence of the cbrA mutant did not result from growth deficiencies on particular carbon substrates observed in vitro. In addition, a mutant in the cognate response regulator CbrB showed no defect in virulence in the peritoneal infection model, ruling out the involvement of certain alterations of virulence properties in the cbrA mutant including defective swarming motility, increased biofilm formation, and cytotoxicity, since these alterations are controlled through CbrB. Further investigations indicated that the mutant was more susceptible to uptake by phagocytes in vitro, resulting in greater overall bacterial killing. Consistent with the virulence defect, it took a smaller number of Dictyostelium discoideum amoebae to kill the cbrA mutant than to kill the wild type. Transcriptional analysis of the cbrA mutant during D. discoideum infection led to the conclusion that CbrA played an important role in the iron metabolism, protection of P. aeruginosa against oxidative stress, and the regulation of certain virulence factors.

  20. Analysis of the Pseudomonas aeruginosa Regulon Controlled by the Sensor Kinase KinB and Sigma Factor RpoN

    PubMed Central

    Damron, F. Heath; Owings, Joshua P.; Okkotsu, Yuta; Varga, John J.; Schurr, Jill R.; Goldberg, Joanna B.; Schurr, Michael J.

    2012-01-01

    Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ22; AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy. PMID:22210761

  1. Reduction of Turgor Is Not the Stimulus for the Sensor Kinase KdpD of Escherichia coli▿

    PubMed Central

    Hamann, Knut; Zimmann, Petra; Altendorf, Karlheinz

    2008-01-01

    Stimulus perception by the KdpD/KdpE two-component system of Escherichia coli is still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting in kdpFABC expression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of the kdpFABC operon revealed that the system responds very effectively to K+-limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K+, Na+, glutamate, proline, glycine, trehalose, putrescine, and spermidine under K+-limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K+-limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K+ concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K+ concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD. PMID:18245296

  2. Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1

    PubMed Central

    Scharein, Benjamin; Groth, Georg

    2011-01-01

    The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1−AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta. PMID:21912672

  3. Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS.

    PubMed

    Kilmury, Sara L N; Burrows, Lori L

    2016-05-24

    Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa Transcription of the major pilin gene-pilA-is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS's phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA-PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression. PMID:27162347

  4. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.

    PubMed

    Reimann, T; Büscher, D; Hipskind, R A; Krautwald, S; Lohmann-Matthes, M L; Baccarini, M

    1994-12-15

    Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production. PMID:7989771

  5. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.

    PubMed

    Reimann, T; Büscher, D; Hipskind, R A; Krautwald, S; Lohmann-Matthes, M L; Baccarini, M

    1994-12-15

    Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production.

  6. Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

    PubMed

    Dupré, Elian; Herrou, Julien; Lensink, Marc F; Wintjens, René; Vagin, Alexey; Lebedev, Andrey; Crosson, Sean; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-03-01

    Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions. PMID:25738876

  7. Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

    PubMed

    Dupré, Elian; Herrou, Julien; Lensink, Marc F; Wintjens, René; Vagin, Alexey; Lebedev, Andrey; Crosson, Sean; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-03-01

    Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

  8. The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface.

    PubMed

    Affandi, Trisiani; Issaian, Aaron V; McEvoy, Megan M

    2016-09-20

    In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Two-component systems generally consist of a sensor histidine kinase and a response regulator, which work together through histidyl-aspartyl phosphorelay to result in gene regulation. One of the two-component systems in Escherichia coli, CusS-CusR, is known to induce expression of cusCFBA genes at increased periplasmic Cu(I) and Ag(I) concentrations to help maintain metal ion homeostasis. CusS is a membrane-associated histidine kinase with a periplasmic sensor domain connected to the cytoplasmic ATP binding and catalytic domains through two transmembrane helices. The mechanism of how CusS senses increasing metal ion concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. The structure reveals that CusS forms a homodimer with four Ag(I) binding sites per dimeric complex. Two symmetric metal binding sites are found at the dimeric interface, which are each formed by two histidines and one phenylalanine with an unusual cation-π interaction. The other metal ion binding sites are in a nonconserved region within each monomer. Functional analyses of CusS variants with mutations in the metal sites suggest that the metal ion binding site at the dimer interface is more important for function. The structural and functional data provide support for a model in which metal-induced dimerization results in increases in kinase activity in the cytoplasmic domains of CusS.

  9. The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface

    PubMed Central

    Affandi, Trisiani; Issaian, Aaron V.; McEvoy, Megan M.

    2016-01-01

    In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Two-component systems generally consist of a sensor histidine kinase and a response regulator, which work together through histidyl-aspartyl phospho-relay to result in gene regulation. One of the two-component systems in Escherichia coli, CusS-CusR, is known to induce expression of cusCFBA genes under increased periplasmic Cu(I) and Ag(I) concentrations to help maintain metal ion homeostasis. CusS is a membrane-associated histidine kinase with a periplasmic sensor domain connected to the cytoplasmic ATP-binding and catalytic domains through two transmembrane helices. The mechanism of how CusS senses increasing metal ion concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. The structure reveals that CusS forms a homodimer with four Ag(I) binding sites per dimeric complex. Two symmetric metal binding sites are found at the dimeric interface, which are each formed by two histidines and one phenylalanine with an unusual cation-π interaction. The other metal ion binding sites are in a non-conserved region within each monomer. Functional analyses of CusS variants with mutations in the metal sites suggest that the metal ion binding site at the dimer interface is more important for function. The structural and functional data provide support for a model in which metal-induced dimerization results in increases in kinase activity in the cytoplasmic domains of CusS. PMID:27583660

  10. Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS – “missing” resonances at the dimer interface

    PubMed Central

    Buchko, Garry W.; Edwards, Thomas E.; Hewitt, Stephen N.; Phan, Isabelle Q.H.; Van Voorhis, Wesley C.; Miller, Samuel I.; Mylera, Peter J.

    2015-01-01

    Using a deuterated sample, all the observable backbone 1HN, 15N, 13Cα, and 13C′ chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are “missing” in the 1H-15N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (μs – ms) and/or is heterogeneous. PMID:25957069

  11. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

    PubMed

    Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

    2014-10-01

    Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure.

  12. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

    PubMed

    Kessler, Aline; Schell, Ursula; Sahr, Tobias; Tiaden, André; Harrison, Christopher; Buchrieser, Carmen; Hilbi, Hubert

    2013-02-01

    Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a

  13. Visualizing autophosphorylation in histidine kinases.

    PubMed

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

  14. AGCVIII Kinases: at the crossroads of cellular signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

  15. A PAS domain with an oxygen labile [4Fe-4S](2+) cluster in the oxygen sensor kinase NreB of Staphylococcus carnosus.

    PubMed

    Müllner, Martin; Hammel, Oliver; Mienert, Bernd; Schlag, Steffen; Bill, Eckhard; Unden, Gottfried

    2008-12-30

    The cytoplasmic histidine sensor kinase NreB of Staphylococcus carnosus responds to O(2) and controls together with the response regulator NreC the expression of genes of nitrate/nitrite respiration. nreBC homologous genes were found in Staphylococcus strains and Bacillus clausii, and a modified form was found in some Lactobacillus strains. NreB contains a sensory domain with similarity to heme B binding PAS domains. Anaerobically prepared NreB of S. carnosus exhibited a (diamagnetic) [4Fe-4S](2+) cluster when assessed by Mossbauer spectroscopy. Upon reaction with air, the cluster was degraded with a half-life of approximately 2.5 min. No significant amounts of Mossbauer or EPR detectable intermediates were found during the decay, but magnetic Mossbauer spectra revealed formation of diamagnetic [2Fe-2S](2+) clusters. After extended exposure to air, NreB was devoid of a FeS cluster. Photoreduction with deazaflavin produced small amounts of [4Fe-4S](+), which were degraded subsequently. The magnetically perturbed Mossbauer spectrum of the [4Fe-4S](2+) cluster corroborated the S = 0 spin state and revealed uniform electric field gradient tensors of the iron sites, suggesting full delocalization of the valence electrons and binding of each of the Fe ions by four S ligands, including the ligand to the protein. Mutation of each of the four Cys residues inactivated NreB function in vivo in accordance with their role as ligands. [4Fe-4S](2+) cluster-containing NreB had high kinase activity. Exposure to air decreased the kinase activity and content of the [4Fe-4S](2+) cluster with similar half-lives. We conclude that the sensory domain of NreB represents a new type of PAS domain containing a [4Fe-4S](2+) cluster for sensing and function. PMID:19102705

  16. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2

    PubMed Central

    Monzel, Christian; Unden, Gottfried

    2015-01-01

    The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase. PMID:26283365

  17. The KdpD Sensor Kinase of Escherichia coli Responds to Several Distinct Signals To Turn on Expression of the Kdp Transport System

    PubMed Central

    2015-01-01

    ABSTRACT Kdp, one of three saturable K+ uptake systems in Escherichia coli, is the system with the highest affinity for K+ and the only one whose expression is strongly controlled by medium K+ concentration. Expression is controlled by a two-component system of KdpD, the sensor kinase, and KdpE, the response regulator. There is general agreement that expression occurs when the growth rate of cells begins to become limited by K+ availability. How K+ limitation results in expression has been controversial. Studying the roles of the major components of the growth medium shows that KdpD senses at least two distinct signals inside the cell, those of Na+ and NH4+, and it probably senses other monovalent cations in the cell. KdpD does not sense turgor. IMPORTANCE The expression of the Kdp K+ transport system of E. coli occurs when cells become limited in their growth rate by the availability of K+. Cells sense limited K+ and try to compensate by taking up other monovalent cations, particularly Na+ and NH4+. These cations are sensed in the cytoplasm by the KdpD response regulator, presumably to stimulate its kinase activity. It is shown that KdpD does not sense turgor, as was suggested earlier. PMID:26350129

  18. Structural characterization and modeling of the Borrelia burgdorferi hybrid histidine kinase Hk1 periplasmic sensor: A system for sensing small molecules associated with tick feeding.

    PubMed

    Bauer, William J; Luthra, Amit; Zhu, Guangyu; Radolf, Justin D; Malkowski, Michael G; Caimano, Melissa J

    2015-10-01

    Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats.

  19. Structural characterization and modeling of the Borrelia burgdorferi hybrid histidine kinase Hk1 periplasmic sensor: a system for sensing small molecules associated with tick feeding

    PubMed Central

    Zhu, Guangyu; Radolf, Justin D.; Malkowski, Michael G.; Caimano, Melissa J.

    2015-01-01

    Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in B. burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanlyate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats. PMID:26321039

  20. HIV and Cocaine Impact Glial Metabolism: Energy Sensor AMP-activated protein kinase Role in Mitochondrial Biogenesis and Epigenetic Remodeling

    PubMed Central

    Samikkannu, Thangavel; Atluri, Venkata S. R.; Nair, Madhavan P. N.

    2016-01-01

    HIV infection and cocaine use have been identified as risk factors for triggering neuronal dysfunction. In the central nervous system (CNS), energy resource and metabolic function are regulated by astroglia. Glia is the major reservoir of HIV infection and disease progression in CNS. However, the role of cocaine in accelerating HIV associated energy deficit and its impact on neuronal dysfunction has not been elucidated yet. The aim of this study is to elucidate the molecular mechanism of HIV associated neuropathogenesis in cocaine abuse and how it accelerates the energy sensor AMPKs and its subsequent effect on mitochondrial oxidative phosphorylation (OXPHOS), BRSKs, CDC25B/C, MAP/Tau, Wee1 and epigenetics remodeling complex SWI/SNF. Results showed that cocaine exposure during HIV infection significantly increased the level of p24, reactive oxygen species (ROS), ATP-utilization and upregulated energy sensor AMPKs, CDC25B/C, MAP/Tau and Wee1 protein expression. Increased ROS production subsequently inhibits OCR/ECAR ratio and OXPHOS, and eventually upregulate epigenetics remodeling complex SWI/SNF in CHME-5 cells. These results suggest that HIV infection induced energy deficit and metabolic dysfunction is accelerated by cocaine inducing energy sensor AMPKs, mitochondrial biogenesis and chromatin remodeling complex SWI/SNF activation, which may lead to neuroAIDS disease progression. PMID:27535703

  1. HIV and Cocaine Impact Glial Metabolism: Energy Sensor AMP-activated protein kinase Role in Mitochondrial Biogenesis and Epigenetic Remodeling.

    PubMed

    Samikkannu, Thangavel; Atluri, Venkata S R; Nair, Madhavan P N

    2016-01-01

    HIV infection and cocaine use have been identified as risk factors for triggering neuronal dysfunction. In the central nervous system (CNS), energy resource and metabolic function are regulated by astroglia. Glia is the major reservoir of HIV infection and disease progression in CNS. However, the role of cocaine in accelerating HIV associated energy deficit and its impact on neuronal dysfunction has not been elucidated yet. The aim of this study is to elucidate the molecular mechanism of HIV associated neuropathogenesis in cocaine abuse and how it accelerates the energy sensor AMPKs and its subsequent effect on mitochondrial oxidative phosphorylation (OXPHOS), BRSKs, CDC25B/C, MAP/Tau, Wee1 and epigenetics remodeling complex SWI/SNF. Results showed that cocaine exposure during HIV infection significantly increased the level of p24, reactive oxygen species (ROS), ATP-utilization and upregulated energy sensor AMPKs, CDC25B/C, MAP/Tau and Wee1 protein expression. Increased ROS production subsequently inhibits OCR/ECAR ratio and OXPHOS, and eventually upregulate epigenetics remodeling complex SWI/SNF in CHME-5 cells. These results suggest that HIV infection induced energy deficit and metabolic dysfunction is accelerated by cocaine inducing energy sensor AMPKs, mitochondrial biogenesis and chromatin remodeling complex SWI/SNF activation, which may lead to neuroAIDS disease progression. PMID:27535703

  2. Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD

    PubMed Central

    Wu, R; Gu, M; Wilton, R; Babnigg, G; Kim, Y; Pokkuluri, P R; Szurmant, H; Joachimiak, A; Schiffer, M

    2013-01-01

    The Bacillus subtilis KinD signal-transducing histidine kinase is a part of the sporulation phosphorelay known to regulate important developmental decisions such as sporulation and biofilm formation. We have determined crystal structures of the extracytoplasmic sensing domain of KinD, which was copurified and crystallized with a pyruvate ligand. The structure of a ligand-binding site mutant was also determined; it was copurified and crystallized with an acetate ligand. The structure of the KinD extracytoplasmic segment is similar to that of several other sensing domains of signal transduction proteins and is composed of tandem Per-Arnt-Sim (PAS)-like domains. The KinD ligand-binding site is located on the membrane distal PAS-like domain and appears to be highly selective; a single mutation, R131A, abolishes pyruvate binding and the mutant binds acetate instead. Differential scanning fluorimetry, using a variety of monocarboxylic and dicarboxylic acids, identified pyruvate, propionate, and butyrate but not lactate, acetate, or malate as KinD ligands. A recent report found that malate induces biofilm formation in a KinD-dependent manner. It was suggested that malate might induce a metabolic shift and increased secretion of the KinD ligand of unknown identity. The structure and binding assays now suggests that this ligand is pyruvate and/or other small monocarboxylic acids. In summary, this study gives a first insight into the identity of a molecular ligand for one of the five phosphorelay kinases of B. subtilis. PMID:23436677

  3. An Analysis of the Solution Structure and Signaling Mechanism of LovK, a Sensor Histidine Kinase Integrating Light and Redox Signals

    SciTech Connect

    Purcell, Erin B.; McDonald, Claudia A.; Palfey, Bruce A.; Crosson, Sean

    2010-12-07

    Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria. These {approx}100-residue photosensory modules are generally encoded within larger, multidomain proteins that control a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes a soluble LOV-histidine kinase, LovK, that regulates the adhesive properties of the cell. Full-length LovK is dimeric as are a series of systematically truncated LovK constructs containing only the N-terminal LOV sensory domain. Nonconserved sequence flanking the LOV domain functions to tune the signaling lifetime of the protein. Size exclusion chromatography and small-angle X-ray scattering (SAXS) demonstrate that the LOV sensor domain does not undergo a large conformational change in response to photon absorption. However, limited proteolysis identifies a sequence flanking the C-terminus of the LOV domain as a site of light-induced change in protein conformation and dynamics. On the basis of SAXS envelope reconstruction and bioinformatic prediction, we propose this dynamic region of structure is an extended C-terminal coiled coil that links the LOV domain to the histidine kinase domain. To test the hypothesis that LOV domain signaling is affected by cellular redox state in addition to light, we measured the reduction potential of the LovK FMN cofactor. The measured potential of -258 mV is congruent with the redox potential of Gram-negative cytoplasm during logarithmic growth (-260 to -280 mV). Thus, a fraction of LovK in the cytosol may be in the reduced state under typical growth conditions. Chemical reduction of the FMN cofactor of LovK attenuates the light-dependent ATPase activity of the protein in vitro, demonstrating that LovK can function as a conditional photosensor that is regulated by the oxidative state of the cellular environment.

  4. A calcium sensor - protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species.

    PubMed

    Beckmann, Linda; Edel, Kai H; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca(2+)) signaling is a universal mechanism of signal transduction and involves Ca(2+) signal formation and decoding of information by Ca(2+) binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca(2+) binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca(2+) signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca(2+) signaling specificity. PMID:27538881

  5. A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance.

    PubMed

    Dörr, Tobias; Alvarez, Laura; Delgado, Fernanda; Davis, Brigid M; Cava, Felipe; Waldor, Matthew K

    2016-01-12

    The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall-acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall-acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

  6. A calcium sensor – protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species

    PubMed Central

    Beckmann, Linda; Edel, Kai H.; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca2+) signaling is a universal mechanism of signal transduction and involves Ca2+ signal formation and decoding of information by Ca2+ binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca2+ binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca2+ signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca2+ signaling specificity. PMID:27538881

  7. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

    PubMed

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

    2015-07-24

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.

  8. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

    PubMed

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

    2015-07-24

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

  9. The photoreaction of the photoactive yellow protein domain in the light sensor histidine kinase Ppr is influenced by the C-terminal domains.

    PubMed

    Kamikubo, Hironari; Koyama, Tomonori; Hayashi, Michihiro; Shirai, Kumiko; Yamazaki, Yoichi; Imamoto, Yasushi; Kataoka, Mikio

    2008-01-01

    To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p-coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p-coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYP(L)/dark and PYP(M)/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYP(L)- and PYP(M)-like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYP(L) compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains.

  10. Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply

    SciTech Connect

    Spoden, Gilles A.; Rostek, Ursula; Lechner, Stefan; Mitterberger, Maria; Mazurek, Sybille; Zwerschke, Werner

    2009-10-01

    The glycolytic key regulator pyruvate kinase M2 (M2-PK or PKM2) can switch between a highly active tetrameric and an inactive dimeric form. The transition between the two conformations regulates the glycolytic flux in tumor cells. We developed specific M2-PK-binding peptide aptamers which inhibit M2-PK, but not the 96% homologous M1-PK isoenzyme. In this study we demonstrate that, at normal blood glucose concentrations, peptide aptamer-mediated inhibition of M2-PK induces a significant decrease of the population doubling (PDL rate) and cell proliferation rate as well as an increase in cell size, whereas under glucose restriction an increase in PDL and cell proliferation rates but a decrease in cell size was observed. Moreover, M2-PK inhibition rescues cells from glucose starvation-induced apoptotic cell death by increasing the metabolic activity. These findings suggest that M2-PK is a metabolic sensor which regulates cell proliferation, cell growth and apoptotic cell death in a glucose supply-dependent manner.

  11. A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

    PubMed Central

    Dörr, Tobias; Alvarez, Laura; Delgado, Fernanda; Davis, Brigid M.; Cava, Felipe; Waldor, Matthew K.

    2016-01-01

    The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enables Vibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-type V. cholerae, mutants lacking wigR fail to recover following exposure to cell-wall–acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression of wigR leads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall–acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well. PMID:26712007

  12. δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Olianas, Alessandra; Onali, Pierluigi

    2012-02-01

    AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake. PMID:22031472

  13. Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro.

    PubMed

    Miyahara, Akira; Hirani, Tripty A; Oakes, Marie; Kereszt, Attila; Kobe, Bostjan; Djordjevic, Michael A; Gresshoff, Peter M

    2008-09-12

    The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant NARK proteins showed autophosphorylation activity in vitro. Several regions of the NARK kinase domain were shown by mass spectrometry to possess phosphoresidues. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole plant autoregulation of nodulation. A wild-type NARK fusion protein transphosphorylated a kinase-inactive mutant NARK fusion protein, suggesting that it is capable of intermolecular autophosphorylation in vitro. In addition, Ser-861 and Thr-963 in the NARK kinase catalytic domain were identified as phosphorylation sites through site-directed mutagenesis. The genes coding for the kinase-associated protein phosphatases KAPP1 and KAPP2, two putative interacting components of NARK, were isolated. NARK kinase domain phosphorylated recombinant KAPP proteins in vitro. Autophosphorylated NARK kinase domain was, in turn, dephosphorylated by both KAPP1 and KAPP2. Our results suggest a model for signal transduction involving NARK in the control of nodule development.

  14. Mechanism of the pH-Induced Conformational Change in the Sensor Domain of the DraK Histidine Kinase via the E83, E105, and E107 Residues

    PubMed Central

    Jee, Jun-Goo; Lee, Jae Kyoung; Kim, Hyo Jeong; Park, Jin-Wan; Kim, Eun-Hee; Hwang, Eunha; Kim, Sang-Yoon; Lee, Eun-Gyeong; Kwon, Ohsuk; Cheong, Hae-Kap

    2014-01-01

    The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces. PMID:25203403

  15. The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

    PubMed

    Jakobs, Mareike; Hoffmann, Kerstin; Liesegang, Heiko; Volland, Sonja; Meinhardt, Friedhelm

    2015-03-01

    In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

  16. A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

    PubMed

    Hirano, Naoko; Marukawa, Yuka; Abe, Jun; Hashiba, Sayuri; Ichikawa, Machiko; Tanabe, Yoichi; Ito, Motomi; Nishii, Ichiro; Tsuchikane, Yuki; Sekimoto, Hiroyuki

    2015-07-01

    Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release. PMID:25941232

  17. A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

    PubMed

    Hirano, Naoko; Marukawa, Yuka; Abe, Jun; Hashiba, Sayuri; Ichikawa, Machiko; Tanabe, Yoichi; Ito, Motomi; Nishii, Ichiro; Tsuchikane, Yuki; Sekimoto, Hiroyuki

    2015-07-01

    Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

  18. The putative oligosaccharide translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri

    PubMed Central

    Miyashiro, Tim; Oehlert, Dane; Ray, Valerie A; Visick, Karen L; Ruby, Edward G

    2014-01-01

    Quorum signaling (QS) describes how bacteria can use small signaling molecules (autoinducers) to coordinate group-level behaviors. In Vibrio fischeri, QS is achieved through a complex regulatory network that ultimately controls bioluminescence, motility, and host colonization. We conducted a genetic screen focused on qrr1, which encodes a small regulatory RNA that is necessary for the core quorum-signaling cascade to transduce autoinducer information into cellular responses. We isolated unique mutants with a transposon inserted into one of two genes within the syp locus, which is involved in biofilm formation. We found that overexpression of sypK, which encodes a putative oligosaccharide translocase, is sufficient to activate qrr1, and, in addition, this effect appears to depend on the kinase activity of the sensor LuxQ. Consistent with the established model for QS in V. fischeri, enhanced expression of qrr1 by the overexpression of sypK resulted in reduced bioluminescence and increased motility. Finally, we found that induction of the syp locus by overexpression of sypG was sufficient to activate qrr1 levels. Together, our results show how conditions that promote biofilm formation impact the quorum-signaling network in V. fischeri, and further highlight the integrated nature of the regulatory circuits involved in complex bacterial behaviors. PMID:25257018

  19. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

    PubMed

    Hempel, Niels; Görisch, Helmut; Mern, Demissew S

    2013-09-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

  20. Synthesis of putative uniflorine A.

    PubMed

    Davis, Andrew S; Pyne, Stephen G; Skelton, Brian W; White, Allan H

    2004-04-30

    A diastereoselective synthesis of the putative structure of the natural product uniflorine A has been achieved by using the Petasis borono-Mannich reaction and ring-closing metathesis as key steps. The NMR data of the synthetic material did not match that reported for the natural product. The structure of the final synthetic product was unequivocally determined by single-crystal X-ray study of its pentaacetate derivative. Thus it was concluded that the proposed structure of uniflorine A is incorrect.

  1. The Tomato Calcium Sensor Cbl10 and Its Interacting Protein Kinase Cipk6 Define a Signaling Pathway in Plant Immunity[C][W

    PubMed Central

    de la Torre, Fernando; Gutiérrez-Beltrán, Emilio; Pareja-Jaime, Yolanda; Chakravarthy, Suma; Martin, Gregory B.; del Pozo, Olga

    2013-01-01

    Ca2+ signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca2+-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca2+, suggesting that tomato Cbl10 and Cipk6 constitute a Ca2+-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca2+ and ROS signaling in plant immunity. PMID:23903322

  2. The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.

    PubMed

    Yegutkin, Gennady G; Mikhailov, Andrey; Samburski, Sergei S; Jalkanen, Sirpa

    2006-08-01

    Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

  3. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  4. Structural identification of putative USPs in Catharanthus roseus.

    PubMed

    Bahieldin, Ahmed; Atef, Ahmed; Shokry, Ahmed M; Al-Karim, Saleh; Al Attas, Sanaa G; Gadallah, Nour O; Edris, Sherif; Al-Kordy, Magdy A; Omer, Abdulkader M Shaikh; Sabir, Jamal S M; Ramadan, Ahmed M; Al-Hajar, Abdulrahman S M; Makki, Rania M; Hassan, Sabah M; El-Domyati, Fotouh M

    2015-10-01

    Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), (tyr)PK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences. PMID:26318047

  5. An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control

    PubMed Central

    2012-01-01

    Background The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR), the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD), which contains motifs of serine (S) or threonine (T) followed by a glutamine (Q). However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown. Results To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. Conclusions Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new putative targets for these

  6. Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization.

    PubMed

    Luo, L; Lee, T; Tsai, L; Tang, G; Jan, L Y; Jan, Y N

    1997-11-25

    The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

  7. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  8. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  9. Early ALS-type gait abnormalities in AMP-dependent protein kinase-deficient mice suggest a role for this metabolic sensor in early stages of the disease.

    PubMed

    Vergouts, Maxime; Marinangeli, Claudia; Ingelbrecht, Caroline; Genard, Geraldine; Schakman, Olivier; Sternotte, Anthony; Calas, André-Guilhem; Hermans, Emmanuel

    2015-12-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motoneurons. While the principal cause of the disease remains so far unknown, the onset and progression of the pathology are increasingly associated with alterations in the control of cell metabolism. On the basis of the well-known key roles of 5'-adenosine monophosphate-activated protein kinase (AMPK) in sensing and regulating the intracellular energy status, we hypothesized that mice with a genetic deletion of AMPK would develop locomotor abnormalities that bear similarity with those detected in the very early disease stage of mice carrying the ALS-associated mutated gene hSOD1(G93A). Using an automated gait analysis system (CatWalk), we here show that hSOD1(G93A) mice and age-matched mice lacking the neuronal and skeletal muscle predominant α2 catalytic subunit of AMPK showed an altered gait, clearly different from wild type control mice. Double mutant mice lacking AMPK α2 and carrying hSOD1(G93A) showed the same early gait abnormalities as hSOD1(G93A) mice over an age span of 8 to 16 weeks. Taken together, these data support the concept that altered AMPK function and associated bioenergetic abnormalities could constitute an important component in the early pathogenesis of ALS. Therapeutic interventions acting on metabolic pathways could prove beneficial on early locomotor deficits, which are sensitively detectable in rodent models using the CatWalk system. PMID:26152932

  10. pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

    SciTech Connect

    Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung; Kwon, Ohsuk; Hong, Young-Soo; Cheong, Chaejoon; Cheong, Hae-Kap

    2013-02-15

    Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.

  11. Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.

    PubMed

    Maierhofer, Tobias; Diekmann, Marion; Offenborn, Jan Niklas; Lind, Christof; Bauer, Hubert; Hashimoto, Kenji; S Al-Rasheid, Khaled A; Luan, Sheng; Kudla, Jörg; Geiger, Dietmar; Hedrich, Rainer

    2014-09-09

    Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

  12. Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase β′ Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins

    PubMed Central

    MacGregor, Barbara J.

    2015-01-01

    The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. “Maribeggiatoa” filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. “Maribeggiatoa” Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in

  13. Design and synthesis of inositolphosphoglycan putative insulin mediators.

    PubMed

    López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

    2005-03-01

    The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators. PMID:15731862

  14. Design and synthesis of inositolphosphoglycan putative insulin mediators.

    PubMed

    López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

    2005-03-01

    The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators.

  15. Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.

    PubMed

    Ma, G T; Hong, Y S; Ives, D H

    1995-03-24

    Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

  16. Systematic deletion analysis of fission yeast protein kinases.

    PubMed

    Bimbó, Andrea; Jia, Yonghui; Poh, Siew Lay; Karuturi, R Krishna Murthy; den Elzen, Nicole; Peng, Xu; Zheng, Liling; O'Connell, Matthew; Liu, Edison T; Balasubramanian, Mohan K; Liu, Jianhua

    2005-04-01

    Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome. PMID:15821139

  17. Protein kinase A signalling in Schistosoma mansoni cercariae and schistosomules.

    PubMed

    Hirst, Natasha L; Lawton, Scott P; Walker, Anthony J

    2016-06-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24h in vitro cultured somules of the blood parasite, Schistosoma mansoni, which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal 'stem' cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu(31),Pro(34)]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni, many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. PMID:26777870

  18. Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter

    PubMed Central

    Moscoso, Joana A.; Schramke, Hannah; Tosi, Tommaso; Dehbi, Amina; Jung, Kirsten

    2015-01-01

    ABSTRACT Nucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory

  19. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching. PMID:25731909

  20. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

  1. Phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR.

    PubMed

    Schell, Ursula; Kessler, Aline; Hilbi, Hubert

    2014-06-01

    The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' disease, a life-threatening pneumonia. For cell-cell communication the bacteria employ the autoinducer LAI-1 (3-hydroxypentadecane-4-one), which is produced and detected by the Lqs (Legionella quorum sensing) system. The system comprises the autoinducer synthase LqsA, the putative sensor kinases LqsS and LqsT, and the prototypic response regulator LqsR. Lqs-regulated processes include L. pneumophila-phagocyte interactions, production of extracellular filaments, and natural competence. Using biochemical approaches we show here that LqsS and LqsT are autophosphorylated by [γ-(32) P]-ATP at a conserved histidine residue (H200 or H204 ) located in their cytoplasmic histidine kinase domain. Pull-down assays revealed that LqsS and LqsT are bound by LqsR or phospho-LqsR. Dependent on the conserved receiver domain aspartate (D108 ), the response regulator prevented autophosphorylation of both sensor kinases by catalysing the dephosphorylation of phospho-LqsS or phospho-LqsT. Moreover, LqsR formed dimers upon phosphorylation at D108 by either acetyl-phosphate or phospho-LqsT. Finally, upon heterologous production in Escherichia coli, LqsT (but not LqsS) was autophosphorylated by ATP, and LqsR prevented the autophosphorylation by catalysing the dephosphorylation of phospho-LqsT. In summary, these results indicate that phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR.

  2. Two Kinase Family Dramas

    PubMed Central

    Leonard, Thomas A.; Hurley, James H.

    2007-01-01

    In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. PMID:17574014

  3. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  4. Fractal Dimension Analysis of Putative Martian Coastlines

    NASA Astrophysics Data System (ADS)

    Gianelli, G. A.

    2005-08-01

    Prior research is equivocal on the existence and location of Martian coastlines. This study proposes a novel method of analyzing putative coastlines; fractal dimensions provide a quantitative measurement of the complexity and nature of a fractal. Geological evidence points to a coastline at the elevation of -3790 meters, called the Deuteronilus contact. It is hypothesized that the fractal dimensions of this putative Martian coastline will be comparable to those of Earth shorelines. A topographic map with a contour line at -3790 meters was obtained from the U. S. Geological Survey, reflecting the most recent Mars Orbiter Laser Altimeter data. The map was cropped into sixty and twenty degree segments, and the putative coastline was isolated from extraneous features. A program which used the box-counting method calculated the fractal dimensions of the putative shorelines. The 22 results were tabulated and compared to 17 fractal dimensions of Earth shorelines, collected from published articles. Ranges were 1.07 to 1.66 for Earth and 1.141 to 1.436 for Mars. The mean was 1.28 for the Mars data and 1.22 for the Earth data, a slight difference that asteroid craters could account for. An unpaired t-test could not prove that the two data sets were significantly different. Although the past existence of a coastline at the Deuteronilus contact cannot be definitively proven without on site investigations, the hypothesis that the fractal dimensions of the putative Martian coastline would be comparable to those of Earth's was accepted, thereby substantiating the claims for the existence of a large northern ocean.

  5. A Glycine soja ABA-responsive receptor-like cytoplasmic kinase, GsRLCK, positively controls plant tolerance to salt and drought stresses.

    PubMed

    Sun, XiaoLi; Sun, Mingzhe; Luo, Xiao; Ding, XiaoDong; Ji, Wei; Cai, Hua; Bai, Xi; Liu, XiaoFei; Zhu, YanMing

    2013-06-01

    Receptor such as protein kinases are proposed to work as sensors to initiate signaling cascades in higher plants. However, little is known about the precise functions of receptor such as protein kinases in abiotic stress response in plants, especially in wild soybean. Here, we focused on characterization of the biological functions of a receptor-like cytoplasmic serine/threonine protein kinase gene, GsRLCK, which was previously identified as a putative salt-alkali stress-related gene from the transcriptome profiles of Glycine soja. Bioinformatic analysis showed that GsRLCK protein contained a conserved kinase catalytic domain and two transmembrane domains at the N-terminus, but no typical extracellular domain. Consistently, GsRLCK-eGFP fusion protein was observed on the plasma membrane, but eGFP alone was distributing throughout the cytoplasm in onion epidermal cells. Quantitative real-time PCR analysis revealed the induced expression of GsRLCK by ABA, salt, alkali, and drought stresses. However, the expression levels of GsRLCK seemed to be similar in different tissues, except soybean pod. Phenotypic assays demonstrated that GsRLCK overexpression decreased ABA sensitivity and altered expression levels of ABA-responsive genes. Furthermore, we also found that GsRLCK conferred increased tolerance to salt and drought stresses and increased expression levels of a handful of stress-responsive genes, when overexpressing in Arabidopsis. In a word, we gave exact evidence that GsRLCK was a novel receptor-like cytoplasmic protein kinase and played a crucial role in plant responses to ABA, salt, and drought stresses.

  6. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

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

    SciTech Connect

    Boel, Gaby-Fleur . E-mail: boel@mail.dife.de; Jurrmann, Nadine; Brigelius-Flohe, Regina

    2005-06-24

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

  8. Putative Drugs and Targets for Bipolar Disorder

    PubMed Central

    Zarate, Carlos A.; Manji, Husseini K.

    2009-01-01

    Current pharmacotherapy for bipolar disorder (BPD) is generally unsatisfactory for a large number of patients. Even with adequate modern bipolar pharmacological therapies, many afflicted individuals continue to have persistent mood episode relapses, residual symptoms, functional impairment and psychosocial disability. Creating novel therapeutics for BPD is urgently needed. Promising drug targets and compounds for BPD worthy of further study involve the following systems: purinergic, dynorphin opioid neuropeptide, cholinergic (muscarinic and nicotinic), melatonin and serotonin (5-HT2C receptor), glutamatergic, hypothalamic-pituitary adrenal (HPA) axis have all been implicated. Intracellular pathways and targets worthy of further study include glycogen synthase kinase-3 protein, protein kinase C, arachidonic acid cascade. PMID:18704977

  9. Identification of the putative staphylococcal AgrB catalytic residues involving the proteolytic cleavage of AgrD to generate autoinducing peptide.

    PubMed

    Qiu, Rongde; Pei, Wuhong; Zhang, Linsheng; Lin, Jianqun; Ji, Guangyong

    2005-04-29

    The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

  10. Putative BRAF activating fusion in a medullary thyroid cancer.

    PubMed

    Kasaian, Katayoon; Wiseman, Sam M; Walker, Blair A; Schein, Jacqueline E; Hirst, Martin; Moore, Richard A; Mungall, Andrew J; Marra, Marco A; Jones, Steven J M

    2016-03-01

    Medullary thyroid cancer (MTC) is a malignancy of the calcitonin-producing parafollicular cells of the thyroid gland. Surgery is the only curative treatment for this cancer. External beam radiation therapy is reserved for adjuvant treatment of MTC with aggressive features. Targeted therapeutics vandetanib and cabozantinib are approved for the treatment of aggressive and metastatic tumors that are not amenable to surgery. The use of these multikinase inhibitors are supported by the observed overactivation of the RET oncoprotein in a large subpopulation of MTCs. However, not all patients carry oncogenic alterations of this kinase. Hence, there is still a need for comprehensive molecular characterization of MTC utilizing whole-genome and transcriptome-sequencing methodologies with the aim of identifying targetable mutations. Here, we describe the genomic profiles of two medullary thyroid cancers and report the presence of a putative oncogenic BRAF fusion in one. Such alterations, previously observed in other malignancies and known targets of available drugs, can benefit patients who currently have no treatment options. PMID:27148585

  11. Magnetism and the putative early Martian life

    NASA Astrophysics Data System (ADS)

    Rochette, P.

    2001-08-01

    A short critical review is provided on three questions linking magnetism and the putative early Mars life. Was there a large internal Martian magnetic field, during which period, and is it a requisite for life? What is the origin of the paleomagnetic signal of Martian meteorites, including ALH84001? What is the present credibility of the case for fossil bacterial magnetite grains in ALH84001?

  12. The Secretory Pathway Kinases

    PubMed Central

    Sreelatha, Anju; Kinch, Lisa N.; Tagliabracci, Vincent S.

    2015-01-01

    Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. PMID:25862977

  13. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

    PubMed Central

    Weng, Q P; Andrabi, K; Klippel, A; Kozlowski, M T; Williams, L T; Avruch, J

    1995-01-01

    The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:7777579

  14. Biochemical and Structural Insights into Doublecortin-like Kinase Domain 1.

    PubMed

    Patel, Onisha; Dai, Weiwen; Mentzel, Mareike; Griffin, Michael D W; Serindoux, Juliette; Gay, Yoann; Fischer, Stefanie; Sterle, Shoukat; Kropp, Ashleigh; Burns, Christopher J; Ernst, Matthias; Buchert, Michael; Lucet, Isabelle S

    2016-09-01

    Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that belongs to the family of microtubule-associated proteins. Originally identified for its role in neurogenesis, DCLK1 has recently been shown to regulate biological processes outside of the CNS. DCLK1 is among the 15 most common putative driver genes for gastric cancers and is highly mutated across various other human cancers. However, our present understanding of how DCLK1 dysfunction leads to tumorigenesis is limited. Here, we provide evidence that DCLK1 kinase activity negatively regulates microtubule polymerization. We present the crystal structure of the DCLK1 kinase domain at 1.7 Å resolution, providing detailed insight into the ATP-binding site that will serve as a framework for future drug design. This structure also allowed for the mapping of cancer-causing mutations within the kinase domain, suggesting that a loss of kinase function may contribute to tumorigenesis. PMID:27545623

  15. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  16. AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes1[w

    PubMed Central

    Reumann, Sigrun; Ma, Changle; Lemke, Steffen; Babujee, Lavanya

    2004-01-01

    To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783–800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in β-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid α-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes. PMID:15333753

  17. Putative Excitatory and Putative Inhibitory Inputs Localize to Different Dendritic Domains in a Drosophila Flight Motoneuron

    PubMed Central

    Kuehn, Claudia; Duch, Carsten

    2012-01-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the targeting of input synapses to specific parts of their dendrites. However, only few examples exist where dendritic architecture can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical, and confocal laser scanning methods this study estimates the location of the spike initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with more than 4000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provided only an estimate of putative input synapse distributions, the data indicated that inhibitory and excitatory synapses were targeted preferentially to different dendritic domains of MN5, and thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  18. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  19. Preliminary X-ray crystallographic analysis of SMU.573, a putative sugar kinase from Streptococcus mutans

    SciTech Connect

    Zhou, Yan-Feng; Li, Lan-Fen; Yang, Cheng; Su, Xiao-Dong

    2008-01-01

    SMU.573 from S. mutans was expressed in E. coli and crystallized. The crystals belong to space group I4 and 2.5 Å resolution diffraction data were collected at an in-house chromium radiation source. SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 Å resolution diffraction data set was collected using an in-house chromium radiation source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 Å, α = β = γ = 90°.

  20. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  1. Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase 4

    PubMed Central

    Hothorn, Michael; Dabi, Tsegaye; Chory, Joanne

    2011-01-01

    Cytokinins are classic plant hormones that orchestrate growth, development and the integrity of stem cell populations. Cytokinin receptors are eukaryotic sensor histidine kinases that are activated both by naturally occurring adenine-type cytokinins and by urea-based synthetic compounds. Crystal structures of the Arabidopsis histidine kinase 4 sensor domain in complex with different cytokinin ligands now rationalize the hormone-binding specificity of the receptor and may spur the design of novel cytokinin ligands. PMID:21964459

  2. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

    PubMed Central

    Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin; Stanley-Wall, Nicola

    2014-01-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  3. Putative respiratory chain of Porphyromonas gingivalis.

    PubMed

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  4. Putative neuroprotective agents in neuropsychiatric disorders.

    PubMed

    Dodd, Seetal; Maes, Michael; Anderson, George; Dean, Olivia M; Moylan, Steven; Berk, Michael

    2013-04-01

    In many individuals with major neuropsychiatric disorders including depression, bipolar disorder and schizophrenia, their disease characteristics are consistent with a neuroprogressive illness. This includes progressive structural brain changes, cognitive and functional decline, poorer treatment response and an increasing vulnerability to relapse with chronicity. The underlying molecular mechanisms of neuroprogression are thought to include neurotrophins and regulation of neurogenesis and apoptosis, neurotransmitters, inflammatory, oxidative and nitrosative stress, mitochondrial dysfunction, cortisol and the hypothalamic-pituitary-adrenal axis, and epigenetic influences. Knowledge of the involvement of each of these pathways implies that specific agents that act on some or multiple of these pathways may thus block this cascade and have neuroprotective properties. This paper reviews the potential of the most promising of these agents, including lithium and other known psychotropics, aspirin, minocycline, statins, N-acetylcysteine, leptin and melatonin. These agents are putative neuroprotective agents for schizophrenia and mood disorders.

  5. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  6. SUMOylation regulates the SNF1 protein kinase.

    PubMed

    Simpson-Lavy, Kobi J; Johnston, Mark

    2013-10-22

    The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK's homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source.

  7. Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

    PubMed

    Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

    2006-01-01

    Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite

  8. Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes.

    PubMed

    Hresko, Richard C; Murata, Haruhiko; Mueckler, Mike

    2003-06-13

    By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt. PMID:12682057

  9. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  10. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  11. Regulation of mouse gamete interaction by a sperm tyrosine kinase.

    PubMed

    Leyton, L; LeGuen, P; Bunch, D; Saling, P M

    1992-12-15

    A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.

  12. Biogenic Origin for Earth's Oldest Putative Microfossils

    SciTech Connect

    De Gregorio, B.; Sharp, T; Flynn, G; Wirick, S; Hervig, R

    2009-01-01

    Carbonaceous microbe-like features preserved within a local chert unit of the 3.5 Ga old Apex Basalt in Western Australia may represent some of the oldest evidence of life on Earth. However, the biogenicity of these putative microfossils has been called into question, primarily because the sample collection locality is a black, carbon-rich, brecciated chert dike representing an Archean submarine hydrothermal spring, suggesting a formation via an abiotic organic synthesis mechanism. Here we describe the macromolecular hydrocarbon structure, carbon bonding, functional group chemistry, and biotic element abundance of carbonaceous matter associated with these filamentous features. These characteristics are similar to those of biogenic kerogen from the ca. 1.9 Ga old Gunflint Formation. Although an abiotic origin cannot be entirely ruled out, it is unlikely that known abiotic synthesis mechanisms could recreate both the structural and compositional complexity of this ancient carbonaceous matter. Thus, we find that a biogenic origin for this material is more likely, implying that the Apex microbe-like features represent authentic biogenic organic matter.

  13. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    PubMed

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  14. Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins.

    PubMed

    Del Valle, M E; Cobo, T; Cobo, J L; Vega, J A

    2012-08-01

    The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.

  15. Toddlers’ Duration of Attention towards Putative Threat

    PubMed Central

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  16. Magnetic Pulse Affects a Putative Magnetoreceptor Mechanism

    PubMed Central

    Davila, Alfonso F.; Winklhofer, Michael; Shcherbakov, Valera P.; Petersen, Nikolai

    2005-01-01

    Clusters of superparamagnetic (SP) magnetite crystals have recently been identified in free nerve endings in the upper-beak skin of homing pigeons and are interpreted as being part of a putative magnetoreceptor system. Motivated by these findings, we developed a physical model that accurately predicts the dynamics of interacting SP clusters in a magnetic field. The main predictions are: 1), under a magnetic field, a group of SP clusters self-assembles into a chain-like structure that behaves like a compass needle under slowly rotating fields; 2), in a frequently changing field as encountered by a moving bird, a stacked chain is a structurally more stable configuration than a single chain; 3), chain-like structures of SP clusters disrupt under strong fields applied at oblique angles; and 4), reassemble on a timescale of hours to days (assuming a viscosity of the cell plasma η ∼ 1 P). Our results offer a novel mechanism for magnetic field perception and are in agreement with the response of birds observed after magnetic-pulse treatments, which have been conducted in the past to specifically test if ferrimagnetic material is involved in magnetoreception, but which have defied explanation so far. Our theoretical results are supported by experiments on a technical SP model system using a high-speed camera. We also offer new predictions that can be tested experimentally. PMID:15863473

  17. The Biogeography of Putative Microbial Antibiotic Production

    PubMed Central

    Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  18. Current sensor

    DOEpatents

    Yakymyshyn, Christopher Paul; Brubaker, Michael Allen; Yakymyshyn, Pamela Jane

    2007-01-16

    A current sensor is described that uses a plurality of magnetic field sensors positioned around a current carrying conductor. The sensor can be hinged to allow clamping to a conductor. The current sensor provides high measurement accuracy for both DC and AC currents, and is substantially immune to the effects of temperature, conductor position, nearby current carrying conductors and aging.

  19. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    PubMed

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue. PMID:27032955

  20. Two-Component Signal Transduction Systems of Desulfovibrio Vulgaris: Structural and Phylogenetic Analysis and Deduction of Putative Cognate Pairs

    SciTech Connect

    Zhang, Weiwen; Culley, David E.; Wu, Gang; Brockman, Fred J.

    2006-01-20

    ABSTRACT-Two-component signal transduction systems (TCSTS) composed of sensory histidine kinases (HK) and response regulators (RR), constitute a key element of the mechanism by which bacteria sense and respond to changes in environments. A large number of TCSTSs including 59 putative HKs and 55 RRs were identified from the Desulfovibrio vulgaris genome, indicating their important roles in regulation of cellular metabolism. In this study, the structural and phylogenetic analysis of all putative TCSTSs in D. vulgaris was performed. The results showed D. vulgaris contained an unexpectedly large number of hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in other bacteria and extensive co-evolution between D. vulgaris HKs and RRs was observed, suggesting that the concordance of HKs and RRs in cognate phylogenetic groups could be indicative of cognate TCSTSs...

  1. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  2. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  3. Putative Lineage of Novel African Usutu Virus, Central Europe

    PubMed Central

    Cadar, Daniel; Bosch, Stefan; Jöst, Hanna; Börstler, Jessica; Garigliany, Mutien-Marie; Becker, Norbert

    2015-01-01

    We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe. PMID:26291923

  4. REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

    PubMed Central

    Shimojo, Masahito; Hersh, Louis B.

    2003-01-01

    The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors. PMID:14645515

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  6. Plant 5-Methylthioribose Kinase

    PubMed Central

    Guranowski, Andrzej

    1983-01-01

    Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg2+ and Mn2+ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The Km values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively. Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the γ-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (Ki = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues. PMID:16662931

  7. Bistability of a coupled Aurora B kinase-phosphatase system in cell division.

    PubMed

    Zaytsev, Anatoly V; Segura-Peña, Dario; Godzi, Maxim; Calderon, Abram; Ballister, Edward R; Stamatov, Rumen; Mayo, Alyssa M; Peterson, Laura; Black, Ben E; Ataullakhanov, Fazly I; Lampson, Michael A; Grishchuk, Ekaterina L

    2016-01-01

    Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division. PMID:26765564

  8. Novel interaction partners of the TPR/MET tyrosine kinase.

    PubMed

    Schaaf, Christian P; Benzing, Jörg; Schmitt, Thomas; Erz, Dorothee H R; Tewes, Magdalena; Bartram, Claus R; Janssen, Johannes W G

    2005-02-01

    A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

  9. Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens.

    PubMed

    Lee, Seung-Jin; Song, Ok-Ryul; Lee, Young-Choon; Choi, Yong-Lark

    2003-02-01

    To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.

  10. The kinase domain of mitochondrial PINK1 faces the cytoplasm

    PubMed Central

    Zhou, Chun; Huang, Yong; Shao, Yufang; May, Jessica; Prou, Delphine; Perier, Celine; Dauer, William; Schon, Eric A.; Przedborski, Serge

    2008-01-01

    Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD. PMID:18687899

  11. Epidermal growth factor receptor kinase domain mutations are rare in salivary gland carcinomas

    PubMed Central

    Dahse, R; Driemel, O; Schwarz, S; Dahse, J; Kromeyer-Hauschild, K; Berndt, A; Kosmehl, H

    2009-01-01

    Activating mutations within the epidermal growth factor (EGFR) tyrosine kinase domain identify non-small cell lung cancer patients with improved clinical response to tyrosine kinase inhibitor therapy. Recently, we identified two EGFR mutations in a cohort of 25 salivary gland carcinomas (SGCs) by screening the tumour samples for the both most common hotspot mutations in exons 19 and 21 by allele-specific PCR. Here, we present a comprehensive sequencing analysis of the entire critical EGFR tyrosine kinase domain in 65 SGC of the main histopathological types. We found EGFR mutations in the tyrosine kinase domain to be a rare event in SGCs. No additional mutations other than the two known exon 19 deletions (c.2235_2249del15) in a mucoepidermoid carcinoma and an adenoid cystic carcinoma have been detected. Other putative predictive markers for EGFR-targeted therapy in SGCs might be relevant and should be investigated. PMID:19174819

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    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.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

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

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    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.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    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.

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

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

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    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.

  20. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    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.

  1. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  2. Cyclin-dependent kinases.

    PubMed

    Malumbres, Marcos

    2014-01-01

    Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  3. The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner

    PubMed Central

    Pfannmüller, Andreas; Wagner, Dominik; Sieber, Christian; Schönig, Birgit; Boeckstaens, Mélanie; Marini, Anna Maria; Tudzynski, Bettina

    2015-01-01

    The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to

  4. PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A*

    PubMed Central

    Matsuda, Shinya; Kominato, Kyohei; Koide-Yoshida, Shizuyo; Miyamoto, Kenji; Isshiki, Kinuka; Tsuji, Akihiko; Yuasa, Keizo

    2014-01-01

    PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser12, Ser66, and Ser109 and that PCTK3 activity significantly increased via phosphorylation at Ser12 by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3. PMID:24831015

  5. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  6. Structural Analysis of Ligand Stimulation of the Histidine Kinase NarX

    SciTech Connect

    Cheung, J.; Hendrickson, W

    2009-01-01

    Histidine kinase receptors are a large family of membrane-spanning proteins found in many prokaryotes and some eukaryotes. They are a part of two-component signal transduction systems, which each comprise a sensor kinase and a response regulator and are involved with the regulation of many cellular processes. NarX is a histidine kinase receptor that responds to nitrate and nitrite to effect regulation of anaerobic respiration in various bacteria. We present high-resolution X-ray crystal structures of the periplasmic sensor domain from Escherichia coli NarX in a complex with nitrate and in the apo state. Our analysis reveals that nitrate-binding induces conformation changes that result in a piston-type displacement between the N- and C-terminal helices of the periplasmic domain. Such conformational changes might represent a conserved mechanism of signaling in histidine kinases by which ligand binding is communicated across the lipid bilayer.

  7. Smart sensors

    NASA Astrophysics Data System (ADS)

    Corsi, Carlo

    2006-08-01

    The term "Smart Sensors" refer to sensors which contain both sensing and signal processing capabilities with objectives ranging from simple viewing to sophisticated remote sensing, surveillance, search/track, weapon guidance, robotics, perceptronics and intelligence applications. In a broad sense, they include any sensor systems covering the whole electromagnetic spectrum: this paper deals specifically with a new class of smart sensors in infrared spectral bands whose developments started some years ago, when it was recognized that the rapid advances of "very large scale integration" (VLSI) processor technology and mosaic infrared detector array technology could be combined to develop new generations of infrared smart sensor systems with much improved performance. So, sophisticated signal processing operations have been developed for these new systems by integrating microcomputers and other VLSI signal processors within or next to the sensor arrays on the same focal plane avoiding complex computing located far away from the sensors. Recently this approach is achieving higher goals by a new and revolutionary sensors concept which introduce inside the sensor some of the basic function of living eyes, such as dynamic stare, dishomogenity compensation, spatial and temporal filtering. New objectives and requirements of these new focal plane processors are presented for this type of new infrared smart sensor systems. This paper is concerned with the processing techniques for only the front end of the focal plane processing, namely, the enhancement of target-to-noise ratio by background clutter suppression and the improvement in target detection by "smart" and pattern correlation threshold.

  8. Map kinases in fungal pathogens.

    PubMed

    Xu, J R

    2000-12-01

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

  9. Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

    PubMed Central

    Walker, Anthony J.; Ressurreição, Margarida; Rothermel, Rolf

    2014-01-01

    Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavor, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behavior, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis. PMID:25132840

  10. Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

    SciTech Connect

    Kitano-Takahashi, Michiko; Morita, Hiroyuki; Kondo, Shin; Tomizawa, Kayoko; Kato, Ryohei; Tanio, Michikazu; Shirota, Yoshiko; Takahashi, Hiroshi; Sugio, Shigetoshi; Kohno, Toshiyuki

    2007-07-01

    The kinase domain (residues 1–331) of human tau-tubulin kinase 2 was expressed in insect cells, purified and crystallized. Diffraction data have been collected to 2.9 Å resolution. Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1–331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  11. Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

    PubMed

    Kim, Eun-Mi; Kim, Jaehi; Kim, Yun-Gon; Lee, Peter; Shin, Dong-Sik; Kim, Mira; Hahn, Ji-Sook; Lee, Yoon-Sik; Kim, Byung-Gee

    2011-05-01

    Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the 'high-throughput phosphorylation profiling (HTPP)' method was developed on the basis of a fully randomized one-bead one-compound (OBOC) combinatorial ladder type peptide library and MALDI-TOF MS. The OBOC ladder peptide library was constructed by the 'split and pool' method on a HiCore resin. The peptide library sequence was Ac-Ala-X-X-X-Ser-X-X-Ala-BEBE-PLL resin. The substrate specificity of murine PKA (cAMP-dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co-activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1-dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay.

  12. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    PubMed Central

    2011-01-01

    Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases

  13. The ANKK1 kinase gene and psychiatric disorders.

    PubMed

    Ponce, Guillermo; Pérez-González, Rocío; Aragüés, María; Palomo, Tomás; Rodríguez-Jiménez, Roberto; Jiménez-Arriero, Miguel Angel; Hoenicka, Janet

    2009-07-01

    The TaqIA single nucleotide polymorphism (SNP, rs1800497), which is located in the gene that codes for the putative kinase ANKK1 (ANKK1) near the termination codon of the D2 dopamine receptor gene (DRD2; chromosome 11q22-q23), is the most studied genetic variation in a broad range of psychiatric disorders and personality traits. A large number of individual genetic association studies have found that the TaqIA SNP is linked to alcoholism and antisocial traits. In addition, it has also been related to other conditions such as schizophrenia, eating disorders, and some behavioral childhood disorders. The TaqIA A1 allele is mainly associated with addictions, antisocial disorders, eating disorders, and attention-deficit/hyperactivity disorders, while the A2 allele occurs more frequently in schizophrenic and obsessive-compulsive patients. Current data show that the TaqIA polymorphism may be a marker of both DRD2 and ANKK1 genetic variants. ANKK1 would belong to a family of kinases involved in signal transduction. This raises the question of whether signaling players intervene in the pathophysiology of psychiatric disorders. Basic research on the ANKK1 protein and its putative interaction with the D2 dopamine receptor could shed light on this issue. PMID:19526298

  14. The mitogen-activated protein kinase kinase kinase BcOs4 is required for vegetative differentiation and pathogenicity in Botrytis cinerea.

    PubMed

    Yang, Qianqian; Yan, Leiyan; Gu, Qin; Ma, Zhonghua

    2012-10-01

    The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.

  15. CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development.

    PubMed

    Diévart, Anne; Dalal, Monica; Tax, Frans E; Lacey, Alexzandria D; Huttly, Alison; Li, Jianming; Clark, Steven E

    2003-05-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

  16. CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

    PubMed Central

    Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.

    2003-01-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain. PMID:12724544

  17. Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.

    PubMed

    Etzkorn, Manuel; Kneuper, Holger; Dünnwald, Pia; Vijayan, Vinesh; Krämer, Jens; Griesinger, Christian; Becker, Stefan; Unden, Gottfried; Baldus, Marc

    2008-10-01

    The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.

  18. Sensor web

    NASA Technical Reports Server (NTRS)

    Delin, Kevin A. (Inventor); Jackson, Shannon P. (Inventor)

    2011-01-01

    A Sensor Web formed of a number of different sensor pods. Each of the sensor pods include a clock which is synchronized with a master clock so that all of the sensor pods in the Web have a synchronized clock. The synchronization is carried out by first using a coarse synchronization which takes less power, and subsequently carrying out a fine synchronization to make a fine sync of all the pods on the Web. After the synchronization, the pods ping their neighbors to determine which pods are listening and responded, and then only listen during time slots corresponding to those pods which respond.

  19. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  20. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    USGS Publications Warehouse

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  1. Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway.

    PubMed

    Ehleben, W; Bölling, B; Merten, E; Porwol, T; Strohmaier, A R; Acker, H

    1998-10-01

    This study applies biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent superoxide anion (O2-) formation and localisation of hydroxyl radicals (*OH) by 3-dimensional (3D) confocal laser scanning microscopy to reveal in human cells putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia. A cell membrane localised non-mitochondrial cytochrome b558 seems to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563 probably probing additionally metabolic changes. *OH the putative second messenger of the oxygen sensing pathway generated by a Fenton reaction could be visualized in the perinuclear space of the three human cell lines used. Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia induced gene expression, interact on various sides of the oxygen sensing pathway confirming the importance of b-type cytochromes and the Fenton reaction.

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

    PubMed Central

    2011-01-01

    biomarker and a putative functional site for kinase-unrelated activities of Cdk4. PMID:21668989

  3. Mechanism of Focal Adhesion Kinase Mechanosensing

    PubMed Central

    Sturm, Sebastian; Bullerjahn, Jakob Tómas; Bronowska, Agnieszka; Gräter, Frauke

    2015-01-01

    Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments. PMID:26544178

  4. Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1.

    PubMed

    Verma, Anita; Maurelli, Anthony T

    2003-10-01

    Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.

  5. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    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. PMID:26579181

  6. Chemical sensors

    DOEpatents

    Lowell, J.R. Jr.; Edlund, D.J.; Friesen, D.T.; Rayfield, G.W.

    1991-07-02

    Sensors responsive to small changes in the concentration of chemical species are disclosed. The sensors comprise a mechanochemically responsive polymeric film capable of expansion or contraction in response to a change in its chemical environment. They are operatively coupled to a transducer capable of directly converting the expansion or contraction to a measurable electrical response. 9 figures.

  7. Temperature Sensor

    NASA Technical Reports Server (NTRS)

    1980-01-01

    Weed Instrument Inc. produces a line of thermocouples - temperature sensors - for a variety of industrial and research uses. One of the company's newer products is a thermocouple specially designed for high accuracy at extreme temperatures above 3,000 degrees Fahrenheit. Development of sensor brought substantial increases in Weed Instrument sales and employment.

  8. Chemical sensors

    SciTech Connect

    Hubbard, C.W.; Gordon, R.L.

    1987-05-01

    The revolution in analytical chemistry promised by recent developments in the field of chemical sensors has potential for significant positive impact on both research and production activities conducted by and for the Department of Energy. Analyses which were, in the past, performed only with a roomful of expensive equipment can now be performed with miniature solid-state electronic devices or small optical probes. Progress in the development of chemical sensors has been rapid, and the field is currently growing at a great rate. In accordance, Pacific Northwest Laboratory initiated a survey of recent literature so that contributors to active programs in research on analytical methods could be made aware of principles and applications of this new technology. This report presents the results of that survey. The sensors discussed here are divided into three types: micro solid-state devices, optical sensors, and piezoelectric crystal devices. The report is divided into three corresponding sections. The first section, ''Micro Solid-State Devices,'' discusses the design, operation, and application of electronic sensors that are produced in much the same way as standard solid-state electronic devices. The second section, ''Optrodes,'' covers the design and operation of chemical sensors that use fiber optics to detect chemically induced changes in optical properties. The final section, ''Piezoelectric Crystal Detectors,'' discusses two types of chemical sensors that depend on the changes in the properties of an oscillating piezoelectric crystal to detect the presence of certain materials. Advantages and disadvantages of each type of sensor are summarized in each section.

  9. Smart Sensors

    NASA Astrophysics Data System (ADS)

    Corsi, C.

    2007-01-01

    The term "Smart Sensors" refers to sensors which contain both sensing and signal processing capabilities with objectives ranging from simple viewing to sophisticated remote sensing, surveillance, search/track, weapon guidance, robotics, perceptronics and intelligence applications. Recently this approach is achieving higher goals by a new and revolutionary sensors concept which introduced inside the sensor some of the basic functions of living eyes, such as dynamic stare, non-uniformity compensation, spatial and temporal filtering. New objectives and requirements are presented for this type of new infrared smart sensor systems. This paper is concerned with the front end of FPA microbolometers processing, namely, the enhancement of target-to-noise ratio by background clutter suppression and the improvement in target detection by "smart" and pattern correlation thresholding.

  10. Intramolecular conformational changes optimize protein kinase C signaling.

    PubMed

    Antal, Corina E; Violin, Jonathan D; Kunkel, Maya T; Skovsø, Søs; Newton, Alexandra C

    2014-04-24

    Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly synthesized PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling.

  11. Intramolecular conformational changes optimize protein kinase C signaling.

    PubMed

    Antal, Corina E; Violin, Jonathan D; Kunkel, Maya T; Skovsø, Søs; Newton, Alexandra C

    2014-04-24

    Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly synthesized PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling. PMID:24631122

  12. Focal adhesion kinase

    PubMed Central

    Stone, Rebecca L; Baggerly, Keith A; Armaiz-Pena, Guillermo N; Kang, Yu; Sanguino, Angela M; Thanapprapasr, Duangmani; Dalton, Heather J; Bottsford-Miller, Justin; Zand, Behrouz; Akbani, Rehan; Diao, Lixia; Nick, Alpa M; DeGeest, Koen; Lopez-Berestein, Gabriel; Coleman, Robert L; Lutgendorf, Susan; Sood, Anil K

    2014-01-01

    This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors. PMID:24755674

  13. Protein kinase C mu is located at the Golgi compartment

    PubMed Central

    1996-01-01

    Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes. PMID:8830770

  14. Putative neurosecretory cells of the cestode Hymenolepis microstoma.

    PubMed

    Webb, R A

    1976-10-01

    Putative neurosecretory cells were observed with the electron microscope in the nerve cords of the neck region of Hymenolepis microstoma. The cells show evidence of glandular activity by the large numbers of dense-core vesicles produced by the Golgi apparatus. The axons contain synaptoidlike structures with surrounding clouds of vesicles; features analogous to known neurosecretory release sites.

  15. Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

    EPA Science Inventory

    Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

  16. Bartonella henselae AS A PUTATIVE CAUSE OF CONGENITAL CHOLESTASIS

    PubMed Central

    VELHO, Paulo Eduardo Neves Ferreira; BELLOMO-BRANDÃO, Maria Ângela; DRUMMOND, Marina Rovani; MAGALHÃES, Renata Ferreira; HESSEL, Gabriel; BARJAS-CASTRO, Maria de Lourdes; ESCANHOELA, Cecília Amélia Fazzio; NEGRO, Gilda Maria Barbaro DEL; OKAY, Thelma Suely

    2016-01-01

    SUMMARY Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin. PMID:27410916

  17. Construct Validity of Putative Causes in Psychosocial Rehabilitation.

    ERIC Educational Resources Information Center

    Dellario, Donald J.

    1991-01-01

    Many variations of psychosocial rehabilitation programs and their unclear relationship to the psychosocial rehabilitation construct have increased the probability of threats to construct validity of putative causes, resulting in potential confounding in investigator interpretation. As a minimum, comprehensive and concise operational definitions of…

  18. Sulfur Isotope Composition of Putative Primary Troilite in Chondrules

    NASA Technical Reports Server (NTRS)

    Tachibana, Shogo; Huss, Gary R.

    2002-01-01

    Sulfur isotope compositions of putative primary troilites in chondrules from Bishunpur were measured by ion probe. These primary troilites have the same S isotope compositions as matrix troilites and thus appear to be isotopically unfractionated. Additional information is contained in the original extended abstract.

  19. Bivalent Inhibitors of Protein Kinases

    PubMed Central

    Gower, Carrie M.; Chang, Matthew E. K.; Maly, Dustin J.

    2015-01-01

    Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described. PMID:24564382

  20. Elucidating the role of the TRPM7 alpha-kinase: TRPM7 kinase inactivation leads to magnesium deprivation resistance phenotype in mice.

    PubMed

    Ryazanova, Lillia V; Hu, Zhixian; Suzuki, Sayuri; Chubanov, Vladimir; Fleig, Andrea; Ryazanov, Alexey G

    2014-12-23

    TRPM7 is an unusual bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation.

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  4. Evidence that phytochrome functions as a protein kinase in plant light signalling

    PubMed Central

    Shin, Ah-Young; Han, Yun-Jeong; Baek, Ayoung; Ahn, Taeho; Kim, Soo Young; Nguyen, Thai Son; Son, Minky; Lee, Keun Woo; Shen, Yu; Song, Pill-Soon; Kim, Jeong-Il

    2016-01-01

    It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants. PMID:27173885

  5. Evidence that phytochrome functions as a protein kinase in plant light signalling.

    PubMed

    Shin, Ah-Young; Han, Yun-Jeong; Baek, Ayoung; Ahn, Taeho; Kim, Soo Young; Nguyen, Thai Son; Son, Minky; Lee, Keun Woo; Shen, Yu; Song, Pill-Soon; Kim, Jeong-Il

    2016-01-01

    It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants. PMID:27173885

  6. Automotive sensors

    NASA Astrophysics Data System (ADS)

    Marek, Jiri; Illing, Matthias

    2003-01-01

    Sensors are an essential component of most electronic systems in the car. They deliver input parameters for comfort features, engine and emission control as well as for the active and passive safety systems. New technologies such as silicon micromachining play an important role for the introduction of these sensors in all vehicle classes. The importance and use of these sensor technologies in today"s automotive applications will be shown in this article. Finally an outlook on important current developments and new functions in the car will be given.

  7. Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans

    PubMed Central

    Lee, Kyung-Tae; So, Yee-Seul; Yang, Dong-Hoon; Jung, Kwang-Woo; Choi, Jaeyoung; Lee, Dong-Gi; Kwon, Hyojeong; Jang, Juyeong; Wang, Li Li; Cha, Soohyun; Meyers, Gena Lee; Jeong, Eunji; Jin, Jae-Hyung; Lee, Yeonseon; Hong, Joohyeon; Bang, Soohyun; Ji, Je-Hyun; Park, Goun; Byun, Hyo-Jeong; Park, Sung Woo; Park, Young-Min; Adedoyin, Gloria; Kim, Taeyup; Averette, Anna F.; Choi, Jong-Soon; Heitman, Joseph; Cheong, Eunji; Lee, Yong-Hwan; Bahn, Yong-Sun

    2016-01-01

    Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets. PMID:27677328

  8. DSTYK kinase domain ablation impaired the mice capabilities of learning and memory in water maze test.

    PubMed

    Li, Kui; Liu, Ji-Wei; Zhu, Zhi-Chuan; Wang, Hong-Tao; Zu, Yong; Liu, Yong-Jie; Yang, Yan-Hong; Xiong, Zhi-Qi; Shen, Xu; Chen, Rui; Zheng, Jing; Hu, Ze-Lan

    2014-01-01

    DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.

  9. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    PubMed

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  10. Wireless sensor

    DOEpatents

    Lamberti, Vincent E.; Howell, JR, Layton N.; Mee, David K.; Sepaniak, Michael J.

    2016-02-09

    Disclosed is a sensor for detecting a target material. The sensor includes a ferromagnetic metal and a molecular recognition reagent coupled to the ferromagnetic metal. The molecular recognition reagent is operable to expand upon exposure to vapor or liquid from the target material such that the molecular recognition reagent changes a tensile stress upon the ferromagnetic metal. The target material is detected based on changes in the magnetic switching characteristics of the ferromagnetic metal caused by the changes in the tensile stress.

  11. Environmental and Metabolic Sensors That Control T Cell Biology

    PubMed Central

    Ramsay, George; Cantrell, Doreen

    2015-01-01

    The T lymphocyte response to pathogens is shaped by the microenvironment. Environmental sensors in T cells include the nutrient-sensing serine/threonine kinases, adenosine monophosphate-activated protein kinase and mammalian target of rapamycin complex 1. Other environmental sensors are transcription factors such as hypoxia-inducible factor-1 and the aryl hydrocarbon receptor. The present review explores the molecular basis for the impact of environmental signals on the differentiation of conventional T cell receptor αβ T cells and how the T cell response to immune stimuli can coordinate the T cell response to environmental cues. PMID:25852681

  12. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  13. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  14. Characterization of a putative S locus encoded receptor protein and its role in self-incompatibility

    SciTech Connect

    Not Available

    1991-01-01

    Monoclonal antibodies (MAb) were raised against purified SLSG and polyclonal antisera were raised against purified trpE/SLR1 fusion proteins. MAbH8 reacts with a protein epitope on SLSG. MAbH8 and the anit-SLR1 antisera were used with immunogold labeling to show SLSG and SLR1 proteins are localized in papillar cell walls in the stigma. MAbH8 reacts with SLSG from CRM+ cells but not CRM-cells; amino acid sequence identity between the two classes was only 65%, vs. 80% within the CRM+ class. SLSG is necessary but not sufficient for self-incompatibility. Variable molecular weight (MW) SLSG proteins are derived from the same SLG gene. MW variations in both SLSG and SLR1 are due to changes in glycosylation and phosphorylation state. SLSG is not detectable in mature pollen, but is expressed during microspore development. Using a SLG probe, a gene for a putative receptor with protein kinase activity was identified.

  15. Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

    PubMed Central

    Neiman, A. M.; Mhaiskar, V.; Manus, V.; Galibert, F.; Dean, N.

    1997-01-01

    The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. PMID:9055074

  16. A fragmented alignment method detects a putative phosphorylation site and a putative BRC repeat in the Drosophila melanogaster BRCA2 protein.

    PubMed

    Chakraborty, Sandeep

    2013-01-01

    Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html.

  17. Tyrosine kinase gene rearrangements in epithelial malignancies.

    PubMed

    Shaw, Alice T; Hsu, Peggy P; Awad, Mark M; Engelman, Jeffrey A

    2013-11-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as 'druggable' targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours.

  18. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  19. Putative melatonin receptors in a human biological clock

    SciTech Connect

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  20. tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors.

    PubMed

    Dumont, D J; Yamaguchi, T P; Conlon, R A; Rossant, J; Breitman, M L

    1992-08-01

    A search for protein tyrosine kinases expressed during murine cardiogenesis resulted in the isolation of a novel tyrosine kinase, designated tek, which maps to mouse chromosome 4 between the brown and pmv-23 loci. The deduced amino acid sequence of tek predicts that it encodes a putative receptor tyrosine kinase that contains a 21 amino acid kinase insert and which is most closely related in its catalytic domains to FGFR1 and the product of the ret proto-oncogene. In situ hybridization analysis of adult tissues, as well as sectioned and whole-mount embryos, showed that tek is specifically expressed in the endocardium, the leptomeninges and the endothelial lining of the vasculature from the earliest stages of their development. Moreover, examination of the morphology of tek-expressing cells, and staging of tek expression relative to that of the endothelial cell marker von Willebrand factor, revealed that tek is expressed prior to von Willebrand factor and appears to mark the embryonic progenitors of mature endothelial cells. tek encodes a novel putative receptor tyrosine kinase that may be critically involved in the determination and/or maintenance of cells of the endothelial lineage.

  1. In vitro activity of rodogyl against putative periodontopathic bacteria.

    PubMed Central

    Quee, T C; Roussou, T; Chan, E C

    1983-01-01

    The minimal inhibitory concentrations of Rodogyl (composite tablet of metronidazole and spiramycin), metronidazole-spiramycin mixture, spiramycin, metronidazole, and tetracycline were determined for selected putative periodontopathic microorganisms. Rodogyl was active against almost all strains, including Bacteroides species and the anaerobic spirochetes. Synergism of the component drugs in the Rodogyl combination was noted against Propionibacterium species. Spiramycin activity against Actinomyces species was enhanced in the presence of metronidazole. PMID:6639002

  2. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.

    PubMed

    Rininsland, Frauke; Xia, Wensheng; Wittenburg, Shannon; Shi, Xiaobo; Stankewicz, Casey; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-10-26

    An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation.

  3. A biochemical and genetic study of Leishmania donovani pyruvate kinase.

    PubMed

    Sandoval, Will; Isea, Raúl; Rodriguez, Evelyn; Ramirez, Jose Luis

    2008-11-15

    Here we present a biochemical and molecular biology study of the enzyme pyruvate kinase (PYK) from the parasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and its kinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated by the effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector binding site of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior: Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, which deployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics (MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but also changed the folding of its domain C. PMID:18725273

  4. PINCH proteins regulate cardiac contractility by modulating integrin-linked kinase-protein kinase B signaling.

    PubMed

    Meder, Benjamin; Huttner, Inken G; Sedaghat-Hamedani, Farbod; Just, Steffen; Dahme, Tillman; Frese, Karen S; Vogel, Britta; Köhler, Doreen; Kloos, Wanda; Rudloff, Jessica; Marquart, Sabine; Katus, Hugo A; Rottbauer, Wolfgang

    2011-08-01

    Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

  5. Water Sensors

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Mike Morris, former Associate Director of STAC, formed pHish Doctor, Inc. to develop and sell a pH monitor for home aquariums. The monitor, or pHish Doctor, consists of a sensor strip and color chart that continually measures pH levels in an aquarium. This is important because when the level gets too high, ammonia excreted by fish is highly toxic; at low pH, bacteria that normally break down waste products stop functioning. Sales have run into the tens of thousands of dollars. A NASA Tech Brief Technical Support Package later led to a salt water version of the system and a DoE Small Business Innovation Research (SBIR) grant for development of a sensor for sea buoys. The company, now known as Ocean Optics, Inc., is currently studying the effects of carbon dioxide buildup as well as exploring other commercial applications for the fiber optic sensor.

  6. Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644.

    PubMed

    Robinson, P J; Lovenberg, W

    1988-01-01

    IC(50) for P63 was 290 ?M. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [(3)H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C.

  7. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  8. A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

    PubMed

    DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

    2014-07-15

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.

  9. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    PubMed

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

  10. Characterization of a tomato protein kinase gene induced by infection by Potato spindle tuber viroid.

    PubMed

    Hammond, R W; Zhao, Y

    2000-09-01

    Viroids--covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases. PMID:10975647

  11. Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon Pyrobaculum calidifontis.

    PubMed

    Hayashi, Maria; Nosaka, Kazuto

    2015-01-01

    Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process.

  12. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    PubMed

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb. PMID:26858255

  13. Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase.

    PubMed

    Nemoto, S; DiDonato, J A; Lin, A

    1998-12-01

    IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

  14. Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region.

    PubMed

    Ma, Y; Cunningham, M E; Wang, X; Ghosh, I; Regan, L; Longley, B J

    1999-05-01

    KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp-557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic alpha-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted alpha-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of alpha-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative alpha-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor. PMID:10224103

  15. Regulation of phosphorylation level and distribution of PTP36, a putative protein tyrosine phosphatase, by cell-substrate adhesion.

    PubMed

    Ogata, M; Takada, T; Mori, Y; Uchida, Y; Miki, T; Okuyama, A; Kosugi, A; Sawada, M; Oh-hora, M; Hamaoka, T

    1999-07-16

    Recently we have cloned a putative protein tyrosine phosphatase, PTP36/PTPD2/pez, which possesses a domain homologous to the N-terminal half of band 4.1 protein. In mouse fibroblasts adhered to substrates, PTP36 was phosphorylated on serine residues. PTP36 was found to make complexes with serine/threonine kinase(s), which phosphorylated PTP36 in vitro. PTP36 was dephosphorylated rapidly when the cell-substrate adhesion was disrupted and it was phosphorylated again along with the reattachment of the cells to fibronectin. Rephosphorylation of PTP36 seemed to depend on actin polymerization since it was inhibited by cytochalasin D. The cell detachment also induced the translocation of PTP36 into the membrane-associated cytoskeletal fraction. Staurosporine and ML-9, which inhibited the phosphorylation of PTP36 in vivo, induced the translocation of PTP36 too. On the contrary, when the dephosphorylation of PTP36 was inhibited by okadaic acid, no translocation of PTP36 was induced by the cell detachment. These results demonstrate that the cell-substrate adhesion and cell spreading regulates the intracellular localization of PTP36 most likely through its phosphorylation and therefore, PTP36 may play important roles in the signal transduction pathway of cell-adhesion. PMID:10400706

  16. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  17. Chemical sensors

    DOEpatents

    Lowell, Jr., James R.; Edlund, David J.; Friesen, Dwayne T.; Rayfield, George W.

    1991-01-01

    Sensors responsive to small changes in the concentration of chemical species are disclosed, comprising (a) a mechanochemically responsive polymeric film capable of expansion or contraction in response to a change in its chemical environment, operatively coupled to (b) a transducer capable of directly converting said expansion or contraction to a measurable electrical response.

  18. Gas sensor

    DOEpatents

    Schmid, Andreas K.; Mascaraque, Arantzazu; Santos, Benito; de la Figuera, Juan

    2014-09-09

    A gas sensor is described which incorporates a sensor stack comprising a first film layer of a ferromagnetic material, a spacer layer, and a second film layer of the ferromagnetic material. The first film layer is fabricated so that it exhibits a dependence of its magnetic anisotropy direction on the presence of a gas, That is, the orientation of the easy axis of magnetization will flip from out-of-plane to in-plane when the gas to be detected is present in sufficient concentration. By monitoring the change in resistance of the sensor stack when the orientation of the first layer's magnetization changes, and correlating that change with temperature one can determine both the identity and relative concentration of the detected gas. In one embodiment the stack sensor comprises a top ferromagnetic layer two mono layers thick of cobalt deposited upon a spacer layer of ruthenium, which in turn has a second layer of cobalt disposed on its other side, this second cobalt layer in contact with a programmable heater chip.

  19. Microcantilever Sensors

    NASA Astrophysics Data System (ADS)

    Lang, Hans Peter; Gerber, Christoph

    Microfabricated cantilevers have been used in atomic force microscopy for the topography imaging of non-conductive surfaces for more than 20 years. Cantilever beams without tips have proved their applicability in recent years as miniaturized, ultrasensitive, and fast-responding sensors for applications in chemistry, physics, biochemistry, and medicine. Microcantilever sensors respond by bending due to the absorption of molecules. A shift in resonance frequency also occurs. They can be operated in different environments such as gaseous environment, liquids, or vacuum. In gas, microcantilever sensors can be operated as an artificial nose, whereby the bending pattern of a microfabricated array of eight polymer-coated silicon cantilevers is characteristic of the different vapors from solvents, flavors, and beverages. When operated in a liquid, microcantilever sensors are able to detect biochemical reactions. Each cantilever is functionalized with a specific biochemical probe receptor, sensitive for detection of the corresponding target molecule. Applications lie in the fields of label- and amplification-free detection of DNA hybridization, the detection of proteins as well as antigen-antibody reactions, and the detection of larger entities, such as bacteria and fungi.

  20. Sensor apparatus

    DOEpatents

    Deason, Vance A [Idaho Falls, ID; Telschow, Kenneth L [Idaho Falls, ID

    2009-12-22

    A sensor apparatus and method for detecting an environmental factor is shown that includes an acoustic device that has a characteristic resonant vibrational frequency and mode pattern when exposed to a source of acoustic energy and, futher, when exposed to an environmental factor, produces a different resonant vibrational frequency and/or mode pattern when exposed to the same source of acoustic energy.

  1. Chemical sensor

    NASA Technical Reports Server (NTRS)

    Rauh, R. David (Inventor)

    1990-01-01

    A sensor for detecting a chemical substance includes an insertion element having a structure which enables insertion of the chemical substance with a resulting change in the bulk electrical characteristics of the insertion element under conditions sufficient to permit effective insertion; the change in the bulk electrical characteristics of the insertion element is detected as an indication of the presence of the chemical substance.

  2. Assaying the Kinase Activity of LRRK2 in vitro

    PubMed Central

    Lewis, Patrick A.

    2012-01-01

    outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates. PMID:22301813

  3. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca sup 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Gandy, S.; Czernik, A.J.; Greengard, P. )

    1988-08-01

    The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggest a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP. Ca{sup 2+}/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. Using rat cerebral cortex synaptosomes prelabeled with {sup 32}P{sub i}, a {sup 32}P-labeled phosphoprotein of {approx}135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.

  4. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases.

    PubMed

    Lai, Shenshen; Safaei, Javad; Pelech, Steven

    2016-03-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.

  5. ERK5 MAP Kinase Regulates Neurogenin1 during Cortical Neurogenesis

    PubMed Central

    Cundiff, Paige; Liu, Lidong; Wang, Yupeng; Zou, Junhui; Pan, Yung-Wei; Abel, Glen; Duan, Xin; Ming, Guo-li; Englund, Chris; Hevner, Robert; Xia, Zhengui

    2009-01-01

    The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors. PMID:19365559

  6. Interaction of the Ras-related protein associated with diabetes Rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation

    PubMed Central

    Zhu, Jianhua; Tseng, Yu-Hua; Kantor, Jason D.; Rhodes, Christopher J.; Zetter, Bruce R.; Moyers, Julie S.; Kahn, C. Ronald

    1999-01-01

    Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP⋅Rad was also reconverted to GTP⋅Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation. PMID:10611312

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

    PubMed

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

    2010-01-01

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

  8. Pressure sensor

    SciTech Connect

    Mee, David K.; Ripley, Edward B.; Nienstedt, Zachary C.; Nienstedt, Alex W.; Howell, Jr., Layton N.

    2015-09-29

    Disclosed is a passive, in-situ pressure sensor. The sensor includes a sensing element having a ferromagnetic metal and a tension inducing mechanism coupled to the ferromagnetic metal. The tension inducing mechanism is operable to change a tensile stress upon the ferromagnetic metal based on a change in pressure in the sensing element. Changes in pressure are detected based on changes in the magnetic switching characteristics of the ferromagnetic metal when subjected to an alternating magnetic field caused by the change in the tensile stress. The sensing element is embeddable in a closed system for detecting pressure changes without the need for any penetrations of the system for power or data acquisition by detecting changes in the magnetic switching characteristics of the ferromagnetic metal caused by the tensile stress.

  9. Corrosion sensor

    DOEpatents

    Glass, Robert S.; Clarke, Jr., Willis L.; Ciarlo, Dino R.

    1994-01-01

    A corrosion sensor array incorporating individual elements for measuring various elements and ions, such as chloride, sulfide, copper, hydrogen (pH), etc. and elements for evaluating the instantaneous corrosion properties of structural materials. The exact combination and number of elements measured or monitored would depend upon the environmental conditions and materials used which are subject to corrosive effects. Such a corrosion monitoring system embedded in or mounted on a structure exposed to the environment would serve as an early warning system for the onset of severe corrosion problems for the structure, thus providing a safety factor as well as economic factors. The sensor array is accessed to an electronics/computational system, which provides a means for data collection and analysis.

  10. Corrosion sensor

    DOEpatents

    Glass, R.S.; Clarke, W.L. Jr.; Ciarlo, D.R.

    1994-04-26

    A corrosion sensor array is described incorporating individual elements for measuring various elements and ions, such as chloride, sulfide, copper, hydrogen (pH), etc. and elements for evaluating the instantaneous corrosion properties of structural materials. The exact combination and number of elements measured or monitored would depend upon the environmental conditions and materials used which are subject to corrosive effects. Such a corrosion monitoring system embedded in or mounted on a structure exposed to the environment would serve as an early warning system for the onset of severe corrosion problems for the structure, thus providing a safety factor as well as economic factors. The sensor array is accessed to an electronics/computational system, which provides a means for data collection and analysis. 7 figures.

  11. Tuberculosis and nature's pharmacy of putative anti-tuberculosis agents.

    PubMed

    Chinsembu, Kazhila C

    2016-01-01

    Due to the growing problem of drug resistant Mycobacterium tuberculosis strains, coupled with the twinning of tuberculosis (TB) to human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), the burden of TB is now difficult to manage. Therefore, new antimycobacterial agents are being sought from natural sources. This review focuses on natural antimycobacterial agents from endophytes and medicinal plants of Africa, Europe, Asia, South America and Canada. In the countries mentioned in this review, numerous plant species display putative anti-TB activity. Several antimycobacterial chemical compounds have also been isolated, including: ellagitannin punicalagin, allicin, anthraquinone glycosides, iridoids, phenylpropanoids, beta-sitosterol, galanthimine, crinine, friedelin, gallic acid, ellagic acids, anthocyanidin, taraxerol, termilignan B, arjunic acid, glucopyranosides, 1-epicatechol, leucopelargonidol, hydroxybenzoic acids, benzophenanthridine alkaloids, neolignans, and decarine. These compounds may provide leads to novel and more efficacious drugs to lessen the global burden of TB and drug-resistant M. tuberculosis strains. If there is a long-term remedy for TB, it must lie in nature's pharmacy of putative antimycobacterial agents.

  12. Understanding putative risk factors for schizophrenia: retrospective and prospective studies

    PubMed Central

    King, Suzanne; Laplante, David; Joober, Ridha

    2005-01-01

    This paper describes a research program intended to provide a better understanding of the influence of several putative risk factors for schizophrenia on child development and psychosis. Two related components of the overall program are described: the retrospective EnviroGen projects, which use a variety of putative risk factors to explain variance in several dimensions of schizophrenia and in psychotic symptoms in community controls, and Project Ice Storm, which prospectively examines the effects of prenatal maternal stress in the children of women who were exposed to the 1998 Quebec ice storm during their pregnancies. The EnviroGen projects have been successful in explaining variance in several dimensions of illness, including premorbid adjustment and severity of dissociative symptoms. Project Ice Storm has demonstrated the noxious effects of prenatal stress on cognitive and language development in children. We have also found that “ice storm children” exposed in specific weeks of gestation show greater dermatoglyphic asymmetry, as has been reported for samples of patients with schizophrenia. In both studies, prenatal maternal stress has been associated with more severe childhood behaviour problems. The combination of retrospective and prospective studies is a rich source of triangulated results providing information about developmental psychopathology. PMID:16151539

  13. Hundreds of putatively functional small open reading frames in Drosophila

    PubMed Central

    2011-01-01

    Background The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization. Results Using bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data. Conclusions The combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist. PMID:22118156

  14. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    SciTech Connect

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  15. Putative cryptoendolithic life in Devonian pillow basalt, Rheinisches Schiefergebirge, Germany.

    PubMed

    Peckmann, J; Bach, W; Behrens, K; Reitner, J

    2008-03-01

    Middle Devonian (Givetian) pillow basalt and inter-pillow breccia from the Rheinisches Schiefergebirge in Germany were found to contain putative biogenic filaments that indicate that life once proliferated within these volcanic rocks. Mineralized filaments are found in carbonate amygdules (vesicles filled by carbonate cement) in the volcanic rock, where they started to form on the internal surface of the once water-filled vesicles. Biogenicity of the filaments is indicated by (1) their size and shape resembling modern microorganisms including a constant diameter along the length of curved filaments, (2) their independence of crystal faces or cleavage planes, (3) branching patterns reminiscent of modern microorganisms, and (4) their spatial clustering and preferential occurrence close to the margin of pillows and in the inter-pillow breccias. A time lag between the deposition of pillow basalt and the activity of endoliths is revealed by the sequence of carbonate cements filling the amygdules. The putative filamentous microorganisms thrived after the formation of early fibrous rim cement, but before later equant calcite spar filled most of the remaining porosity. Microbial clay authigenesis analogous to the encrustation of prokaryotes in modern iron-rich environments led to the preservation of filaments. The filaments predominantly consist of the clay minerals chamosite and illite. Having dwelled in water-filled vesicles, the Devonian basalt-hosted filaments apparently represent cryptoendoliths. This finding suggests that a previously unrecognized niche for life exists within volcanic rock.

  16. Tuberculosis and nature's pharmacy of putative anti-tuberculosis agents.

    PubMed

    Chinsembu, Kazhila C

    2016-01-01

    Due to the growing problem of drug resistant Mycobacterium tuberculosis strains, coupled with the twinning of tuberculosis (TB) to human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), the burden of TB is now difficult to manage. Therefore, new antimycobacterial agents are being sought from natural sources. This review focuses on natural antimycobacterial agents from endophytes and medicinal plants of Africa, Europe, Asia, South America and Canada. In the countries mentioned in this review, numerous plant species display putative anti-TB activity. Several antimycobacterial chemical compounds have also been isolated, including: ellagitannin punicalagin, allicin, anthraquinone glycosides, iridoids, phenylpropanoids, beta-sitosterol, galanthimine, crinine, friedelin, gallic acid, ellagic acids, anthocyanidin, taraxerol, termilignan B, arjunic acid, glucopyranosides, 1-epicatechol, leucopelargonidol, hydroxybenzoic acids, benzophenanthridine alkaloids, neolignans, and decarine. These compounds may provide leads to novel and more efficacious drugs to lessen the global burden of TB and drug-resistant M. tuberculosis strains. If there is a long-term remedy for TB, it must lie in nature's pharmacy of putative antimycobacterial agents. PMID:26464047

  17. Characterisation of putative oxygen chemoreceptors in bowfin (Amia calva).

    PubMed

    Porteus, Cosima S; Wright, Patricia A; Milsom, William K

    2014-04-15

    Serotonin containing neuroepithelial cells (NECs) are putative oxygen sensing cells found in different locations within the gills of fish. In this study we wished to determine the effect of sustained internal (blood) hypoxaemia versus external (aquatic) hypoxia on the size and density of NECs in the first gill arch of bowfin (Amia calva), a facultative air breather. We identified five different populations of serotonergic NECs in this species (Types I-V) based on location, presence of synaptic vesicles (SV) that stain for the antibody SV2, innervation and labelling with the neural crest marker HNK-1. Cell Types I-III were innervated, and these cells, which participate in central O2 chemoreflexes, were studied further. Although there was no change in the density of any cell type in bowfin after exposure to sustained hypoxia (6.0 kPa for 7 days) without access to air, all three of these cell types increased in size. In contrast, only Type II and III cells increased in size in bowfin exposed to sustained hypoxia with access to air. These data support the suggestion that NECs are putative oxygen-sensing cells, that they occur in several locations, and that Type I cells monitor only hypoxaemia, whereas both other cell types monitor hypoxia and hypoxaemia.

  18. Distribution of putative xenogeneic silencers in prokaryote genomes.

    PubMed

    Perez-Rueda, Ernesto; Ibarra, J Antonio

    2015-10-01

    Gene silencing is an important function as it keeps newly acquired foreign DNA repressed, thereby avoiding possible deleterious effects in the host organism. Known transcriptional regulators associated with this process are called xenogeneic silencers (XS) and belong to either the H-NS, Lsr2, MvaT or Rok families. In the work described here we looked for XS-like regulators and their distribution in prokaryotic organisms was evaluated. Our analysis showed that putative XS regulators similar to H-NS, Lsr2, MvaT or Rok are present only in bacteria (31.7%). This does not exclude the existence of alternative XS in the rest of the organisms analyzed. Additionally, of the four XS groups evaluated in this work, those from the H-NS family have diversified more than the other groups. In order to compare the distribution of these putative XS regulators we also searched for other nucleoid-associated proteins (NAPs) not included in this group such as Fis, EbfC/YbaB, HU/IHF and Alba. Results showed that NAPs from the Fis, EbfC/YbaB, HU/IHF and Alba families are widely (94%) distributed among prokaryotes. These NAPs were found in multiple combinations with or without XS-like proteins. In regard with XS regulators, results showed that only XS proteins from one family were found in those organisms containing them. This suggests specificity for this type of regulators and their corresponding genomes.

  19. Chemical sensors

    DOEpatents

    Lowell, J.R. Jr.; Edlund, D.J.; Friesen, D.T.; Rayfield, G.W.

    1992-06-09

    Sensors responsive to small changes in the concentration of chemical species are disclosed, comprising a mechanicochemically responsive polymeric film capable of expansion or contraction in response to a change in its chemical environment, either operatively coupled to a transducer capable of directly converting the expansion or contraction to a measurable electrical or optical response, or adhered to a second inert polymeric strip, or doped with a conductive material. 12 figs.

  20. Sensor assembly

    DOEpatents

    Bennett, Thomas E.; Nelson, Drew V.

    2004-04-13

    A ribbon-like sensor assembly is described wherein a length of an optical fiber embedded within a similar lengths of a prepreg tow. The fiber is ""sandwiched"" by two layers of the prepreg tow which are merged to form a single consolidated ribbon. The consolidated ribbon achieving a generally uniform distribution of composite filaments near the embedded fiber such that excess resin does not ""pool"" around the periphery of the embedded fiber.

  1. Chemical sensors

    DOEpatents

    Lowell, Jr., James R.; Edlund, David J.; Friesen, Dwayne T.; Rayfield, George W.

    1992-01-01

    Sensors responsive to small changes in the concentration of chemical species are disclosed, comprising a mechanicochemically responsive polymeric film capable of expansion or contraction in response to a change in its chemical environment, either operatively coupled to a transducer capable of directly converting the expansion or contraction to a measurable electrical or optical response, or adhered to a second inert polymeric strip, or doped with a conductive material.

  2. Position sensor

    NASA Technical Reports Server (NTRS)

    Auer, Siegfried (Inventor)

    1988-01-01

    A radiant energy angle sensor is provided wherein the sensitive portion thereof comprises a pair of linear array detectors with each detector mounted normal to the other to provide X and Y channels and a pair of slits spaced from the pair of linear arrays with each of the slits positioned normal to its associated linear array. There is also provided electrical circuit means connected to the pair of linear array detectors and to separate X and Y axes outputs.

  3. KID, a Kinase Inhibitor Database project.

    PubMed

    Collin, O; Meijer, L

    1999-01-01

    The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.

  4. Recognition-Domain Focused (RDF) Chemosensors: Versatile and Efficient Reporters of Protein Kinase Activity

    PubMed Central

    Luković, Elvedin; González-Vera, Juan A.; Imperiali, Barbara

    2009-01-01

    Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a β-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg2+ and undergoes chelation-enhanced fluorescence (CHEF). While the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy has been developed that also relies on CHEF. In this approach, the requirement for the constrained β-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKCα, PKCβI, PKCδ, Pim2, Akt1, MK2 and PKA), as well as receptor (IRK) and non-receptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower KM when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKCβI and PKCδ, which were previously unattainable using the BTF approach. PMID:18759402

  5. An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO.

    PubMed

    Moore, Jason O; Hendrickson, Wayne A

    2012-04-01

    The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.

  6. An Asymmetry-to-Symmetry Switch in Signal Transmission by the Histidine Kinase Receptor for TMAO

    SciTech Connect

    Moore, Jason O.; Hendrickson, Wayne A.

    2012-06-28

    The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.

  7. Cell fate regulation governed by a repurposed bacterial histidine kinase

    DOE PAGESBeta

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

  8. Cell fate regulation governed by a repurposed bacterial histidine kinase

    SciTech Connect

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  9. Structure and functions of plant calcium-dependent protein kinases.

    PubMed

    Klimecka, Maria; Muszyńska, Grazyna

    2007-01-01

    Calcium ions as second messengers play an essential role in many important cellular processes. In plants, transient changes in calcium content in the cytosol (calcium signatures) have been observed during growth, development and under stress conditions. Such diverse functions require many different calcium sensors. One of the largest and most differentiated group of calcium sensors are protein kinases, among them calcium-dependent protein kinases (CDPKs) which were identified only in plants and protists. CDPKs have a regulatory domain which is able to bind calcium ions. For regulation of CDPKs activities not only calcium ions but also specific phospholipids and autophosphorylation are responsible. CDPKs have many different substrates, which reflects the diversity of their functions. Potential protein substrates of CDPK are involved in carbon and nitrogen metabolism, phospholipid synthesis, defense responses, ion and water transport, cytoskeleton organization, transcription and hormone responses. Presently, participation of CDPKs in stress signal transduction pathways (e.g., cold, drought, high salinity, wounding) is intensively studied in many laboratories. An intriguing, but still not fully clarified problem is the cross-talk via CDPKs among different signaling pathways that enables signal integration at different levels and ensure appropriate downstream responses.

  10. Assessing Kinase Activity in Plants with In-Gel Kinase Assays.

    PubMed

    Wang, Pengcheng; Zhu, Jian-Kang

    2016-01-01

    The in-gel protein kinase assay is a powerful method to measure the protein phosphorylation activity of specific protein kinases. Any protein substrate can be embedded in polyacrylamide gels where they can be phosphorylated by protein kinases that are separated in the gel under denaturing conditions and then renatured. The kinase activity can be visualized in situ in the gels by autoradiography. This method has been used to compare the activities of protein kinases in parallel samples or to identify their potential substrates. Here, we describe in detail an in-gel kinase assay to measure the activity of some protein kinases in plants.

  11. Epidermal growth factor receptor in the prawn Macrobrachium rosenbergii: function and putative signaling cascade.

    PubMed

    Sharabi, Omri; Ventura, Tomer; Manor, Rivka; Aflalo, Eliahu D; Sagi, Amir

    2013-09-01

    Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene is expressed in most tested tissues. The role of Mr-EGFR was revealed by temporarily silencing the transcript through weekly injections of double-stranded Mr-EGFR RNA. Such treatment resulted in a significant reduction in growth and a delay in the appearance of a male secondary sexual characteristic, namely the appendix masculina. An additional function of Mr-EGFR was revealed with respect to eye development. Although the optic ganglion appeared to have retained its normal morphology, Mr-EGFR-silenced individuals developed abnormal eyes that presented irregular organization of the ommatidia, reflected by unorganized receptor cells occupying large areas of the dioptric portion and by a shortened crystalline tract layer.

  12. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    SciTech Connect

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  13. A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1

    PubMed Central

    DeMille, Desiree; Bikman, Benjamin T.; Mathis, Andrew D.; Prince, John T.; Mackay, Jordan T.; Sowa, Steven W.; Hall, Tacie D.; Grose, Julianne H.

    2014-01-01

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. PMID:24850888

  14. Bistability of a coupled Aurora B kinase-phosphatase system in cell division

    PubMed Central

    Zaytsev, Anatoly V; Segura-Peña, Dario; Godzi, Maxim; Calderon, Abram; Ballister, Edward R; Stamatov, Rumen; Mayo, Alyssa M; Peterson, Laura; Black, Ben E; Ataullakhanov, Fazly I; Lampson, Michael A; Grishchuk, Ekaterina L

    2016-01-01

    Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division. DOI: http://dx.doi.org/10.7554/eLife.10644.001 PMID:26765564

  15. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    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, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. 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.

  16. Exceptional error minimization in putative primordial genetic codes

    PubMed Central

    2009-01-01

    Background The standard genetic code is redundant and has a highly non-random structure. Codons for the same amino acids typically differ only by the nucleotide in the third position, whereas similar amino acids are encoded, mostly, by codon series that differ by a single base substitution in the third or the first position. As a result, the code is highly albeit not optimally robust to errors of translation, a property that has been interpreted either as a product of selection directed at the minimization of errors or as a non-adaptive by-product of evolution of the code driven by other forces. Results We investigated the error-minimization properties of putative primordial codes that consisted of 16 supercodons, with the third base being completely redundant, using a previously derived cost function and the error minimization percentage as the measure of a code's robustness to mistranslation. It is shown that, when the 16-supercodon table is populated with 10 putative primordial amino acids, inferred from the results of abiotic synthesis experiments and other evidence independent of the code's evolution, and with minimal assumptions used to assign the remaining supercodons, the resulting 2-letter codes are nearly optimal in terms of the error minimization level. Conclusion The results of the computational experiments with putative primordial genetic codes that contained only two meaningful letters in all codons and encoded 10 to 16 amino acids indicate that such codes are likely to have been nearly optimal with respect to the minimization of translation errors. This near-optimality could be the outcome of extensive early selection during the co-evolution of the code with the primordial, error-prone translation system, or a result of a unique, accidental event. Under this hypothesis, the subsequent expansion of the code resulted in a decrease of the error minimization level that became sustainable owing to the evolution of a high-fidelity translation system

  17. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  18. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  19. Putative Genes Involved in Saikosaponin Biosynthesis in Bupleurum Species

    PubMed Central

    Lin, Tsai-Yun; Chiou, Chung-Yi; Chiou, Shu-Jiau

    2013-01-01

    Alternative medicinal agents, such as the herb Bupleurum, are increasingly used in modern medicine to supplement synthetic drugs. First, we present a review of the currently known effects of triterpene saponins-saikosaponins of Bupleurum species. The putative biosynthetic pathway of saikosaponins in Bupleurum species is summarized, followed by discussions on identification and characterization of genes involved in the biosynthesis of saikosaponins. The purpose is to provide a brief review of gene extraction, functional characterization of isolated genes and assessment of expression patterns of genes encoding enzymes in the process of saikosaponin production in Bupleurum species, mainly B. kaoi. We focus on the effects of MeJA on saikosaponin production, transcription patterns of genes involved in biosynthesis and on functional depiction. PMID:23783277

  20. Secretive ciliates and putative asexuality in microbial eukaryotes.

    PubMed

    Dunthorn, Micah; Katz, Laura A

    2010-05-01

    Facultative sexuality is assumed to have occurred in the ancestor of all extant eukaryotes, but the distribution and maintenance of sex among microbial eukaryotes is still under debate. In this paper, we address the purported asexuality in colpodean ciliates as an exemplary lineage. Colpodeans are a primarily terrestrial clade thought to have arisen up to 900 MYA and contain one known derived sexual species. We conclude that the putative asexuality of this lineage is an observational artifact. We suggest that the same might hold for other microbial eukaryotes, and that many are secretively sexual as well. Theoretical work from the distantly related plants and animals suggests that both the evolutionary success of ancient asexuals and the reversal of the loss of sex are highly unlikely, further suggesting that colpodeans are secretively sexual. However, it remains to be seen to what extent sexual theories and predictions derived from macro-organismic lineages apply also to microbial eukaryotes.

  1. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    PubMed

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  2. Functional Analysis of a Putative Dothistromin Toxin MFS Transporter Gene

    PubMed Central

    Bradshaw, Rosie E.; Feng, Zhilun; Schwelm, Arne; Yang, Yongzhi; Zhang, Shuguang

    2009-01-01

    Dothistromin is a non-host selective toxin produced by the pine needle pathogen Dothistroma septosporum. Dothistromin is not required for pathogenicity, but may have a role in competition and niche protection. To determine how D. septosporum tolerates its own toxin, a putative dothistromin transporter, DotC, was investigated. Studies with mutants lacking a functional dotC gene, overproducing DotC, or with a DotC-GFP fusion gene, did not provide conclusive evidence of a role in dothistromin efflux. The mutants revealed a major effect of DotC on dothistromin biosynthesis but were resistant to exogenous dothistromin. Intracellular localization studies suggest that compartmentalization may be important for dothistromin tolerance. PMID:22069539

  3. Influenza Sensor

    DOEpatents

    Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

    2006-03-28

    A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

  4. Influenza Sensor

    DOEpatents

    Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

    2005-05-17

    A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

  5. Influenza sensor

    DOEpatents

    Swanson, Basil I.; Song, Xuedong; Unkefer, Clifford; Silks, III, Louis A.; Schmidt, Jurgen G.

    2003-09-30

    A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

  6. Hydrogen sensor

    DOEpatents

    Duan, Yixiang; Jia, Quanxi; Cao, Wenqing

    2010-11-23

    A hydrogen sensor for detecting/quantitating hydrogen and hydrogen isotopes includes a sampling line and a microplasma generator that excites hydrogen from a gas sample and produces light emission from excited hydrogen. A power supply provides power to the microplasma generator, and a spectrometer generates an emission spectrum from the light emission. A programmable computer is adapted for determining whether or not the gas sample includes hydrogen, and for quantitating the amount of hydrogen and/or hydrogen isotopes are present in the gas sample.

  7. Astrocytes in the optic nerve head express putative mechanosensitive channels

    PubMed Central

    Choi, Hee Joo; Sun, Daniel

    2015-01-01

    Purpose To establish whether optic nerve head astrocytes express candidate molecules to sense tissue stretch. Methods We used conventional PCR, quantitative PCR, and single-cell reverse transcription PCR (RT–PCR) to assess the expression of various members of the transient receptor potential (TRP) channel family and of the recently characterized mechanosensitive channels Piezo1 and 2 in optic nerve head tissue and in single, isolated astrocytes. Results Most TRP subfamilies (TRPC, TRPM, TRPV, TRPA, and TRPP) and Piezo1 and 2 were expressed in the optic nerve head of the mouse. Quantitative real-time PCR analysis showed that TRPC1, TRPM7, TRPV2, TRPP2, and Piezo1 are the dominant isoforms in each subfamily. Single-cell RT–PCR revealed that many TRP isoforms, TRPC1–2, TRPC6, TRPV2, TRPV4, TRPM2, TRPM4, TRPM6–7, TRPP1–2, and Piezo1–2, are expressed in astrocytes of the optic nerve head, and that most astrocytes express TRPC1 and TRPP1–2. Comparisons of the TRPP and Piezo expression levels between different tissue regions showed that Piezo2 expression was higher in the optic nerve head and the optic nerve proper than in the brain and the corpus callosum. TRPP2 also showed higher expression in the optic nerve head. Conclusions Astrocytes in the optic nerve head express multiple putative mechanosensitive channels, in particular the recently identified channels Piezo1 and 2. The expression of putative mechanosensitive channels in these cells may contribute to their responsiveness to traumatic or glaucomatous injury. PMID:26236150

  8. Putative Regulatory Factors Associated with Intramuscular Fat Content

    PubMed Central

    Cesar, Aline S. M.; Regitano, Luciana C. A.; Koltes, James E.; Fritz-Waters, Eric R.; Lanna, Dante P. D.; Gasparin, Gustavo; Mourão, Gerson B.; Oliveira, Priscila S. N.; Reecy, James M.; Coutinho, Luiz L.

    2015-01-01

    Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption. PMID:26042666

  9. Putative regulatory factors associated with intramuscular fat content.

    PubMed

    Cesar, Aline S M; Regitano, Luciana C A; Koltes, James E; Fritz-Waters, Eric R; Lanna, Dante P D; Gasparin, Gustavo; Mourão, Gerson B; Oliveira, Priscila S N; Reecy, James M; Coutinho, Luiz L

    2015-01-01

    Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption.

  10. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  11. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    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.

  12. Cloning and expression analyses of Sucrose non-fermenting-1-Related Kinase 1 (SnRK1b) gene during development of sorghum and maize endosperm, and its implicated role in sugar-to-starch metabolic transition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A full-length cDNA clone, SbSnRK1b (1530 bp, GenBank accession no. EF544393), encoding a putative serine/threonine protein kinase homologue of yeast (Saccharomyces cerevisiae) SNF1, was isolated from developing endosperm of sorghum [Sorghum bicolor (Moench) L.]. Multiple sequence alignment data show...

  13. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.

  14. Semiconductor sensors

    NASA Technical Reports Server (NTRS)

    Gatos, Harry C. (Inventor); Lagowski, Jacek (Inventor)

    1977-01-01

    A semiconductor sensor adapted to detect with a high degree of sensitivity small magnitudes of a mechanical force, presence of traces of a gas or light. The sensor includes a high energy gap (i.e., .about. 1.0 electron volts) semiconductor wafer. Mechanical force is measured by employing a non-centrosymmetric material for the semiconductor. Distortion of the semiconductor by the force creates a contact potential difference (cpd) at the semiconductor surface, and this cpd is determined to give a measure of the force. When such a semiconductor is subjected to illumination with an energy less than the energy gap of the semiconductors, such illumination also creates a cpd at the surface. Detection of this cpd is employed to sense the illumination itself or, in a variation of the system, to detect a gas. When either a gas or light is to be detected and a crystal of a non-centrosymmetric material is employed, the presence of gas or light, in appropriate circumstances, results in a strain within the crystal which distorts the same and the distortion provides a mechanism for qualitative and quantitative evaluation of the gas or the light, as the case may be.

  15. Force sensor

    DOEpatents

    Grahn, A.R.

    1993-05-11

    A force sensor and related method for determining force components is described. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

  16. Force sensor

    DOEpatents

    Grahn, Allen R.

    1993-01-01

    A force sensor and related method for determining force components. The force sensor includes a deformable medium having a contact surface against which a force can be applied, a signal generator for generating signals that travel through the deformable medium to the contact surface, a signal receptor for receiving the signal reflected from the contact surface, a generation controller, a reception controller, and a force determination apparatus. The signal generator has one or more signal generation regions for generating the signals. The generation controller selects and activates the signal generation regions. The signal receptor has one or more signal reception regions for receiving signals and for generating detections signals in response thereto. The reception controller selects signal reception regions and detects the detection signals. The force determination apparatus measures signal transit time by timing activation and detection and, optionally, determines force components for selected cross-field intersections. The timer which times by activation and detection can be any means for measuring signal transit time. A cross-field intersection is defined by the overlap of a signal generation region and a signal reception region.

  17. Microcantilever sensor

    DOEpatents

    Thundat, Thomas G.; Wachter, Eric A.

    1998-01-01

    An improved microcantilever sensor is fabricated with at least one microcantilever attached to a piezoelectric transducer. The microcantilever is partially surface treated with a compound selective substance having substantially exclusive affinity for a targeted compound in a monitored atmosphere. The microcantilever sensor is also provided with a frequency detection means and a bending detection means. The frequency detection means is capable of detecting changes in the resonance frequency of the vibrated microcantilever in the monitored atmosphere. The bending detection means is capable of detecting changes in the bending of the vibrated microcantilever in the monitored atmosphere coactively with the frequency detection means. The piezoelectric transducer is excited by an oscillator means which provides a signal driving the transducer at a resonance frequency inducing a predetermined order of resonance on the partially treated microcantilever. Upon insertion into a monitored atmosphere, molecules of the targeted chemical attach to the treated regions of the microcantilever resulting in a change in oscillating mass as well as a change in microcantilever spring constant thereby influencing the resonant frequency of the microcantilever oscillation. Furthermore, the molecular attachment of the target chemical to the treated regions induce areas of mechanical strain in the microcantilever consistent with the treated regions thereby influencing microcantilever bending. The rate at which the treated microcantilever accumulates the target chemical is a function of the target chemical concentration. Consequently, the extent of microcantilever oscillation frequency change and bending is related to the concentration of target chemical within the monitored atmosphere.

  18. Microcantilever sensor

    DOEpatents

    Thundat, T.G.; Wachter, E.A.

    1998-02-17

    An improved microcantilever sensor is fabricated with at least one microcantilever attached to a piezoelectric transducer. The microcantilever is partially surface treated with a compound selective substance having substantially exclusive affinity for a targeted compound in a monitored atmosphere. The microcantilever sensor is also provided with a frequency detection means and a bending detection means. The frequency detection means is capable of detecting changes in the resonance frequency of the vibrated microcantilever in the monitored atmosphere. The bending detection means is capable of detecting changes in the bending of the vibrated microcantilever in the monitored atmosphere coactively with the frequency detection means. The piezoelectric transducer is excited by an oscillator means which provides a signal driving the transducer at a resonance frequency inducing a predetermined order of resonance on the partially treated microcantilever. Upon insertion into a monitored atmosphere, molecules of the targeted chemical attach to the treated regions of the microcantilever resulting in a change in oscillating mass as well as a change in microcantilever spring constant thereby influencing the resonant frequency of the microcantilever oscillation. Furthermore, the molecular attachment of the target chemical to the treated regions induce areas of mechanical strain in the microcantilever consistent with the treated regions thereby influencing microcantilever bending. The rate at which the treated microcantilever accumulates the target chemical is a function of the target chemical concentration. Consequently, the extent of microcantilever oscillation frequency change and bending is related to the concentration of target chemical within the monitored atmosphere. 16 figs.

  19. Design of Targeted Inhibitors of Polo-like Kinase 1 (Plk1)

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    2011-03-01

    Computational design of small molecule inhibitors of Polo-like Kinase 1 (Plk1) is presented. Plk1, which regulates cell cycle, is often overexpressed in cancers. Its downregulation was shown to inhibit cancer progression. Most inhibitors of kinases' interact with the highly conserved ATP binding site. This makes the development of Plk1-specific inhibitors challenging, since different kinases have similar ATP sites. However, Plk1 also contains the polo-box domain (PBD), which is absent from other kinases. In this study, the PBD site was used as a target for designed Plk1 inhibitors. Common structural features of experimentally known Plk1 ligands were first identified. The information was used to design putative small molecules that specifically bonded Plk1. Druglikeness and possible toxicities of the designed molecules were determined. Molecules with no implied toxicities and optimal druglikeness were used for docking studies. The docking studies identified several molecules that made stable complexes with the Plk1 PBD site. Possible utilization of the designed molecules in drugs against cancers with overexpressed Plk1 is discussed.

  20. Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

    PubMed Central

    Itakura, Eisuke; Kishi, Chieko; Inoue, Kinji

    2008-01-01

    Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways. PMID:18843052

  1. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes.

    PubMed

    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2008-02-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes. PMID:18054038

  2. Trypanosoma cruzi arginine kinase characterization and cloning. A novel energetic pathway in protozoan parasites.

    PubMed

    Pereira, C A; Alonso, G D; Paveto, M C; Iribarren, A; Cabanas, M L; Torres, H N; Flawiá, M M

    2000-01-14

    This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates L-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mM L-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease. PMID:10625703

  3. Mevalonate kinase deficiency: current perspectives

    PubMed Central

    Favier, Leslie A; Schulert, Grant S

    2016-01-01

    Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder with a spectrum of manifestations, including the well-defined clinical phenotypes of hyperimmunoglobulinemia D and periodic fever syndrome and mevalonic aciduria. Patients with MKD have recurrent attacks of hyperinflammation associated with fever, abdominal pain, arthralgias, and mucocutaneous lesions, and more severely affected patients also have dysmorphisms and central nervous system anomalies. MKD is caused by mutations in the gene encoding mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. Mevalonate kinase is essential for the biosynthesis of nonsterol isoprenoids, which mediate protein prenylation. Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation. Given the emerging understanding of MKD as an autoinflammatory disorder, recent treatment approaches have largely focused on cytokine-directed biologic therapy. Herein, we review the current genetic and pathologic understanding of MKD, its various clinical phenotypes, and the evolving treatment approach for this multifaceted disorder. PMID:27499643

  4. Sensors for Entertainment.

    PubMed

    Lamberti, Fabrizio; Sanna, Andrea; Rokne, Jon

    2016-07-15

    Sensors are becoming ubiquitous in all areas of science, technology, and society. In this Special Issue on "Sensors for Entertainment", developments in progress and the current state of application scenarios for sensors in the field of entertainment is explored.

  5. Sensors, Update 1

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Göpel, Wolfgang; Hesse, Joachim

    1996-12-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Treatments include current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Furthermore, the sensor market as well as peripheral aspects such as standards are covered. Each volume is divided into four sections. Sensor Technology, reviews highlights in applied and basic research, Sensor Applications, covers new or improved applications of sensors, Sensor Markets, provides an overview of suppliers and market trends for a particular section, and Sensor Standards, reviews recent legislation and requirements for sensors. With this unique combination of information in each volume, Sensors Update will be of value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  6. Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

    PubMed Central

    Mongan, Maureen; Tan, Zongqing; Chen, Liang; Peng, Zhimin; Dietsch, Maggie; Su, Bing; Leikauf, George; Xia, Ying

    2008-01-01

    Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury. PMID:18467339

  7. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  8. Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

    PubMed Central

    Trajtenberg, Felipe; Albanesi, Daniela; Ruétalo, Natalia; Botti, Horacio; Mechaly, Ariel E.; Nieves, Marcos; Aguilar, Pablo S.; Cybulski, Larisa; Larrieux, Nicole; de Mendoza, Diego

    2014-01-01

    ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. PMID:25406381

  9. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals.

    PubMed

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years. PMID:26788065

  10. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals

    PubMed Central

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years. PMID:26788065

  11. A Putative Multiple-Demand System in the Macaque Brain

    PubMed Central

    Bell, Andrew H.; Buckley, Mark J.; Mitchell, Anna S.; Sallet, Jerome; Duncan, John

    2016-01-01

    In humans, cognitively demanding tasks of many types recruit common frontoparietal brain areas. Pervasive activation of this “multiple-demand” (MD) network suggests a core function in supporting goal-oriented behavior. A similar network might therefore be predicted in nonhuman primates that readily perform similar tasks after training. However, an MD network in nonhuman primates has not been described. Single-cell recordings from macaque frontal and parietal cortex show some similar properties to human MD fMRI responses (e.g., adaptive coding of task-relevant information). Invasive recordings, however, come from limited prespecified locations, so they do not delineate a macaque homolog of the MD system and their positioning could benefit from knowledge of where MD foci lie. Challenges of scanning behaving animals mean that few macaque fMRI studies specifically contrast levels of cognitive demand, so we sought to identify a macaque counterpart to the human MD system using fMRI connectivity in 35 rhesus macaques. Putative macaque MD regions, mapped from frontoparietal MD regions defined in humans, were found to be functionally connected under anesthesia. To further refine these regions, an iterative process was used to maximize their connectivity cross-validated across animals. Finally, whole-brain connectivity analyses identified voxels that were robustly connected to MD regions, revealing seven clusters across frontoparietal and insular cortex comparable to human MD regions and one unexpected cluster in the lateral fissure. The proposed macaque MD regions can be used to guide future electrophysiological investigation of MD neural coding and in task-based fMRI to test predictions of similar functional properties to human MD cortex. SIGNIFICANCE STATEMENT In humans, a frontoparietal “multiple-demand” (MD) brain network is recruited during a wide range of cognitively demanding tasks. Because this suggests a fundamental function, one might expect a similar

  12. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    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

  13. Proteomic identification of putative plasmodesmatal proteins from Chara corallina.

    PubMed

    Faulkner, Christine R; Blackman, Leila M; Cordwell, Stuart J; Overall, Robyn L

    2005-07-01

    Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.

  14. Putative role of brain acetaldehyde in ethanol addiction

    PubMed Central

    Deng, Xin-sheng; Deitrich, Richard A.

    2008-01-01

    The putative contribution of brain acetaldehyde (AcH) to ethanol (EtOH) tolerance and dependence (addiction) is reviewed. Although the role of AcH in EtOH addiction has been controversial, there are data showing a relationship. AcH can be formed in the brain tissues through the peroxidatic activity of catalase and by oxidation via other oxidizing enzymes such as cytochrome P-4502E1. Significant formation of AcH occurs in vitro in brain tissue at concentrations of EtOH that can be achieved by voluntary consumption of EtOH by rodents. AcH itself possesses reinforcing properties, which suggests that some of the behavioral pharmacological effects attributed to EtOH may be a result of the formation of AcH, and supports the involvement of AcH in EtOH addiction. Modulation of aldehyde dehydrogenase (ALDH) and brain catalase activity can change EtOH-related addictive behaviors presumably by changing AcH levels. Moreover, some condensation reaction products of AcH may promote some actions of EtOH and its consumption. On the basis of the findings, it can be concluded that AcH may mediate some of the CNS actions of EtOH including tolerance and dependence, although further exploration the involvement of AcH in EtOH addiction is warranted. PMID:19122804

  15. Putative impact of RNA editing on drug discovery.

    PubMed

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  16. Putative mycobacterial efflux inhibitors from the seeds of Aframomum melegueta.

    PubMed

    Gröblacher, Barbara; Maier, Veronika; Kunert, Olaf; Bucar, Franz

    2012-07-27

    In order to identify new putative efflux pump inhibitors that represent an appropriate target in antimycobacterial chemotherapy, nine paradol- and gingerol-related compounds (1-9) isolated from the seeds of Aframomum melegueta were assessed for their potential to inhibit ethidium bromide (EtBr) efflux in a Mycobacterium smegmatis model. Five of the compounds from A. melegueta and NMR spectroscopic data of the diketone 6-gingerdione (2) and its enolic tautomers, methyl-6-gingerol (5) and rac-6-dihydroparadol (7), are presented herein for the first time. After determination of their antimycobacterial activities and modulatory effects on the MIC of antibiotics as well as their synergistic effects in combination with antibiotics against M. smegmatis mc(2) 155, their impact on EtBr accumulation and efflux was evaluated using a microtiter plate-based fluorometric assay. The compounds exhibited moderate to weak antimycobacterial activities, and the best modulators induced a 4- to 16-fold decrease of the MICs of EtBr and rifampicin as well as a reduction of the MIC of isoniazid with fractional inhibitory concentration index values indicating synergistic activities in some cases. 6-Paradol (3), 8-gingerol (6), and rac-6-dihydroparadol (7) were the most potent EtBr efflux inhibitors in M. smegmatis mc(2) 155, displaying EtBr efflux inhibiting activities comparable to reference inhibitors.

  17. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    SciTech Connect

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-04-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with (/sup 14/C)-5-aminolevulinic acid (/sup 14/C)-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H/sub 2/O/sub 2/ stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with (/sup 14/C)-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed.

  18. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

    PubMed Central

    Kierny, Michael R.; Cunningham, Thomas D.; Bouhenni, Rachida A.; Edward, Deepak P.; Kay, Brian K.

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots. PMID:25902199

  19. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems

    PubMed Central

    Lomnitz, Jason G.; Savageau, Michael A.

    2016-01-01

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values. PMID:27578053

  20. The inducible CAM plants in putative lunar lander experiments

    NASA Astrophysics Data System (ADS)

    Burlak, Olexii; Zaetz, Iryna; Soldatkin, Olexii; Rogutskyy, Ivan; Danilchenko, Boris; Mikheev, Olexander; de Vera, Jean-Pierre; Vidmachenko, Anatolii; Foing, Bernard H.; Kozyrovska, Natalia

    Precursory lunar lander experiments on growing plants in locker-based chambers will increase our understanding of effect of lunar conditions on plant physiology. The inducible CAM (Cras-sulacean Acid Metabolism)-plants are reasonable model for a study of relationships between environmental challenges and changes in plant/bacteria gene expression. In inducible CAM-plants the enzymatic machinery for the environmentally activated CAM switches on from a C3-to a full-CAM mode of photosynthesis in response to any stresses (Winter et al., 2008). In our study, Kalanchoe spp. are shown to be promising candidates for putative lunar experiments as resistant to irradiation and desiccation, especially after inoculation with a bacterial consortium (Boorlak et al., 2010). Within frames of the experiment we expect to get information about the functional activity of CAM-plants, in particular, its organogenesis, photosystem, the circadian regulation of plant metabolism on the base of data gaining with instrumental indications from expression of the reporter genes fused to any genes involved in vital functions of the plant (Kozyrovska et al., 2009). References 1. Winter K., Garcia M., Holtum J. (2008) J. Exp. Bot. 59(7):1829-1840 2. Bourlak O., Lar O., Rogutskyy I., Mikheev A., Zaets I., Chervatyuk N., de Vera J.-P., Danilchenko A.B. Foing B.H., zyrovska N. (2010) Space Sci. Technol. 3. Kozyrovska N.O., Vidmachenko A.P., Foing B.H. et al. Exploration/call/estec/ESA. 2009.

  1. A putative corticosteroid hormone in Pacific lamprey, Entosphenus tridentatus.

    PubMed

    Rai, Satbir; Szeitz, András; Roberts, Brent W; Christie, Quill; Didier, Wesley; Eom, Junho; Yun, Sang-Seon; Close, David A

    2015-02-01

    Great efforts have been put forth to elucidate the mechanisms of the stress response in vertebrates and demonstrate the conserved response across different vertebrate groups, ranging from similarities in the activation of the hypothalamic-pituitary-adrenal axis to the release and role of corticosteroids. There is however, still very little known about stress physiology in the Pacific lamprey (Entosphenus tridentatus), descendants of the earliest vertebrate lineage, the agnathans. In this paper we demonstrate that 11-deoxycortisol, a steroid precursor to cortisol in the steroidogenic pathway, may be a functional corticosteroid in Pacific lamprey. We identified the putative hormone in Pacific lamprey plasma by employing an array of methods such as RIA, HPLC and mass spectrometry analysis. We demonstrated that plasma levels of 11-deoxycortisol significantly increased in Pacific lamprey 0.5 and 1 h after stress exposure and that lamprey corticotropin releasing hormone injections increased circulating levels of 11-deoxycortisol, suggesting that the stress response is under the control of the HPA/I axis as it is in higher vertebrates. A comprehensive understanding of vertebrate stress physiology may help shed light on the evolution of the corticosteroid signaling system within the vertebrate lineage.

  2. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems.

    PubMed

    Lomnitz, Jason G; Savageau, Michael A

    2016-01-01

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values. PMID:27578053

  3. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    SciTech Connect

    Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J.

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  4. Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

    PubMed

    Kawakami, Yuko; Nishimoto, Hajime; Kitaura, Jiro; Maeda-Yamamoto, Mari; Kato, Roberta M; Littman, Dan R; Leitges, Michael; Rawlings, David J; Kawakami, Toshiaki

    2004-11-12

    Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2. PMID:15364915

  5. Protein Kinase D family kinases: roads start to segregate.

    PubMed

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. PMID:24847910

  6. Tec family kinases in inflammation and disease.

    PubMed

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

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis. PMID:22449071

  7. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  8. Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

    PubMed Central

    Rich, J J; Kinscherf, T G; Kitten, T; Willis, D K

    1994-01-01

    Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity. Images PMID:8002569

  9. Computational analysis of AnmK-like kinase: New insights into the cell wall metabolism of fungi.

    PubMed

    Dai, Jianghong; Qu, Hong; Yu, Zhisheng; Yang, Jiangke; Zhang, Hongxun

    2015-08-21

    1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is the unique enzyme that marks the recycling of the cell wall of Escherichia coli. Here, 81 fungal AnmK-like kinase sequences from 57 fungal species were searched in the NCBI database and a phylogenetic tree was constructed. The three-dimensional structure of an AnmK-like kinase, levoglucosan kinase (LGK) of the yeast Lipomyces starkeyi, was modeled; molecular docking revealed that AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively. Analysis suggests that 1,6-anhydro-N-acetylglucosamine (anhGlcNAc) and/or 1,6-anhydro-β-d-glucosamine (anhGlcN) would be the appropriate substrates of AnmK-like kinases. Also, the counterparts of other characteristic enzymes of cell wall recycling of bacteria were found in fungi. Taken together, it is proposed that a putative recycling of anhGlcNAc/anhGlcN, which is associated with the hydrolysis of cell walls, exists in fungi. This computational analysis will provide new insights into the metabolism of fungal cell walls. PMID:25979372

  10. Computational analysis of AnmK-like kinase: New insights into the cell wall metabolism of fungi.

    PubMed

    Dai, Jianghong; Qu, Hong; Yu, Zhisheng; Yang, Jiangke; Zhang, Hongxun

    2015-08-21

    1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is the unique enzyme that marks the recycling of the cell wall of Escherichia coli. Here, 81 fungal AnmK-like kinase sequences from 57 fungal species were searched in the NCBI database and a phylogenetic tree was constructed. The three-dimensional structure of an AnmK-like kinase, levoglucosan kinase (LGK) of the yeast Lipomyces starkeyi, was modeled; molecular docking revealed that AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively. Analysis suggests that 1,6-anhydro-N-acetylglucosamine (anhGlcNAc) and/or 1,6-anhydro-β-d-glucosamine (anhGlcN) would be the appropriate substrates of AnmK-like kinases. Also, the counterparts of other characteristic enzymes of cell wall recycling of bacteria were found in fungi. Taken together, it is proposed that a putative recycling of anhGlcNAc/anhGlcN, which is associated with the hydrolysis of cell walls, exists in fungi. This computational analysis will provide new insights into the metabolism of fungal cell walls.

  11. A unifying mechanism for WNK kinase regulation of sodium-chloride cotransporter.

    PubMed

    Huang, Chou-Long; Cheng, Chih-Jen

    2015-11-01

    Mammalian with-no-lysine [K] (WNK) kinases are a family of four serine-threonine protein kinases, WNK1-4. Mutations of WNK1 and WNK4 in humans cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Increased Na(+) reabsorption through Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule plays an important role in the pathogenesis of hypertension in patients with PHA2. However, how WNK1 and WNK4 regulate NCC and how mutations of WNKs cause activation of NCC have been controversial. Here, we review current state of literature supporting a compelling model that WNK1 and WNK4 both contribute to stimulation of NCC. The precise combined effects of WNK1 and WNK4 on NCC remain unclear but likely are positive rather than antagonistic. The recent discovery that WNK kinases may function as an intracellular chloride sensor adds a new dimension to the physiological role of WNK kinases. Intracellular chloride-dependent regulation of WNK's may underlie the mechanism of regulation of NCC by extracellular K(+). Definite answer yet will require future investigation by tubular perfusion in mice with altered WNK kinase expression.

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

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  13. Sensor response rate accelerator

    DOEpatents

    Vogt, Michael C.

    2002-01-01

    An apparatus and method for sensor signal prediction and for improving sensor signal response time, is disclosed. An adaptive filter or an artificial neural network is utilized to provide predictive sensor signal output and is further used to reduce sensor response time delay.

  14. Focal adhesion kinase is involved in mechanosensing during fibroblast migration

    NASA Technical Reports Server (NTRS)

    Wang, H. B.; Dembo, M.; Hanks, S. K.; Wang, Y.

    2001-01-01

    Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

  15. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    PubMed

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  16. Fiber optic sensors

    NASA Technical Reports Server (NTRS)

    Hesse, J.; Sohler, W.

    1984-01-01

    A survey of the developments in the field of fiber optics sensor technology is presented along with a discussion of the advantages of optical measuring instruments as compared with electronic sensors. The two primary types of fiber optics sensors, specifically those with multiwave fibers and those with monowave fibers, are described. Examples of each major sensor type are presented and discussed. Multiwave detectors include external and internal fiber optics sensors. Among the monowave detectors are Mach-Zender interferometers, Michelson interferometers, Sagnac interferometers (optical gyroscopes), waveguide resonators, and polarimeter sensors. Integrated optical sensors and their application in spectroscopy are briefly discussed.

  17. Crossflow vorticity sensor

    NASA Technical Reports Server (NTRS)

    Holmes, Bruce J. (Inventor); Carraway, Debra L. (Inventor); Holmes, Harlan K. (Inventor); Moore, Thomas C. (Inventor)

    1988-01-01

    A crossflow vorticity sensor for the detection of crossflow vorticity characteristics is described. The sensor is comprised of crossflow sensors which are noninvasively adhered to a swept wing laminar surface either singularly, in multi-element strips, in polar patterns, or in orthogonal patterns. These crossflow sensors are comprised of hot-film sensor elements which operate as a constant temperature anemometer circuit to detect heat transfer rate changes. Accordingly, crossflow vorticity characteristics are determined via cross-correlation. In addition, the crossflow sensors have a thickness which does not exceed a maximum value h in order to avoid contamination of downstream crossflow sensors.

  18. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    SciTech Connect

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  19. Development of Magnetostrictive Sensors

    NASA Astrophysics Data System (ADS)

    Kleinke, Darrell Kenneth

    1995-11-01

    The behavior of Magnetostrictive sensors was investigated through experimentation and mathematical modeling. Two prototype sensors were designed, built and tested. A generalized design procedure is proposed and applied. The experimental data shows the viability of the magnetostrictive sensor as an alternative to conventional sensors. The experimental data was used to uncover the limitations of existing mathematical models, and to validate a new model. The prototype sensors demonstrate the ruggedness and cost effectiveness of the magnetostrictive sensor. The mathematical model accurately predicted the behavior of two different sensor configurations, thus illustrating the model's flexibility. The design procedure can be used by sensor designers as a general guideline. The application of the design procedure is a unique application of magnetostrictive sensors, illustrating the viability, practicality and flexibility of magnetostrictive sensor technology.

  20. Sensors, Update 2

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Göpel, Wolfgang; Hesse, Joachim

    1996-10-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Coverage includes current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Furthermore, the sensor market as well as peripheral aspects such as standards are covered. Each volume is divided into four sections. Sensor Technology, reviews highlights in applied and basic research, Sensor Applications, covers new or improved applications of sensors, Sensor Markets, provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update will be of value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  1. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    ERIC Educational Resources Information Center

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  2. EDITORIAL: Humidity sensors Humidity sensors

    NASA Astrophysics Data System (ADS)

    Regtien, Paul P. L.

    2012-01-01

    produced at relatively low cost. Therefore, they find wide use in lots of applications. However, the method requires a material that possesses some conflicting properties: stable and reproducible relations between air humidity, moisture uptake and a specific property (for instance the length of a hair, the electrical impedance of the material), fast absorption and desorption of the water vapour (to obtain a short response time), small hysteresis, wide range of relative humidity (RH) and temperature-independent output (only responsive to RH). For these reasons, much research is done and is still going on to find suitable materials that combine high performance and low price. In this special feature, three of the four papers report on absorption sensors, all with different focus. Aziz et al describe experiments with newly developed materials. The surface structure is extensively studied, in view of its ability to rapidly absorb water vapour and exhibit a reproducible change in the resistance and capacitance of the device. Sanchez et al employ optical fibres coated with a thin moisture-absorbing layer as a sensitive humidity sensor. They have studied various coating materials and investigated the possibility of using changes in optical properties of the fibre (here the lossy mode resonance) due to a change in humidity of the surrounding air. The third paper, by Weremczuk et al, focuses on a cheap fabrication method for absorption-based humidity sensors. The inkjet technology appears to be suitable for mass fabrication of such sensors, which is demonstrated by extensive measurements of the electrical properties (resistance and capacitance) of the absorbing layers. Moreover, they have developed a model that describes the relation between humidity and the electrical parameters of the moisture-sensitive layer. Despite intensive research, absorption sensors still do not meet the requirements for high accuracy applications. The dew-point temperature method is more appropriate

  3. Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator

    PubMed Central

    O’Grady, Joseph F.; Hoelters, Laura S.; Swain, Martin T.

    2016-01-01

    Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our

  4. Regulation of the Putative TRPV1t Salt Taste Receptor by Phosphatidylinositol 4, 5-Bisphosphate

    PubMed Central

    Phan, Tam-Hao T.; Ren, ZuoJun; Mummalaneni, Shobha; Melone, Pamela; Mahavadi, Sunila; Murthy, Karnam S.; DeSimone, John A.

    2010-01-01

    Regulation of the putative amiloride and benzamil (Bz)-insensitive TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate (PIP2) was studied by monitoring chorda tympani (CT) taste nerve responses to 0.1 M NaCl solutions containing Bz (5 × 10−6 M; a specific ENaC blocker) and resiniferatoxin (RTX; 0–10 × 10−6 M; a specific TRPV1 agonist) in Sprague-Dawley rats and in wildtype (WT) and TRPV1 knockout (KO) mice. In rats and WT mice, RTX elicited a biphasic effect on the NaCl + Bz CT response, increasing the CT response between 0.25 × 10−6 and 1 × 10−6 M. At concentrations >1 × 10−6 M, RTX inhibited the CT response. An increase in PIP2 by topical lingual application of U73122 (a phospholipase C blocker) or diC8-PIP2 (a short chain synthetic PIP2) inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. A decrease in PIP2 by topical lingual application of phenylarsine oxide (a phosphoinositide 4 kinase blocker) enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 × 10−6 M. The ENaC-dependent NaCl CT responses were not altered by changes in PIP2. An increase in PIP2 enhanced CT responses to sweet (0.3 M sucrose) and bitter (0.01 M quinine) stimuli. RTX produced the same increase in the Bz-insensitive Na+response when present in salt solutions containing 0.1 M NaCl + Bz, 0.1 M monosodium glutamate + Bz, 0.1 M NaCl + Bz + 0.005 M SC45647, or 0.1 M NaCl + Bz + 0.01 M quinine. No effect of RTX was observed on CT responses in WT mice and rats in the presence of the TRPV1 blocker N-(3-methoxyphenyl)-4-chlorocinnamide (1 × 10−6 M) or in TRPV1 KO mice. We conclude that PIP2 is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste, although in the last case, PIP2 seems to directly regulate the taste receptor protein itself, i.e., the TRPV1 ion channel or its taste

  5. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

    PubMed

    Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2008-08-01

    We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

  6. The aerosols' fate in a putative ammonia ocean on Titan

    NASA Astrophysics Data System (ADS)

    Ramírez, S. I.; Coll, P.; Buch, A.; Brassé, C.; Poch, O.; Raulin, F.

    2010-04-01

    A laboratory study on the chemical transformation of Titan's aerosol analogues placed under putative surface conditions of the satellite was performed. The surface of Titan was one of the targets of the Cassini-Huygens mission and of several of the Cassini orbiter instruments, especially ISS, VIMS and Radar. The first images revealed an interesting solid surface with features that suggest aeolian, tectonic, fluvial processes and even an impact structure[1]. Since then, more detailed descriptions of dunes, channels, lakes, impact craters and cryovolcanic structures have been documented[2]. The existence of an internal liquid water ocean, containing a few percent ammonia has been proposed[2, 3]. It has also been proposed that ammonia-water mixtures can erupt from the putative subsurface ocean leading to cryovolcanism[4]. The Cassini Titan Radar Mapper obtained Synthetic Aperture Radar (SAR) images during 2004 and 2005 that revealed a highly complex geology occurring at Titan's surface[5], among which cryovolcanic features play a central role. The composition of the cryomagma is mainly proposed to be a mixture of water ice and ammonia[6, 7, 8], although ammonia has not been directly detected on Titan, but suggested by recent Cassini-VIMS observations[9]. In order to understand the role that ammonia may play on the chemical transformation of atmospheric aerosols once they reach the surface, we designed the following protocol: laboratory analogues of Titan's aerosols were synthesized from a N2:CH4 (98:2) mixture irradiated under a continuous flow regime of 845 sccm inside which, a cold plasma of 180 W was established. The synthesized analogues were recovered and partitioned in several 10.0 mg samples that were placed in 4.0 mL-volume of aqueous ammonia solutions (25.00, 12.50, 6.25 and 3.125%) at different temperatures (298, 277, 253 and 93 K) for 10 weeks. After a derivatization process performed to the aerosols' refractory phase with N

  7. Tissue Factor Residues That Putatively Interact with Membrane Phospholipids

    PubMed Central

    Ke, Ke; Yuan, Jian; Morrissey, James H.

    2014-01-01

    Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit), bound to the integral membrane protein, tissue factor (the regulatory subunit). Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma). Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165) predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor. PMID:24516673

  8. Detection of putative virulence genes of Lactococcus garvieae.

    PubMed

    Ture, Mustafa; Altinok, Ilhan

    2016-04-12

    Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae.

  9. Credibility Analysis of Putative Disease-Causing Genes Using Bioinformatics

    PubMed Central

    Abel, Olubunmi; Powell, John F.; Andersen, Peter M.; Al-Chalabi, Ammar

    2013-01-01

    Background Genetic studies are challenging in many complex diseases, particularly those with limited diagnostic certainty, low prevalence or of old age. The result is that genes may be reported as disease-causing with varying levels of evidence, and in some cases, the data may be so limited as to be indistinguishable from chance findings. When there are large numbers of such genes, an objective method for ranking the evidence is useful. Using the neurodegenerative and complex disease amyotrophic lateral sclerosis (ALS) as a model, and the disease-specific database ALSoD, the objective is to develop a method using publicly available data to generate a credibility score for putative disease-causing genes. Methods Genes with at least one publication suggesting involvement in adult onset familial ALS were collated following an exhaustive literature search. SQL was used to generate a score by extracting information from the publications and combined with a pathogenicity analysis using bioinformatics tools. The resulting score allowed us to rank genes in order of credibility. To validate the method, we compared the objective ranking with a rank generated by ALS genetics experts. Spearman's Rho was used to compare rankings generated by the different methods. Results The automated method ranked ALS genes in the following order: SOD1, TARDBP, FUS, ANG, SPG11, NEFH, OPTN, ALS2, SETX, FIG4, VAPB, DCTN1, TAF15, VCP, DAO. This compared very well to the ranking of ALS genetics experts, with Spearman's Rho of 0.69 (P = 0.009). Conclusion We have presented an automated method for scoring the level of evidence for a gene being disease-causing. In developing the method we have used the model disease ALS, but it could equally be applied to any disease in which there is genotypic uncertainty. PMID:23755159

  10. Identification of Putative Natriuretic Hormones Isolated from Human Urine.

    PubMed

    Kramer, Herbert J

    2015-01-01

    This brief review describes some representative methodological approaches to the isolation of putative endogenous inhibitors of epithelial sodium transport - i.e., as ouabain-like factors (OLF) that inhibit the sodium transport enzyme Na-K-ATPase or inhibit the epithelial sodium channel (ENaC). Gel chromatography and reverse-phase (RP)-high performance liquid chromatography (HPLC) of lyophilized and reconstituted 24 h-urine from salt-loaded healthy humans led to two active fractions, a hydrophilic OLF-1 and a lipophilic OLF-2, whose mass (Ms)-spectroscopic data indicate a Mr of 391 (1, 2). Further identification was attempted by Ms-, infrared (IR)-, ultraviolet (UV)-, and (1)H-NMR-spectroscopy. OLF-1 and OLF-2 may be closely related if not identical to (di)ascorbic acid or its salts such as vanadium (V)-V(v)-diascorbate with Mr 403 (3) and V(IV)-diascorbate. OLF-1 and V(v)-diascorbate are about 10-fold stronger inhibitors of Na-K-ATPase than OLF-2 and V(IV)-diascorbate, respectively. In conscious rats, i.v. infusion of OLF-1 and OLF-2 resulted in a strong natriuresis. In a similar study, Cain et al. (4) isolated a sodium transport inhibitor from the urine of uremic patients by gel chromatography and RP-HPLC. In uremic rats, a natriuretic response to the injection of the active material was found. Xanthurenic acid 8-O-β-d-glucoside (Mr 368) and xanthurenic acid 8-O-sulfate (Mr 284) were identified as endogenous inhibitors of sodium transport acting, e.g., by ENaC blockade. No definite relation to blood pressure, body fluid volume, or sodium balance has been reported for any of these above factors, and further studies to identify the natriuretic and/or ouabain-like compound(s) or hormone(s) will be needed. PMID:26052310

  11. Detection of putative virulence genes of Lactococcus garvieae.

    PubMed

    Ture, Mustafa; Altinok, Ilhan

    2016-04-12

    Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae. PMID:27068503

  12. Sensor sentinel computing device

    DOEpatents

    Damico, Joseph P.

    2016-08-02

    Technologies pertaining to authenticating data output by sensors in an industrial environment are described herein. A sensor sentinel computing device receives time-series data from a sensor by way of a wireline connection. The sensor sentinel computing device generates a validation signal that is a function of the time-series signal. The sensor sentinel computing device then transmits the validation signal to a programmable logic controller in the industrial environment.

  13. Evaluation of infrasound sensors

    SciTech Connect

    Kromer, R.P.; McDonald, T.S.

    1998-08-01

    Sandia is evaluating the performance of various infrasound sensors that could be used as part of the International Monitoring Systems (IMS). Specifications for infrasound stations are outlined in CTBT/PC/II/1/Add.2. This document specifies minimum requirements for sensor, digitizer and system. The infrasound sensors evaluation task has the following objectives: provide an overview of the sensors presently in use; evaluate these sensors with respect to the requirements of the IMS.

  14. Crystal structure of brain-type creatine kinase at 1.41 A resolution.

    PubMed Central

    Eder, M.; Schlattner, U.; Becker, A.; Wallimann, T.; Kabsch, W.; Fritz-Wolf, K.

    1999-01-01

    Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates. PMID:10595529

  15. A Novel Protein Kinase-Like Domain in a Selenoprotein, Widespread in the Tree of Life

    PubMed Central

    Dudkiewicz, Małgorzata; Szczepińska, Teresa; Grynberg, Marcin; Pawłowski, Krzysztof

    2012-01-01

    structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network. PMID:22359664

  16. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  17. Sensor Authentication in Collaborating Sensor Networks

    SciTech Connect

    Bielefeldt, Jake Uriah

    2014-11-01

    In this thesis, we address a new security problem in the realm of collaborating sensor networks. By collaborating sensor networks, we refer to the networks of sensor networks collaborating on a mission, with each sensor network is independently owned and operated by separate entities. Such networks are practical where a number of independent entities can deploy their own sensor networks in multi-national, commercial, and environmental scenarios, and some of these networks will integrate complementary functionalities for a mission. In the scenario, we address an authentication problem wherein the goal is for the Operator Oi of Sensor Network Si to correctly determine the number of active sensors in Network Si. Such a problem is challenging in collaborating sensor networks where other sensor networks, despite showing an intent to collaborate, may not be completely trustworthy and could compromise the authentication process. We propose two authentication protocols to address this problem. Our protocols rely on Physically Unclonable Functions, which are a hardware based authentication primitive exploiting inherent randomness in circuit fabrication. Our protocols are light-weight, energy efficient, and highly secure against a number of attacks. To the best of our knowledge, ours is the first to addresses a practical security problem in collaborating sensor networks.

  18. A novel viral thymidylate kinase with dual kinase activity.

    PubMed

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  19. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  20. Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1.

    PubMed

    Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

    2015-01-01

    Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite's life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

  1. Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

    PubMed Central

    Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

    2015-01-01

    Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

  2. Huntingtin-associated protein 1 (HAP1) is a cGMP-dependent kinase anchoring protein (GKAP) specific for the cGMP-dependent protein kinase Iβ isoform.

    PubMed

    Corradini, Eleonora; Burgers, Pepijn P; Plank, Michael; Heck, Albert J R; Scholten, Arjen

    2015-03-20

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.

  3. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  4. Multifunctional Abl kinases in health and disease.

    PubMed

    Khatri, Aaditya; Wang, Jun; Pendergast, Ann Marie

    2016-01-01

    The Abelson tyrosine kinases were initially identified as drivers of leukemia in mice and humans. The Abl family kinases Abl1 and Abl2 regulate diverse cellular processes during development and normal homeostasis, and their functions are subverted during inflammation, cancer and other pathologies. Abl kinases can be activated by multiple stimuli leading to cytoskeletal reorganization required for cell morphogenesis, motility, adhesion and polarity. Depending on the cellular context, Abl kinases regulate cell survival and proliferation. Emerging data support important roles for Abl kinases in pathologies linked to inflammation. Among these are neurodegenerative diseases and inflammatory pathologies. Unexpectedly, Abl kinases have also been identified as important players in mammalian host cells during microbial pathogenesis. Thus, the use of Abl kinase inhibitors might prove to be effective in the treatment of pathologies beyond leukemia and solid tumors. In this Cell Science at a Glance article and in the accompanying poster, we highlight the emerging roles of Abl kinases in the regulation of cellular processes in normal cells and diverse pathologies ranging from cancer to microbial pathogenesis.

  5. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... National (UK) Information Centre for Metabolic Diseases National Organization for Rare Disorders (NORD): Pyruvate Kinase Deficiency Genetic Testing Registry (1 link) Pyruvate kinase deficiency of red cells Scientific articles on PubMed (1 link) PubMed OMIM (1 link) ...

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

    PubMed

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

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

  7. Sensors, Update 12

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Fedder, Gary K.; Korvink, Jan G.

    2003-04-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Coverage includes current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Each volume is divided into three sections. Sensor Technology, reviews highlights in applied and basic research, Sensor Applications, covers new or improved applications of sensors, Sensor Markets, provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update will be of value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  8. Sensors, Update 8

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Göpel, Wolfgang; Hesse, Joachim

    2001-02-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Coverage includes current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Each volume is divided into three sections: Sensor Technology reviews highlights in applied and basic research, while Sensor Applications covers new or improved applications of sensors, and Sensor Markets provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update will be invaluable to scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  9. Sensors, Update 11

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Fedder, Gary K.; Korvink, Jan G.

    2003-03-01

    Sensors Update ensures that you stay at the cutting edge of the field, presenting the current highlights of sensor and related microelectromechanical systems technology. Coverage includes most recent developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles based on micro- and nanotechnology. Each volume is divided into three sections: Sensor Technology reviews highlights in applied and basic research, Sensor Applications covers new or improved applications of sensors and Sensor Markets provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update is of must-have value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  10. Sensors, Update 10

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Fedder, Gary K.; Korvink, Jan G.

    2002-04-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Coverage includes current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Each volume is divided into three sections. Sensor Technology, reviews highlights in applied and basic research, Sensor Applications, covers new or improved applications of sensors, Sensor Markets, provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update will be of value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  11. Sensors, Update 9

    NASA Astrophysics Data System (ADS)

    Baltes, Henry; Göpel, Wolfgang; Hesse, Joachim

    2001-10-01

    Sensors Update ensures that you stay at the cutting edge of the field. Built upon the series Sensors, it presents an overview of highlights in the field. Coverage includes current developments in materials, design, production, and applications of sensors, signal detection and processing, as well as new sensing principles. Each volume is divided into three sections. Sensor Technology, reviews highlights in applied and basic research, Sensor Applications, covers new or improved applications of sensors, Sensor Markets, provides a survey of suppliers and market trends for a particular area. With this unique combination of information in each volume, Sensors Update will be of value for scientists and engineers in industry and at universities, to sensors developers, distributors, and users.

  12. Smart Sensor Systems

    NASA Astrophysics Data System (ADS)

    Hunter, G. W.; Stetter, J. R.; Hesketh, P. J.; Liu, C. C.

    Sensors and sensor systems are vital to our awareness of our surroundings and provide safety, security, and surveillance, as well as enable monitoring of our health and environment. A transformative advance in the field of sensor technology has been the development of "Smart Sensor Systems". The definition of a Smart Sensor may vary, but typically at a minimum a Smart Sensor is the combination of a sensing element with processing capabilities provided by a microprocessor. That is, Smart Sensors are basic sensing elements with embedded intelligence. The sensor signal is fed to the microprocessor, which processes the data and provides an informative output to an external user. A more expansive view of a Smart Sensor System, which is used in this article, is illustrated in Fig. 19.1: a complete self-contained sensor system that includes the capabilities for logging, processing with a model of sensor response and other data, self-contained power, and an ability to transmit or display informative data to an outside user. The fundamental idea of a smart sensor is that the integration of silicon microprocessors with sensor technology cannot only provide interpretive power and customized outputs, but also significantly improve sensor system performance and capabilities.

  13. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    SciTech Connect

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-08-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence {sup 916}NFNHIHKRIRRVADKYLSG{sup 934} comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin {alpha}/{beta} mechanism employing importins {alpha}1 and {alpha}3 but not {alpha}5. This transport was inhibited by wheat germ agglutinin and GTP{gamma}S. The sequence {sup 1414}SKKTNRGSQLHKYYMKRRTL{sup 1433}, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin {alpha}1/{beta} or {alpha}3/{beta} complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

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

    PubMed

    Myre, Michael A; O'Day, Danton H

    2004-09-17

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

  15. Tyrosine Kinase Inhibitors and Pregnancy

    PubMed Central

    Abruzzese, Elisabetta; Trawinska, Malgorzata Monika; Perrotti, Alessio Pio; De Fabritiis, Paolo

    2014-01-01

    The management of patients with chronic myeloid leukemia (CML) during pregnancy has become 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; in fact, 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, including the necessity to address issues relating to fertility and pregnancy. Physicians are frequently 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 TKI treated CML patients, as well as how to manage a planned and/or unplanned pregnancy. PMID:24804001

  16. Mind Operational Semantics and Brain Operational Architectonics: A Putative Correspondence

    PubMed Central

    Benedetti, Giulio; Marchetti, Giorgio; Fingelkurts, Alexander A; Fingelkurts, Andrew A

    2010-01-01

    ) of different complexity within OA’s theory: EOMC could correspond to simple OMs, correlators to complex OMs and the correlational network to a set of simple and complex OMs. Finally, a set of experiments is proposed to verify the putative correspondence between OS and OA and prove the existence of an integrated continuum between brain and mind. PMID:21113277

  17. Putative cryomagma interaction with aerosols deposit at Titan's surface

    NASA Astrophysics Data System (ADS)

    Coll, Patrice; Navarro-Gonzalez, Rafael; Raulin, Francois; Coscia, David; Ramirez, Sandra I.; Buch, Arnaud; Szopa, Cyril; Poch, Olivier; Cabane, Michel; Brassé, Coralie

    The largest moon of Saturn, Titan, is known for its dense, nitrogen-rich atmosphere. The organic aerosols which are produced in Titan’s atmosphere are of great astrobiological interest, particularly because of their potential evolution when they reach the surface and may interact with putative ammonia-water cryomagma [1]. In this context we have followed the evolution of alkaline pH hydrolysis (25wt% ammonia-water) of Titan aerosol analogues, that have been qualified as representative of Titan’s aerosols [2]. Indeed the first results obtained by the ACP experiment onboard Huygens probe revealed that the main products obtained after thermolysis of Titan’s collected aerosols, were ammonia (NH3) and hydrogen cyanide (HCN). Then performing a direct comparison of the volatiles produced after a thermal treatment done in conditions similar to the ones used by the ACP experiment, we may estimate that the tholins we used are relevant to chemical analogues of Titan’s aerosols, and to note free of oxygen. Taking into account recent studies proposing that the subsurface ocean may contain a lower fraction of ammonia (about 5wt% or less [3]), and assuming the presence of specific gas species [4, 5], in particular CO2 and H2S, trapped in likely internal ocean, we determine a new probable composition of the cryomagma which could potentially interact with deposited Titan’s aerosols. We then carried out different hydrolyses, taking into account this composition, and we established the influence of the hydrolysis temperature on the organic molecules production. References: [1] Mitri et al., 2008. Resurfacing of Titan by ammonia-water cryomagma. Icarus. 196, 216-224. [2] Coll et al. 2013, Can laboratory tholins mimic the chemistry producing Titan's aerosols? A review in light of ACP experimental results, Planetary and Space Science 77, 91-103. [3] Tobie et al. 2012. Titan’s Bulk Composition Constrained by Cassini-Huygens: implication for internal outgassing. The

  18. Evaluation and critical assessment of putative MCL-1 inhibitors

    PubMed Central

    Varadarajan, S; Vogler, M; Butterworth, M; Dinsdale, D; Walensky, L D; Cohen, G M

    2013-01-01

    High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and

  19. Silicon sensor integration to form smart sensors

    NASA Astrophysics Data System (ADS)

    Gourdeas, Leon; James, Daniel A.; Thiel, David V.; See, Le Lian

    2002-11-01

    The use of silicon-based sensors requires the addition of external support electronics to allow for compatibility with external logging and display instruments. The development of a smart sensor technology, where the support electronics are incorporated into the sensor allows for a simpler interface. To achieve this integration techniques are required for the connection of substrate sensors with drive and support circuitry (operational amplifiers and CMOS circuitry), for effective encapsulation into a single packaged device. In this paper a literature review of basic peripheral and internal interconnect techniques is presented. Three techniques for interconnects were experimentally investigated (wraparound, thermomigration and etched micro via"s) using in-house fabrication equipment and the results presented and discussed. An integrated "smart" light sensor was constructed by forming a schotkey diode on n-type silicon. The sensor was integrated with a commercially available LM324 quad operational amplifier die and etched micro via`s were used to connect between the electronics on one side and the silicon sensor on the other side so forming a smart sensor. The light level sensor was calibrated and tested for suitability as a solar intensity monitor.

  20. Optochemical Activation of Kinase Function in Live Cells

    PubMed Central

    Karginov, Andrei V.; Hahn, Klaus M.; Deiters, Alexander

    2015-01-01

    Summary Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases, and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases (RapR-kinases) in conjunction with a photocaged analog of rapamycin. PMID:24718793

  1. Prioritization of putative metabolite identifications in LC-MS/MS experiments using a computational pipeline.

    PubMed

    Zhou, Bin; Xiao, Jun Feng; Ressom, Habtom W

    2013-01-01

    One of the major bottle-necks in current LC-MS-based metabolomic investigations is metabolite identification. An often-used approach is to first look up metabolites from databases through peak mass, followed by verification of the obtained putative identifications using MS/MS data. However, the mass-based search may provide inappropriate putative identifications when the observed peak is from isotopes, fragments, or adducts. In addition, a large fraction of peaks is often left with multiple putative identifications. To differentiate these putative identifications, manual verification of metabolites through comparison between biological samples and authentic compounds is necessary. However, such experiments are laborious, especially when multiple putative identifications are encountered. It is desirable to use computational approaches to obtain more reliable putative identifications and prioritize them before performing experimental verification of the metabolites. In this article, a computational pipeline is proposed to assist metabolite identification with improved metabolome coverage and prioritization capability. Multiple publicly available software tools and databases, along with in-house developed algorithms, are utilized to fully exploit the information acquired from LC-MS/MS experiments. The pipeline is successfully applied to identify metabolites on the basis of LC-MS as well as MS/MS data. Using accurate masses, retention time values, MS/MS spectra, and metabolic pathways/networks, more appropriate putative identifications are retrieved and prioritized to guide subsequent metabolite verification experiments. PMID:23307777

  2. Prioritization of putative metabolite identifications in LC-MS/MS experiments using a computational pipeline.

    PubMed

    Zhou, Bin; Xiao, Jun Feng; Ressom, Habtom W

    2013-01-01

    One of the major bottle-necks in current LC-MS-based metabolomic investigations is metabolite identification. An often-used approach is to first look up metabolites from databases through peak mass, followed by verification of the obtained putative identifications using MS/MS data. However, the mass-based search may provide inappropriate putative identifications when the observed peak is from isotopes, fragments, or adducts. In addition, a large fraction of peaks is often left with multiple putative identifications. To differentiate these putative identifications, manual verification of metabolites through comparison between biological samples and authentic compounds is necessary. However, such experiments are laborious, especially when multiple putative identifications are encountered. It is desirable to use computational approaches to obtain more reliable putative identifications and prioritize them before performing experimental verification of the metabolites. In this article, a computational pipeline is proposed to assist metabolite identification with improved metabolome coverage and prioritization capability. Multiple publicly available software tools and databases, along with in-house developed algorithms, are utilized to fully exploit the information acquired from LC-MS/MS experiments. The pipeline is successfully applied to identify metabolites on the basis of LC-MS as well as MS/MS data. Using accurate masses, retention time values, MS/MS spectra, and metabolic pathways/networks, more appropriate putative identifications are retrieved and prioritized to guide subsequent metabolite verification experiments.

  3. Induction of thymidine kinase in enzyme-deficient Chinese hamster cells.

    PubMed

    Harris, M

    1982-06-01

    Previous work with Chinese hamster cells suggests that thymidine kinase deficiency and loss of potential for plating in HAT medium may arise by a process of mutation coupled with site-specific repression by bromodeoxyuridine at the tk locus. In this study, tk- Chinese hamster cells were exposed to a series of inductors to determine whether revertants for the putative second stage originate by genetic or epigenetic change. Brief exposure to 5-azacytidine resulted in massive conversion to the HAT+ state, and revertants showed levels of thymidine kinase activity intermediate between those of tk- and wild-type cells. By contrast, incidence of HAT+ cells rose only slightly in populations mutagenized with ethyl methanesulfonate. Large increases in frequency of HAT+ cells were obtained by treatment with n-butyrate and L-ethionine, which affect gene expression in other cell systems but have no known mutagenic potential. Induction of HAT+ revertants seems to be mediated by a stable epigenetic shift, which reverses the gradual extinction of thymidine kinase activity in the parent cells. The data support the view that induction in Chinese hamster cells results from changes in DNA methylation patterns, and suggests studies to define the process in molecular terms.

  4. Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases.

    PubMed

    Wang, Kan; Hackett, John T; Cox, Michael E; Van Hoek, Monique; Lindstrom, Jon M; Parsons, Sarah J

    2004-03-01

    Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.

  5. Growth inhibition by bupivacaine is associated with inactivation of ribosomal protein S6 kinase 1.

    PubMed

    Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

    2014-01-01

    Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation.

  6. Multiple Functions of Let-23, a Caenorhabditis Elegans Receptor Tyrosine Kinase Gene Required for Vulval Induction

    PubMed Central

    Aroian, R. V.; Sternberg, P. W.

    1991-01-01

    The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions. PMID:2071015

  7. Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

    PubMed Central

    Fowler, Matthew L.; Ingram-Smith, Cheryl

    2012-01-01

    Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PPi formation but not in the direction of acetyl phosphate/Pi formation suggests that the physiological direction of the reaction is toward acetate/PPi production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba. PMID:22903977

  8. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  9. Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy

    PubMed Central

    Webb, Thomas R; Slavish, Jake; George, Rani E; Look, A Thomas; Xue, Liquan; Jiang, Qin; Cui, Xiaoli; Rentrop, Walter B; Morris, Stephan W

    2009-01-01

    Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was initially identified in constitutively activated oncogenic fusion forms – the most common being nucleophosmin-ALK – in anaplastic large-cell lymphomas, and subsequent studies have identified ALK fusions in diffuse large B-cell lymphomas, systemic histiocytosis, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas and non-small-cell lung carcinomas. More recently, genomic DNA amplification and protein overexpression, as well as activating point mutations, of ALK have been described in neuroblastomas. In addition to those cancers for which a causative role for aberrant ALK activity is well validated, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies – including glioblastoma and breast cancer – via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with the reported ALK ligands, pleiotrophin and midkine. This review summarizes normal ALK biology, the confirmed and putative roles of ALK in the development of human cancers and efforts to target ALK using small-molecule kinase inhibitors. PMID:19275511

  10. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  11. A comprehensive analysis of LACK (Leishmania homologue of receptors for activated C kinase) in the context of Visceral Leishmaniasis

    PubMed Central

    Sinha, Sukrat; Kumar, Abhay; Sundaram, Shanthy

    2013-01-01

    The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target. PMID:24143055

  12. Multi Sensor Array

    NASA Technical Reports Server (NTRS)

    Immer, Christopher; Voska, Ned (Technical Monitor)

    2002-01-01

    This paper presents viewgraphs on the Multi Sensor Array. The topics include: 1) MSA Algorithm; 2) Types of Sensors for the MSA; 3) How to test the MSA; 4) Monte Carlo Simulation; and 5) Accelerated Life Tests.

  13. Electrochemical Sensors: Functionalized Silica

    SciTech Connect

    Fryxell, Glen E.; Lin, Yuehe; Yantasee, Wassana

    2009-03-24

    This chapter summarizes recent devellopment of electrochemical sensors based on functionlized mesoporous silica materials. The nanomatrials based sensors have been developed for sensitive and selective enrironmental detection of toxic heavy metal and uranium ions.

  14. Neonatal hyperbilirubinemia caused by pyruvate kinase deficiency.

    PubMed

    Hammer, S G; Lewan, R B

    1988-01-01

    We report an infant with neonatal hyperbilirubinemia due to pyruvate kinase deficiency. The initial approach involved rapid evaluation, phototherapy, and close monitoring of serum bilirubin levels. Follow-up included maintenance on folic acid, monitoring blood counts, and educating the parents about the course of pyruvate kinase deficiency, especially aplastic crisis. We suggest that the informed family practitioner can manage neonatal hyperbilirubinemia and pyruvate kinase deficiency with referrals at critical times to pediatric or surgical specialists. The practitioner must be able to recognize quickly the need for exchange transfusion for severe jaundice and for blood transfusions or splenectomy when significant anemia or aplastic crisis occurs.

  15. Functional analysis of anomeric sugar kinases.

    PubMed

    Conway, Louis P; Voglmeir, Josef

    2016-09-01

    Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study. PMID:27351442

  16. CFTR: a hub for kinases and crosstalk of cAMP and Ca2+.

    PubMed

    Kunzelmann, Karl; Mehta, Anil

    2013-09-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). The resulting disease is pleiotropic consistent with the idea that CFTR acts as a node within a network of signalling proteins. CFTR is not only a regulator of multiple transport proteins and controlled by numerous kinases but also participates in many signalling pathways that are disrupted after expression of its commonest mutant (F508del-CFTR). It operates in membrane compartments creating a scaffold for cytoskeletal elements, surface receptors, kinases and phosphodiesterases. CFTR is exposed to membrane-local second messengers such that a CFTR-interacting, low cellular energy sensor kinase (AMP- and ADP-activated kinase, AMPK) signals through a high energy phosphohistidine protein kinase (nucleoside diphosphate kinase, NDPK). CFTR also translocates a Ca(2+)-dependent adenylate cyclase to its proximity so that a rigid separation between cAMP-dependent and Ca(2+)-dependent regulation of Cl(-) transport becomes obsolete. In the presence of wild-type CFTR, parallel activation of CFTR and outwardly rectifying anoctamin 6 Cl(-) channels is observed, while the Ca(2+)-activated anoctamin 1 Cl(-) channel is inhibited. In contrast, in CF cells, CFTR is missing/mislocalized and the outwardly rectifying chloride channel is attenuated while Ca(2+)-dependent Cl(-) secretion (anoctamin 1) appears upregulated. Additionally, we consider the idea that F508del-CFTR when trapped in the endoplasmic reticulum augments IP3-mediated Ca(2+) release by providing a shunt pathway for Cl(-). CFTR and the IP3 receptor share the characteristic that they both assemble their partner proteins to increase the plasticity of their hub responses. In CF, the CFTR hub fails to form at the plasma membrane, with widespread detrimental consequences for cell signalling.

  17. Digital Sensor Technology

    SciTech Connect

    Ted Quinn; Jerry Mauck; Richard Bockhorst; Ken Thomas

    2013-07-01

    The nuclear industry has been slow to incorporate digital sensor technology into nuclear plant designs due to concerns with digital qualification issues. However, the benefits of digital sensor technology for nuclear plant instrumentation are substantial in terms of accuracy, reliability, availability, and maintainability. This report demonstrates these benefits in direct comparisons of digital and analog sensor applications. It also addresses the qualification issues that must be addressed in the application of digital sensor technology.

  18. Giant magnetoresistive sensor

    DOEpatents

    Stearns, Daniel G.; Vernon, Stephen P.; Ceglio, Natale M.; Hawryluk, Andrew M.

    1999-01-01

    A magnetoresistive sensor element with a three-dimensional micro-architecture is capable of significantly improved sensitivity and highly localized measurement of magnetic fields. The sensor is formed of a multilayer film of alternately magnetic and nonmagnetic materials. The sensor is optimally operated in a current perpendicular to plane mode. The sensor is useful in magnetic read/write heads, for high density magnetic information storage and retrieval.

  19. Fiber optic geophysical sensors

    DOEpatents

    Homuth, Emil F.

    1991-01-01

    A fiber optic geophysical sensor in which laser light is passed through a sensor interferometer in contact with a geophysical event, and a reference interferometer not in contact with the geophysical event but in the same general environment as the sensor interferometer. In one embodiment, a single tunable laser provides the laser light. In another embodiment, separate tunable lasers are used for the sensor and reference interferometers. The invention can find such uses as monitoring for earthquakes, and the weighing of objects.

  20. Secure Sensor Platform

    SciTech Connect

    Troy Ross, Barry Schoeneman

    2010-08-25

    The Secure Sensor Platform (SSP) software provides a framework of functionality to support the development of low-power autonomous sensors for nuclear safeguards. This framework provides four primary functional blocks of capabilities required to implement autonomous sensors. The capabilities are: communications, security, power management, and cryptography. Utilizing this framework establishes a common set of functional capabilities for seamless interoperability of any sensor based upon the SSP concept.

  1. Sensors for Entertainment.

    PubMed

    Lamberti, Fabrizio; Sanna, Andrea; Rokne, Jon

    2016-01-01

    Sensors are becoming ubiquitous in all areas of science, technology, and society. In this Special Issue on "Sensors for Entertainment", developments in progress and the current state of application scenarios for sensors in the field of entertainment is explored. PMID:27428981

  2. Touch Sensor for Robots

    NASA Technical Reports Server (NTRS)

    Primus, H. C.

    1986-01-01

    Touch sensor for robot hands provides information about shape of grasped object and force exerted by gripper on object. Pins projecting from sensor create electrical signals when pressed. When grasped object depresses pin, it contacts electrode under it, connecting electrode to common electrode. Sensor indicates where, and how firmly, gripper has touched object.

  3. Sensor readout detector circuit

    DOEpatents

    Chu, D.D.; Thelen, D.C. Jr.

    1998-08-11

    A sensor readout detector circuit is disclosed that is capable of detecting sensor signals down to a few nanoamperes or less in a high (microampere) background noise level. The circuit operates at a very low standby power level and is triggerable by a sensor event signal that is above a predetermined threshold level. A plurality of sensor readout detector circuits can be formed on a substrate as an integrated circuit (IC). These circuits can operate to process data from an array of sensors in parallel, with only data from active sensors being processed for digitization and analysis. This allows the IC to operate at a low power level with a high data throughput for the active sensors. The circuit may be used with many different types of sensors, including photodetectors, capacitance sensors, chemically-sensitive sensors or combinations thereof to provide a capability for recording transient events or for recording data for a predetermined period of time following an event trigger. The sensor readout detector circuit has applications for portable or satellite-based sensor systems. 6 figs.

  4. Sensor readout detector circuit

    DOEpatents

    Chu, Dahlon D.; Thelen, Jr., Donald C.

    1998-01-01

    A sensor readout detector circuit is disclosed that is capable of detecting sensor signals down to a few nanoamperes or less in a high (microampere) background noise level. The circuit operates at a very low standby power level and is triggerable by a sensor event signal that is above a predetermined threshold level. A plurality of sensor readout detector circuits can be formed on a substrate as an integrated circuit (IC). These circuits can operate to process data from an array of sensors in parallel, with only data from active sensors being processed for digitization and analysis. This allows the IC to operate at a low power level with a high data throughput for the active sensors. The circuit may be used with many different types of sensors, including photodetectors, capacitance sensors, chemically-sensitive sensors or combinations thereof to provide a capability for recording transient events or for recording data for a predetermined period of time following an event trigger. The sensor readout detector circuit has applications for portable or satellite-based sensor systems.

  5. Sensors for Entertainment

    PubMed Central

    Lamberti, Fabrizio; Sanna, Andrea; Rokne, Jon

    2016-01-01

    Sensors are becoming ubiquitous in all areas of science, technology, and society. In this Special Issue on “Sensors for Entertainment”, developments in progress and the current state of application scenarios for sensors in the field of entertainment is explored. PMID:27428981

  6. SALAD helicopter integrated sensor

    SciTech Connect

    Soo Hoo, M.S.

    1988-08-01

    The theory and operation of an integrated acoustic and seismic sensor for use with the SALAD helicopter detection system is presented. This sensor incorporates a microphone, geophone, acoustic preamplifier, and tamper indicating features in a buryable, compact aluminum package. This sensor is intended for deployment within a pre-selected, controlled media.

  7. Automotive vehicle sensors

    SciTech Connect

    Sheen, S.H.; Raptis, A.C.; Moscynski, M.J.

    1995-09-01

    This report is an introduction to the field of automotive vehicle sensors. It contains a prototype data base for companies working in automotive vehicle sensors, as well as a prototype data base for automotive vehicle sensors. A market analysis is also included.

  8. Generic EO sensor simulator

    NASA Astrophysics Data System (ADS)

    Akerman, Alexander, III; Hoffman, George A., Jr.

    1988-01-01

    A generic sensor simulation has been developed which emulates the imagery produced by closed circuit television sensors. Applications to date include a monochromatic vidicon and a color CCD camera. The core software program was extracted from the MARSAM II model embedded within the Avionics Laboratory Sensor Performance Model (ALSPM).

  9. High temperature sensor

    DOEpatents

    Tokarz, Richard D.

    1982-01-01

    A high temperature sensor includes a pair of electrical conductors separated by a mass of electrical insulating material. The insulating material has a measurable resistivity within the sensor that changes in relation to the temperature of the insulating material within a high temperature range (1,000 to 2,000 K.). When required, the sensor can be encased within a ceramic protective coating.

  10. Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

    PubMed

    Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap

    2016-04-01

    African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands.

  11. Heat Perception and Aversive Learning in Honey Bees: Putative Involvement of the Thermal/Chemical Sensor AmHsTRPA

    PubMed Central

    Junca, Pierre; Sandoz, Jean-Christophe

    2015-01-01

    The recent development of the olfactory conditioning of the sting extension response (SER) has provided new insights into the mechanisms of aversive learning in honeybees. Until now, very little information has been gained concerning US detection and perception. In the initial version of SER conditioning, bees learned to associate an odor CS with an electric shock US. Recently, we proposed a modified version of SER conditioning, in which thermal stimulation with a heated probe is used as US. This procedure has the advantage of allowing topical US applications virtually everywhere on the honeybee body. In this study, we made use of this possibility and mapped thermal responsiveness on the honeybee body, by measuring workers' SER after applying heat on 41 different structures. We then show that bees can learn the CS-US association even when the heat US is applied on body structures that are not prominent sensory organs, here the vertex (back of the head) and the ventral abdomen. Next, we used a neuropharmalogical approach to evaluate the potential role of a recently described Transient Receptor Potential (TRP) channel, HsTRPA, on peripheral heat detection by bees. First, we applied HsTRPA activators to assess if such activation is sufficient for triggering SER. Second, we injected HsTRPA inhibitors to ask whether interfering with this TRP channel affects SER triggered by heat. These experiments suggest that HsTRPA may be involved in heat detection by bees, and represent a potential peripheral detection system in thermal SER conditioning. PMID:26635613

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  13. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

  14. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    PubMed Central

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  15. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

    PubMed

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-06-12

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

  16. Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor.

    PubMed

    Fong, Desiree H; Xiong, Bing; Hwang, Jiyoung; Berghuis, Albert M

    2011-05-09

    Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

  17. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  18. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  19. Characterization of protein kinases from Blepharisma intermedium.

    PubMed

    Beyer, J

    1975-12-01

    Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.

  20. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... cytoskeleton), gene activity (expression), and protein production and modification. Most MVK gene mutations that cause mevalonate kinase ... What are the different ways in which a genetic condition can be inherited? More about Inheriting Genetic ...

  1. Virtual Sensor Test Instrumentation

    NASA Technical Reports Server (NTRS)

    Wang, Roy

    2011-01-01

    Virtual Sensor Test Instrumentation is based on the concept of smart sensor technology for testing with intelligence needed to perform sell-diagnosis of health, and to participate in a hierarchy of health determination at sensor, process, and system levels. A virtual sensor test instrumentation consists of five elements: (1) a common sensor interface, (2) microprocessor, (3) wireless interface, (4) signal conditioning and ADC/DAC (analog-to-digital conversion/ digital-to-analog conversion), and (5) onboard EEPROM (electrically erasable programmable read-only memory) for metadata storage and executable software to create powerful, scalable, reconfigurable, and reliable embedded and distributed test instruments. In order to maximize the efficient data conversion through the smart sensor node, plug-and-play functionality is required to interface with traditional sensors to enhance their identity and capabilities for data processing and communications. Virtual sensor test instrumentation can be accessible wirelessly via a Network Capable Application Processor (NCAP) or a Smart Transducer Interlace Module (STIM) that may be managed under real-time rule engines for mission-critical applications. The transducer senses the physical quantity being measured and converts it into an electrical signal. The signal is fed to an A/D converter, and is ready for use by the processor to execute functional transformation based on the sensor characteristics stored in a Transducer Electronic Data Sheet (TEDS). Virtual sensor test instrumentation is built upon an open-system architecture with standardized protocol modules/stacks to interface with industry standards and commonly used software. One major benefit for deploying the virtual sensor test instrumentation is the ability, through a plug-and-play common interface, to convert raw sensor data in either analog or digital form, to an IEEE 1451 standard-based smart sensor, which has instructions to program sensors for a wide variety of

  2. A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

    PubMed

    Ota, Hiroko; Sakasegawa, Shin-Ichi; Yasuda, Yuko; Imamura, Shigeyuki; Tamura, Tomohiro

    2008-12-01

    The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

  3. A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum.

    PubMed

    Duan, Yabing; Ge, Changyan; Liu, Shengming; Wang, Jianxin; Zhou, Mingguo

    2013-09-01

    Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.

  4. A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum.

    PubMed

    Duan, Yabing; Ge, Changyan; Liu, Shengming; Wang, Jianxin; Zhou, Mingguo

    2013-09-01

    Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia. PMID:23724858

  5. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues

    PubMed Central

    Mann, Thomas H.; Seth Childers, W.; Blair, Jimmy A.; Eckart, Michael R.; Shapiro, Lucy

    2016-01-01

    All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation. PMID:27117914

  6. C. elegans S6K Mutants Require a Creatine Kinase-Like Effector for Lifespan Extension

    PubMed Central

    McQuary, Philip R.; Liao, Chen-Yu; Chang, Jessica T.; Kumsta, Caroline; She, Xingyu; Davis, Andrew; Chu, Chu-Chiao; Gelino, Sara; Gomez-Amaro, Rafael L.; Petrascheck, Michael; Brill, Laurence M.; Ladiges, Warren C.; Kennedy, Brian K.; Hansen, Malene

    2016-01-01

    Deficiency of S6 kinase (S6K) extends the lifespan of multiple species, but the underlying mechanisms are unclear. To discover potential effectors of S6K-mediated longevity, we performed a proteomics analysis of long-lived rsks-1/S6K C. elegans mutants compared to wild-type animals. We identified the arginine kinase ARGK-1 as the most significantly enriched protein in rsks-1/S6K mutants. ARGK-1 is an ortholog of mammalian creatine kinase, which maintains cellular ATP levels. We found that argk-1 is a selective effector of rsks-1/S6K-mediated longevity, and overexpression of ARGK-1 extends C. elegans lifespan, in part by activating the energy sensor AAK-2/AMPK. argk-1 is also required for the reduced body size and increased stress resistance observed in rsks-1/S6K mutants. Finally, creatine kinase levels are increased in the brains of S6K1 knockout mice. Our study identifies ARGK-1 as a longevity effector in C. elegans with reduced RSKS-1/S6K levels. PMID:26923601

  7. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.

    PubMed

    Rininsland, Frauke; Xia, Wensheng; Wittenburg, Shannon; Shi, Xiaobo; Stankewicz, Casey; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-10-26

    An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation. PMID:15494445

  8. Structural connectivity patterns associated with the putative visual word form area and children's reading ability.

    PubMed

    Fan, Qiuyun; Anderson, Adam W; Davis, Nicole; Cutting, Laurie E

    2014-10-24

    With the advent of neuroimaging techniques, especially functional MRI (fMRI), studies have mapped brain regions that are associated with good and poor reading, most centrally a region within the left occipito-temporal/fusiform region (L-OT/F) often referred to as the visual word form area (VWFA). Despite an abundance of fMRI studies of the putative VWFA, research about its structural connectivity has just started. Provided that the putative VWFA may be connected to distributed regions in the brain, it remains unclear how this network is engaged in constituting a well-tuned reading circuitry in the brain. Here we used diffusion MRI to study the structural connectivity patterns of the putative VWFA and surrounding areas within the L-OT/F in children with typically developing (TD) reading ability and with word recognition deficits (WRD; sometimes referred to as dyslexia). We found that L-OT/F connectivity varied along a posterior-anterior gradient, with specific structural connectivity patterns related to reading ability in the ROIs centered upon the putative VWFA. Findings suggest that the architecture of the putative VWFA connectivity is fundamentally different between TD and WRD, with TD showing greater connectivity to linguistic regions than WRD, and WRD showing greater connectivity to visual and parahippocampal regions than TD. Findings thus reveal clear structural abnormalities underlying the functional abnormalities in the putative VWFA in WRD.

  9. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  10. Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

    PubMed Central

    Müller, André C.; Giambruno, Roberto; Weißer, Juliane; Májek, Peter; Hofer, Alexandre; Bigenzahn, Johannes W.; Superti-Furga, Giulio; Jessen, Henning J.; Bennett, Keiryn L.

    2016-01-01

    Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates. PMID:27346722

  11. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  12. Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

    PubMed Central

    Matsunaga, Yohei; Qadota, Hiroshi; Furukawa, Miho; Choe, Heejoo (Helen); Benian, Guy M.

    2015-01-01

    In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin. PMID:25851606

  13. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  14. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  15. Multifuctional integrated sensors (MFISES).

    SciTech Connect

    Homeijer, Brian D.; Roozeboom, Clifton

    2015-10-01

    Many emerging IoT applications require sensing of multiple physical and environmental parameters for: completeness of information, measurement validation, unexpected demands, improved performance. For example, a typical outdoor weather station measures temperature, humidity, barometric pressure, light intensity, rainfall, wind speed and direction. Existing sensor technologies do not directly address the demand for cost, size, and power reduction in multi-paramater sensing applications. Industry sensor manufacturers have developed integrated sensor systems for inertial measurements that combine accelerometers, gyroscopes, and magnetometers, but do not address environmental sensing functionality. In existing research literature, a technology gap exists between the functionality of MEMS sensors and the real world applications of the sensors systems.

  16. Combustion pressure sensor arrangement

    SciTech Connect

    Sawamoto, K.; Nagaishi, H.; Takeuchi, K.

    1986-07-29

    A combustion pressure sensor arrangement in an internal combustion engine having a cylinder head, comprising: a plug seating formed in the cylinder head; an annular pressure sensor; an ignition plug screwed into the cylinder head in such a manner that the pressure sensor is clamped between the ignition plug and the plug seating; an ignition plug accommodation hole formed in the cylinder head for accommodating therein the ignition plug; and a guide sleeve joined at one end thereof to the outer periphery of the pressure sensor and fitted in the ignition plug accommodation hole, wherein the one end of the guide sleeve is fitted on the outer periphery of the pressure sensor.

  17. Hepatitis C Virus NS5A Inhibits Mixed Lineage Kinase 3 to Block Apoptosis*

    PubMed Central

    Amako, Yutaka; Igloi, Zsofia; Mankouri, Jamel; Kazlauskas, Arunas; Saksela, Kalle; Dallas, Mark; Peers, Chris; Harris, Mark

    2013-01-01

    Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K+ channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K+ homeostasis. PMID:23857585

  18. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

    Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

  19. Sensor mount assemblies and sensor assemblies

    DOEpatents

    Miller, David H.

    2012-04-10

    Sensor mount assemblies and sensor assemblies are provided. In an embodiment, by way of example only, a sensor mount assembly includes a busbar, a main body, a backing surface, and a first finger. The busbar has a first end and a second end. The main body is overmolded onto the busbar. The backing surface extends radially outwardly relative to the main body. The first finger extends axially from the backing surface, and the first finger has a first end, a second end, and a tooth. The first end of the first finger is disposed on the backing surface, and the tooth is formed on the second end of the first finger.

  20. Distributed sensor networks

    NASA Astrophysics Data System (ADS)

    Lacoss, Richard T.

    1986-09-01

    The Distributed Sensor Networks (DSN) program was aimed at developing distributed target surveillance and tracking methods for systems employing multiple spatially distributed sensors and processing resources. Such systems would be made up of sensors, data bases, and processors distributed throughout an area and interconnected by an appropriate digital data communication system. The hypothesis of the program was that through netting and distributed processing, the information from many sensors could be combined to yield effective surveillance systems. The overall concept called for a mix of sensor types as well as geographically distributed sensors. Surveillance and tracking of low-flying aircraft with ground-based acoustic and imaging sensors was used to develop and evaluate DSN concepts in the light of a specific problem. An experimental DSN testbed system was developed and has been used to test and demonstrate DSN techniques. Small arrays of microphones providing directional information were employed as acoustic sensors and visible TV cameras were used as imaging sensors in the testbed system. The primary accomplishment during this final report period was the demonstration of distributed real time tracking using both TV and acoustic sensors. Tracking was implemented as a geographically decentralized confederacy of autonomous cooperating nodes. Thus the feasibility of this organization has been established for a DSN system containing multiple sensor types as well as distributed nodes.