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Sample records for putative sensor kinase

  1. 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

  2. Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris.

    PubMed

    Li, Rui-Fang; Lu, Guang-Tao; Li, Lei; Su, Hui-Zhao; Feng, Guo-fang; Chen, Ya; He, Yong-Qiang; Jiang, Bo-Le; Tang, Dong-Jie; Tang, Ji-Liang

    2014-07-01

    The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.

  3. A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii.

    PubMed

    Rather, P N; Paradise, M R; Parojcic, M M; Patel, S

    1998-06-01

    A recessive mutation, aarG1, has been identified that resulted in an 18-fold increase in the expression of beta-galactosidase from an aac(2')-lacZ fusion. Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2')-Ia. The effects of aarG1 on aac(2')-Ia expression were mediated by aarP-dependent and -independent mechanisms. The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance. This Mar phenotype also resulted from aarP-dependent and -independent mechanisms. Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem. The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ. The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase. The AarG product was functionally similar to PhoQ, as it was able to restore wild-type levels of maganin resistance to a Salmonella typhimurium phoQ mutant. However, expression of the aarP and aac(2')-Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.

  4. 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.

  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. 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

  7. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  8. 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.

  9. 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

  10. 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...

  11. Phosphatidylinositol-3-kinase as a putative target for anticancer action of clotrimazole.

    PubMed

    Furtado, Cristiane M; Marcondes, Mariah C; Carvalho, Renato S; Sola-Penna, Mauro; Zancan, Patricia

    2015-05-01

    Clotrimazole (CTZ) has been proposed as an antitumoral agent because of its properties that inhibit glycolytic enzymes and detach them from the cytoskeleton. However, the broad effects of the drug, e.g., acting on different enzymes and pathways, indicate that CTZ might also affect several signaling pathways. In this study, we show that CTZ interferes with the human breast cancer cell line MCF-7 after a short incubation period (4 h), thereby diminishing cell viability, promoting apoptosis, depolarizing mitochondria, inhibiting key glycolytic regulatory enzymes, decreasing the intracellular ATP content, and permeating plasma membranes. CTZ treatment also interferes with autophagy. Moreover, when the incubation is performed under hypoxic conditions, certain effects of CTZ are enhanced, such as phosphatidylinositol-3-phosphate kinase (PI3K), which is inhibited upon CTZ treatment; this inhibition is potentiated under hypoxia. CTZ-induced PI3K inhibition is not caused by upstream effects of CTZ because the drug does not affect the interaction of the PI3K regulatory subunit and the insulin receptor substrate (IRS)-1. Additionally, CTZ directly inhibits human purified PI3K in a dose-dependent and reversible manner. Pharmacologic and in silico results suggest that CTZ may bind to the PI3K catalytic site. Therefore, we conclude that PI3K is a novel, putative target for the antitumoral effects of CTZ, interfering with autophagy, apoptosis, cell division and viability.

  12. The Neurospora crassa DCC-1 Protein, a Putative Histidine Kinase, Is Required for Normal Sexual and Asexual Development and Carotenogenesis▿

    PubMed Central

    Barba-Ostria, Carlos; Lledías, Fernando; Georgellis, Dimitris

    2011-01-01

    Two-component signaling pathways based on phosphoryl group transfer between histidine kinase and response regulator proteins regulate environmental responses in bacteria, archaea, plants, slime molds, and fungi. Here we characterize a mutant form of DCC-1, a putative histidine kinase encoded by the NCU00939 gene of the filamentous fungus Neurospora crassa. We show that this protein participates in the regulation of processes such as conidiation, perithecial development, and, to a certain degree, carotenogenesis. Furthermore, DCC-1 is suggested to exert its effect by promoting cyclic AMP production, thereby placing this protein within the context of a signaling pathway. PMID:22058142

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

    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.

  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. Characterization of PINK1 (PTEN-induced putative kinase 1) mutations associated with Parkinson disease in mammalian cells and Drosophila.

    PubMed

    Song, Saera; Jang, Seoyeon; Park, Jeehye; Bang, Sunhoe; Choi, Sekyu; Kwon, Kyum-Yil; Zhuang, Xiaoxi; Kim, Eunjoon; Chung, Jongkyeong

    2013-02-22

    Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.

  17. Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis

    PubMed Central

    Contri, Antonella; Brunati, Anna Maria; Trentin, Livio; Cabrelle, Anna; Miorin, Marta; Cesaro, Luca; Pinna, Lorenzo A.; Zambello, Renato; Semenzato, Gianpietro; Donella-Deana, Arianna

    2005-01-01

    B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell–receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target. PMID:15650771

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain and a receiver domain. We have previously demonstrated that, while the Erwinia amylovora rcsC mutant produces more amylovoran than the wild type strain in vitro, the mutant remains avirulent on both immature pea...

  20. 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

    2015-06-09

    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.

  1. 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

  2. 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.

  3. Sphingosine Kinase: A Novel Putative Target for the Prevention of Infection-Triggered Preterm Birth

    PubMed Central

    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

  4. 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.

  5. TodK, a putative histidine protein kinase, regulates timing of fruiting body morphogenesis in Myxococcus xanthus.

    PubMed

    Rasmussen, Anders A; Søgaard-Andersen, Lotte

    2003-09-01

    In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation. These two processes are induced by the cell surface-associated C signal, with aggregation being induced after 6 h and sporulation being induced once cells have completed the aggregation process. We report the identification of TodK, a putative histidine protein kinase of two-component regulatory systems that is important for the correct timing of aggregation and sporulation. Loss of TodK function results in early aggregation and early, as well as increased levels of, sporulation. Transcription of todK decreases 10-fold in response to starvation independently of the stringent response. Loss of TodK function specifically results in increased expression of a subset of C-signal-dependent genes. Accelerated development in a todK mutant depends on the known components in the C-signal transduction pathway. TodK is not important for synthesis of the C signal. From these results we suggest that TodK is part of a signal transduction system which converges on the C-signal transduction pathway to negatively regulate aggregation, sporulation, and the expression of a subset of C-signal-dependent genes. TodK and the SdeK histidine protein kinase, which is part of a signal transduction system that converges on the C-signal transduction pathway to stimulate aggregation, sporulation, and C-signal-dependent gene expression, act in independent genetic pathways. We suggest that the signal transduction pathways defined by TodK and SdeK act in concert with the C-signal transduction pathway to control the timing of aggregation and sporulation.

  6. 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

    2016-09-20

    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.

  7. 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

  8. The histidine kinase CusS senses silver ions through direct binding by its sensor domain

    PubMed Central

    Gudipaty, Swapna A.; McEvoy, Megan M.

    2014-01-01

    The Cus system of Escherichia coli aids in protection of cells from high concentrations of Ag(I) and Cu(I). The histidine kinase CusS of the CusRS two-component system functions as a Ag(I)/Cu(I)-responsive sensor kinase and is essential for induction of the genes encoding the CusCFBA efflux pump. In this study, we have examined the molecular features of the sensor domain of CusS in order to understand how a metal-responsive histidine kinase senses specific metal ions. We find that the predicted periplasmic sensor domain of CusS directly interacts with Ag(I) ions and undergoes a conformational change upon metal binding. Metal binding also enhances the tendency of the domain to dimerize. These findings suggest a model for activation of the histidine kinase through metal binding events in the periplasmic sensor domain. PMID:24948475

  9. A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA.

    PubMed

    Sevvana, Madhumati; Vijayan, Vinesh; Zweckstetter, Markus; Reinelt, Stefan; Madden, Dean R; Herbst-Irmer, Regine; Sheldrick, George M; Bott, Michael; Griesinger, Christian; Becker, Stefan

    2008-03-21

    Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. [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.

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

    PubMed

    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.

  16. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae.

    PubMed Central

    Ramer, S W; Davis, R W

    1993-01-01

    This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways. STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa. Its expression pattern is similar to that of several other protein kinases in the mating response pathway. Deletion of the kinase domain of STE20 causes sterility in both haploid mating types. This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele. A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone. This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12. The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12. Images PMID:8421676

  17. 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

  18. 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

    2016-02-09

    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.

  19. The switch helix: a putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase.

    PubMed

    Moon, Thomas M; Osborne, Brent W; Dostmann, Wolfgang R

    2013-07-01

    For over three decades the isozymes of cGMP-dependent protein kinase (PKG) have been studied using an array of biochemical and biophysical techniques. When compared to its closest cousin, cAMP-dependent protein kinase (PKA), these studies revealed a set of identical domain types, yet containing distinct, sequence-specific features. The recently solved structure of the PKG regulatory domain showed the presence of the switch helix (SW), a novel motif that promotes the formation of a domain-swapped dimer in the asymmetric unit. This dimer is mediated by the interaction of a knob motif on the C-terminal locus of the SW, with a hydrophobic nest on the opposing protomer. This nest sits adjacent to the cGMP binding pocket of the B-site. Priming of this site by cGMP may influence the geometry of the hydrophobic nest. Moreover, this unique interaction may have wide implications for the architecture of the inactive and active forms of PKG. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

  20. 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.

  1. 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.

  2. 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

  3. Regulation of protein kinase Cmu by basic peptides and heparin. Putative role of an acidic domain in the activation of the kinase.

    PubMed

    Gschwendt, M; Johannes, F J; Kittstein, W; Marks, F

    1997-08-15

    Protein kinase Cmu is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCmu have been identified as yet. Moreover, the regulation of PKCmu activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCmu. Here we show that aldolase is phosphorylated by PKCmu in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCmu is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCmu is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms. We postulate that PKCmu contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCmu activity by binding to the acidic domain of the active enzyme.

  4. Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

    PubMed Central

    Liu, Shan-Lu; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway. PMID:12829832

  5. 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

  6. Mutations in Two Putative Phosphorylation Motifs in the Tomato Pollen Receptor Kinase LePRK2 Show Antagonistic Effects on Pollen Tube Length*

    PubMed Central

    Salem, Tamara; Mazzella, Agustina; Barberini, María Laura; Wengier, Diego; Motillo, Viviana; Parisi, Gustavo; Muschietti, Jorge

    2011-01-01

    The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. PMID:21131355

  7. 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.

  8. Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

    PubMed Central

    Goodman, Andrew L.; Merighi, Massimo; Hyodo, Mamoru; Ventre, Isabelle; Filloux, Alain; Lory, Stephen

    2009-01-01

    The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS–GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk. PMID:19171785

  9. 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(-).

  10. 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.

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

    PubMed Central

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

    2017-01-01

    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

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

    PubMed

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

    2015-10-08

    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.

  13. Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    PubMed Central

    Eyüboglu, Banu; Pfister, Karen; Haberer, Georg; Chevalier, David; Fuchs, Angelika; Mayer, Klaus FX; Schneitz, Kay

    2007-01-01

    Background Receptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function. Results We approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF) class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis, and an analysis of the

  14. Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

    PubMed Central

    Yang, C; Kaplan, H B

    1997-01-01

    Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression. PMID:9401035

  15. Genetic analysis of signal integration by the Sinorhizobium meliloti sensor kinase FeuQ.

    PubMed

    VanYperen, Ryan D; Orton, Taylor S; Griffitts, Joel S

    2015-02-01

    Two-component signalling systems allow bacteria to recognize and respond to diverse environmental stimuli. Auxiliary proteins can provide an additional layer of control to these systems. The Sinorhizobium meliloti FeuPQ two-component system is required for symbiotic development and is negatively regulated by the auxiliary small periplasmic protein FeuN. This study explores the mechanistic basis of this regulation. We provide evidence that FeuN directly interacts with the sensor kinase FeuQ. Isolation and characterization of an extensive set of FeuN-insensitive and FeuN-mimicking variants of FeuQ reveal specific FeuQ residues (periplasmic and intracellular) that control the transmission of FeuN-specific signalling information. Similar analysis of the FeuN protein highlights short patches of compatibly charged residues on each protein that probably engage one another, giving rise to the downstream effects on target gene expression. The accumulated evidence suggests that the periplasmic interaction between FeuN and FeuQ introduces an intracellular conformational change in FeuQ, resulting in an increase in its ability to remove phosphate from its cognate response regulator FeuP. These observations underline the complex manner in which membrane-spanning sensor kinases interface with the extracytoplasmic environment and convert that information to changes in intracellular processes.

  16. Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

    PubMed

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Eguchi, Yoko; Yanagihara, Shiho; Edahiro, Keisuke; Inoue, Yuki; Taniguchi, Momoka; Yoshida, Myu; Yamamoto, Kaneyoshi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Utsumi, Ryutaro; Koike, Tohru

    2015-01-01

    Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].

  17. 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.

  18. A putative bifunctional histidine kinase/phosphatase of the HWE family exerts positive and negative control on the Sinorhizobium meliloti general stress response.

    PubMed

    Sauviac, Laurent; Bruand, Claude

    2014-07-01

    The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.

  19. Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants*

    PubMed Central

    He, Cuiwen H.; Xie, Letian X.; Allan, Christopher M.; Tran, UyenPhuong C.; Clarke, Catherine F.

    2014-01-01

    Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. PMID:24406904

  20. Regulation of sialidase production in Clostridium perfringens by the orphan sensor histidine kinase ReeS.

    PubMed

    Hiscox, Thomas J; Harrison, Paul F; Chakravorty, Anjana; Choo, Jocelyn M; Ohtani, Kaori; Shimizu, Tohru; Cheung, Jackie K; Rood, Julian I

    2013-01-01

    Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.

  1. Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase*

    PubMed Central

    Trajtenberg, Felipe; Graña, Martin; Ruétalo, Natalia; Botti, Horacio; Buschiazzo, Alejandro

    2010-01-01

    DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface. PMID:20507988

  2. Regulation of Sialidase Production in Clostridium perfringens by the Orphan Sensor Histidine Kinase ReeS

    PubMed Central

    Hiscox, Thomas J.; Harrison, Paul F.; Chakravorty, Anjana; Choo, Jocelyn M.; Ohtani, Kaori; Shimizu, Tohru

    2013-01-01

    Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens. PMID:24023881

  3. A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

    PubMed

    Du, Jiao; Xu, Qinfeng; Lu, Xiaoquan; Zhang, Chun-yang

    2014-08-19

    Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

  4. 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

  5. PAS kinase as a nutrient sensor in neuroblastoma and hypothalamic cells required for the normal expression and activity of other cellular nutrient and energy sensors.

    PubMed

    Hurtado-Carneiro, Verónica; Roncero, Isabel; Blazquez, Enrique; Alvarez, Elvira; Sanz, Carmen

    2013-12-01

    PAS kinase (PASK) is a nutrient sensor that is highly conserved throughout evolution. PASK-deficient mice reveal a metabolic phenotype similar to that described in S6 kinase-1 S6K1-deficient mice that are protected against obesity. Hypothalamic metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR), play an important role in feeding behavior, the homeostasis of body weight, and energy balance. These sensors respond to changes in nutrient levels in the hypothalamic areas involved in feeding behavior and in neuroblastoma N2A cells, and we have recently reported that those effects are modulated by the anorexigenic peptide glucagon-like peptide-1 (GLP-1). Here, we identified PASK in both N2A cells and rat VMH and LH areas and found that its expression is regulated by glucose and GLP-1. High levels of glucose decreased Pask gene expression. Furthermore, PASK-silenced N2A cells record an impaired response by the AMPK and mTOR/S6K1 pathways to changes in glucose levels. Likewise, GLP-1 effect on the activity of AMPK, S6K1, and other intermediaries of both pathways and the regulatory role at the level of gene expression were also blocked in PASK-silenced cells. The absence of response to low glucose concentrations in PASK-silenced cells correlates with increased ATP content, low expression of mRNA coding for AMPK upstream kinase LKB1, and enhanced activation of S6K1. Our findings indicate that, at least in N2A cells, PASK is a key kinase in GLP-1 actions and exerts a coordinated response with the other metabolic sensors, suggesting that PASK might play an important role in feeding behavior.

  6. 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.

  7. Alkali metals in addition to acidic pH activate the EvgS histidine kinase sensor in Escherichia coli.

    PubMed

    Eguchi, Yoko; Utsumi, Ryutaro

    2014-09-01

    Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception.

  8. CitA (citrate) and DcuS (C4-dicarboxylate) sensor kinases in thermophilic Geobacillus kaustophilus and Geobacillus thermodenitrificans.

    PubMed

    Graf, Sabrina; Broll, Constanze; Wissig, Juliane; Strecker, Alexander; Parowatkin, Maria; Unden, Gottfried

    2016-01-01

    The thermophilic Geobacillus thermodenitrificans and Geobacillus kaustophilus are able to use citrate or C4-dicarboxylates like fumarate or succinate as the substrates for growth. The genomes of the sequenced Geobacillus strains (nine strains) each encoded a two-component system of the CitA family. The sensor kinase of G. thermodenitrificans (termed CitAGt) was able to replace CitA of Escherichia coli (CitAEc) in a heterologous complementation assay restoring expression of the CitAEc-dependent citC-lacZ reporter gene and anaerobic growth on citrate. Complementation was specific for citrate. The sensor kinase of G. kaustophilus (termed DcuSGk) was able to replace DcuSEc of E. coli. It responded in the heterologous expression system to C4-dicarboxylates and to citrate, suggesting that DcuSGk is, like DcuSEc, a C4-dicarboxylate sensor with a side-activity for citrate. DcuSGk, unlike the homologous DctS from Bacillus subtilis, required no binding protein for function in the complementation assay. Thus, the thermophilic G. thermodenitrificans and G. kaustophilus contain citrate and C4-dicarboxylate sensor kinases of the CitA and DcuS type, respectively, and retain function and substrate specificity under mesophilic growth conditions in E. coli.

  9. [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.

  10. Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia*

    PubMed Central

    Khodai-Kalaki, Maryam; Aubert, Daniel F.; Valvano, Miguel A.

    2013-01-01

    AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion, and protease secretion. Here we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine 245 and aspartic acid 536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator. PMID:24014026

  11. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants.

    PubMed

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

    2012-08-01

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

  12. 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

  13. 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

  14. 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.

  15. Transposon-mediated enhancer detection reveals the location, morphology and development of the cupular organs, which are putative hydrodynamic sensors, in the ascidian Ciona intestinalis.

    PubMed

    Ohta, Naoyuki; Horie, Takeo; Satoh, Nori; Sasakura, Yasunori

    2010-11-01

    The adult of the ascidian Ciona intestinalis has cupular organs, i.e., putative hydrodynamic sensors, at the atrial epithelium. The cupular organ consists of support cells and sensory neurons, and it extends a gelatinous matrix, known as a cupula, toward the atrial cavity. These characteristics are shared with sensory hair cells in the vertebrate inner ear and lateral line neuromasts in fish and amphibians, which suggests an evolutionary link between the cupular organ and these vertebrate hydrodynamic sensors. In the present study, we have isolated and investigated two transposon-mediated enhancer detection lines that showed GFP expression in support cells of the cupular organs. Using the enhancer detection lines and neuron marker transgenic lines, we describe the position, morphology, and development of the cupular organs. Cupular organs were found at the atrial epithelium, but not in the branchial epithelium. We found that cupular organs are also present along the dorsal fold and the gonoducts. The cells lining the pre-atrial opening in juveniles are presumably precursor cells of the cupular organ. To our knowledge, the present study is the first precise description of the ascidian cupular organ, providing evidence that may help to resolve discrepancies among previous studies on the organ.

  16. 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

  17. Light-regulated expression of sensor histidine kinase CKI1 controls cytokinin-related development.

    PubMed

    Dobisova, Tereza; Hrdinova, Vendula; Cuesta, Candela; Michlickova, Sarka; Urbankova, Ivana; Hejatkova, Romana; Žádníková, Petra; Pernisova, Marketa; Benkova, Eva; Hejatko, Jan

    2017-03-14

    In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The crosstalk between cytokinin response and light is known for a long time. However, the molecular mechanism underlying the interactionbetween light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT-1 (CKI1), encoding the constitutively active sensor histidine kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE 1 (HY1) which encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertiblephytochromes. Our analysis confirmed the light-dependent regulation oftheCKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlatewithmisregulation of MSP signaling, changedcytokinin sensitivity and developmental aberrations,previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

  18. Role of the energy sensor AMP-activated protein kinase in renal physiology and disease.

    PubMed

    Hallows, Kenneth R; Mount, Peter F; Pastor-Soler, Núria M; Power, David A

    2010-05-01

    The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na(+)-K(+)-2Cl(-) cotransporter (NKCC), and the vacuolar H(+)-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.

  19. Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis.

    PubMed

    Chourashi, Rhishita; Mondal, Moumita; Sinha, Ritam; Debnath, Anusuya; Das, Suman; Koley, Hemanta; Chatterjee, Nabendu Sekhar

    2016-12-01

    Vibrio cholera survival in an aquatic environment depends on chitin utilization pathway that requires two factors, chitin binding protein and chitinases. The chitinases and the chitin utilization pathway are regulated by a two-component sensor histidine kinase ChiS in V. cholerae. In recent studies these two factors are also shown to be involved in V. cholerae pathogenesis. However, the role played by their upstream regulator ChiS in pathogenesis is yet to be known. In this study, we investigated the activation of ChiS in presence of mucin and its functional role in pathogenesis. We found ChiS is activated in mucin supplemented media. The isogenic chiS mutant (ChiS(-)) showed less growth compared to the wild type strain (ChiS(+)) in the presence of mucin supplemented media. The ChiS(-) strain also showed highly retarded motility as well as mucin layer penetration in vitro. Our result also showed that ChiS was important for adherence and survival in HT-29 cell. These observations indicate that ChiS is activated in presence of intestinal mucin and subsequently switch on the chitin utilization pathway. In animal models, our results also supported the in vitro observation. We found reduced fluid accumulation and colonization during infection with ChiS(-) strain. We also found ChiS(-) mutant with reduced expression of ctxA, toxT and tcpA. The cumulative effect of these events made V. cholerae ChiS(-) strain hypovirulent. Hence, we propose that ChiS plays a vital role in V. cholerae pathogenesis.

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

    PubMed

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

    2015-10-07

    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.

  1. 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.

  2. The Extracellular Domain of the Saccharomyces cerevisiae Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity

    PubMed Central

    Ostrander, Darin B.; Gorman, Jessica A.

    1999-01-01

    The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Δ strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (ΔTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the ΔTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD. PMID:10198019

  3. The lipid kinase PI5P4Kβ is an intracellular GTP sensor for metabolism and tumorigenesis

    PubMed Central

    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.

    2016-01-01

    Summary 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

  4. Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor

    PubMed Central

    Salvi, Michele; Schomburg, Benjamin; Giller, Karin; Graf, Sabrina; Unden, Gottfried; Becker, Stefan; Lange, Adam; Griesinger, Christian

    2017-01-01

    Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid- and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal β-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain. PMID:28265100

  5. Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor.

    PubMed

    Salvi, Michele; Schomburg, Benjamin; Giller, Karin; Graf, Sabrina; Unden, Gottfried; Becker, Stefan; Lange, Adam; Griesinger, Christian

    2017-03-21

    Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid- and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal β-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain.

  6. The Sinorhizobium meliloti sensor histidine kinase CbrA contributes to free-living cell cycle regulation.

    PubMed

    Sadowski, Craig S; Wilson, Daniel; Schallies, Karla B; Walker, Graham; Gibson, Katherine E

    2013-08-01

    Sinorhizobium meliloti is alternately capable of colonizing the soil as a free-living bacterium or establishing a chronic intracellular infection with its legume host for the purpose of nitrogen fixation. We previously identified the S. meliloti two-component sensor histidine kinase CbrA as playing an important role in regulating exopolysaccharide production, flagellar motility and symbiosis. Phylogenetic analysis of CbrA has highlighted its evolutionary relatedness to the Caulobacter crescentus sensor histidine kinases PleC and DivJ, which are involved in CtrA-dependent cell cycle regulation through the shared response regulator DivK. We therefore became interested in testing whether CbrA plays a role in regulating S. meliloti cell cycle processes. We find the loss of cbrA results in filamentous cell growth accompanied by cells that contain an aberrant genome complement, indicating CbrA plays a role in regulating cell division and possibly DNA segregation. S. meliloti DivK localizes to the old cell pole during distinct phases of the cell cycle in a phosphorylation-dependent manner. Loss of cbrA results in a significantly decreased rate of DivK polar localization when compared with the wild-type, suggesting CbrA helps regulate cell cycle processes by modulating DivK phosphorylation status as a kinase. Consistent with a presumptive decrease in DivK phosphorylation and activity, we also find the steady-state level of CtrA increased in cbrA mutants. Our data therefore demonstrate that CbrA contributes to free-living cell cycle regulation, which in light of its requirement for symbiosis, points to the potential importance of cell cycle regulation for establishing an effective host interaction.

  7. Blue-light-activated histidine kinases: two-component sensors in bacteria.

    PubMed

    Swartz, Trevor E; Tseng, Tong-Seung; Frederickson, Marcus A; Paris, Gastón; Comerci, Diego J; Rajashekara, Gireesh; Kim, Jung-Gun; Mudgett, Mary Beth; Splitter, Gary A; Ugalde, Rodolfo A; Goldbaum, Fernando A; Briggs, Winslow R; Bogomolni, Roberto A

    2007-08-24

    Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

  8. Peroxide Sensors for the Fission Yeast Stress-activated Mitogen-activated Protein Kinase Pathway

    PubMed Central

    Buck, Vicky; Quinn, Janet; Pino, Teresa Soto; Martin, Humberto; Saldanha, Jose; Makino, Kozo; Morgan, Brian A.; Millar, Jonathan B.A.

    2001-01-01

    The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast. PMID:11179424

  9. Characterization of the PAS domain in the sensor-kinase BvgS: mechanical role in signal transmission

    PubMed Central

    2013-01-01

    Background In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37°C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap domains and a cytoplasmic PAS domain that precedes the kinase domain, which is followed by additional phosphotransfer domains. PAS domains are small, ubiquitous sensing or regulatory domains. The function of the PAS domain in BvgS remains unknown. Results We showed that recombinant BvgS PAS proteins form dimers that are stabilized by α helical regions flanking the PAS core. A structural model of the PAS domain dimer was built and probed by site-directed mutagenesis and by biochemical and functional analyses. Although we found no ligands for the PAS domain cavity, its integrity is required for signaling. We also showed that the structural stability of the PAS core and its proper coupling to its flanking N- and C-terminal α helices are crucial for BvgS activity. Conclusions We propose that a major function of the BvgS PAS domain is to maintain conformational signals arising from mechanical strain generated by the periplasmic domain. The tight structure of the PAS core and its connections with the upstream and downstream helices ensure signaling to the kinase domain, which determines BvgS activity. Many mild substitutions that map to the PAS domain keep BvgS active but make it unresponsive to negative modulators, supporting that modulation increases conformational strain

  10. Mutations in CG8878, a Novel Putative Protein Kinase, Enhance P Element Dependent Silencing (PDS) and Position Effect Variegation (PEV) in Drosophila melanogaster

    PubMed Central

    McCracken, Allen; Locke, John

    2014-01-01

    Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3–7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at wm4, indicating a previously unknown common silencing mechanism between the two. PMID:24614804

  11. 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 ...

  12. 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...

  13. Mutational analysis of the residue at position 48 in the Salmonella enterica Serovar Typhimurium PhoQ sensor kinase.

    PubMed

    Sanowar, Sarah; Martel, Alexandre; Moual, Hervé Le

    2003-03-01

    The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg(2+) depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg(2+), in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states.

  14. Identification of a Ubiquinone-binding Site That Affects Autophosphorylation of the Sensor Kinase RegB*S

    PubMed Central

    Swem, Lee R.; Gong, Xing; Yu, Chang-An; Bauer, Carl E.

    2009-01-01

    Rhodobacter capsulatus regulates many metabolic processes in response to the level of environmental oxygen and the energy state of the cell. One of the key global redox regulators of the cell’s metabolic physiology is the sensor kinase RegB that controls the synthesis of numerous energy generation and utilization processes. In this study, we have succeeded in purifying full-length RegB containing six transmembrane-spanning elements. Exogenous addition of excess oxidized coenzyme Q1 is capable of inhibiting RegB autophosphorylation ~6-fold. However, the addition of reduced coenzyme Q1 exhibits no inhibitory effect on kinase activity. A ubiquinone-binding site, as defined by azidoquinone photo affinity cross-linking, was determined to lie within a periplasmic loop between transmembrane helices 3 and 4 that contains a fully conserved heptapeptide sequence of GGXXNPF. Mutation of the phenylalanine in this heptapeptide renders RegB constitutively active in vivo, indicating that this domain is responsible for sensing the redox state of the ubiquinone pool and subsequently controlling RegB autophosphorylation. PMID:16407278

  15. Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.

    PubMed

    May, Randal; Riehl, Terrence E; Hunt, Clayton; Sureban, Sripathi M; Anant, Shrikant; Houchen, Courtney W

    2008-03-01

    In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase-like-1 (DCAMKL-1) to examine radiation-induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL-1 expression. Immunoreactive DCAMKL-1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL-1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL-1-positive cells. We also observed stem cell apoptosis and mitotic DCAMKL-1-expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL-1-expressing cells were negative for proliferating cell nuclear antigen and nuclear beta-catenin in normal-appearing intestine. However, beta-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of beta-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents.

  16. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  17. 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

  18. Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum

    PubMed Central

    He, Kuang; Dragnea, Vladimira

    2015-01-01

    ABSTRACT Rhodospirillum centenum forms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3 signaling cascade. The Che3 cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3 and CheR3, two CheW3 linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3 cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3 does not serve as a phosphor donor for CheY3; instead, CheA3 inhibits a CheS3→CheY3 two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3 is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro, the phosphorylation level of CheS3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3. PMID:25944862

  19. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3.

    PubMed

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan Dw

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

  20. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

    PubMed Central

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan DW

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed. PMID:24637704

  1. Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry

    PubMed Central

    Sulmann, Stefan; Kussrow, Amanda; Bornhop, Darryl J.; Koch, Karl-Wilhelm

    2017-01-01

    Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase–activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels. PMID:28361875

  2. Genetic and mechanistic analyses of the periplasmic domain of the enterohemorrhagic E. coli (EHEC) QseC histidine sensor kinase.

    PubMed

    Parker, Christopher T; Russell, Regan; Njoroge, Jacqueline W; Jimenez, Angel G; Taussig, Ron; Sperandio, Vanessa

    2017-01-30

    The histidine sensor kinase (HK) QseC, senses autoinducer-3 (AI-3), and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate expression of virulence genes in enterohemorrhagic E. coli (EHEC). The QseB, QseF and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain, and also aligned these sequences from 12 different species to identify the most conserved amino acids. We selected eight conserved aminoacids in all of these QseC homologs. These QseC site directed mutants were expressed and still able to autophosphorylate, albeit four mutants depicted increased basal level of phosphorylation. These mutants have differential flagella and motility, LEE and Shiga toxin expression phenotypes. We selected four mutants for more in depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE and QseF. This suggests that these mutations in the periplasmic sensing domain affected the downstream of the QseC signaling cascade, and therefore, can influence which pathway QseC regulates.

  3. Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

    PubMed

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Tamaki, Keisuke; Iemata, Mika; Warren, Noel; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G; Pangalos, Menelas N; Moss, Stephen J

    2007-01-18

    GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

  4. 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

  5. Phospho-dependent functional modulation of GABAB receptors by the metabolic sensor AMP-dependent protein kinase

    PubMed Central

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Warren, Noel; Tamaki, Keisuke; Iemata, Mika; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G.; Pangalos, Menelas N.; Moss., Stephen J.

    2007-01-01

    GABAB receptors are heterodimeric G-protein coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly-rectifying K+ channels (GIRKs) and inhibiting Ca2+ channels. We demonstrate here that GABAB receptors are intimately associated with 5’AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5’AMP concentration caused by enhanced metabolic activity, anoxia or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABAB receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a novel neuroprotective mechanism, which under conditions of metabolic stress or after ischemia increases GABAB receptor function to reduce excitotoxicity and thereby promoting neuronal survival. PMID:17224405

  6. The cytoplasmic PASC domain of the sensor kinase DcuS of Escherichia coli: role in signal transduction, dimer formation, and DctA interaction

    PubMed Central

    Monzel, Christian; Degreif-Dünnwald, Pia; Gröpper, Christina; Griesinger, Christian; Unden, Gottfried

    2013-01-01

    The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC/PASC, nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC. Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology. PMID:24039243

  7. The Structure of the C-Terminal Domain of the Protein Kinase AtSOS2 Bound to the Calcium Sensor AtSOS3

    PubMed Central

    Sánchez-Barrena, María José; Fujii, Hiroaki; Angulo, Ivan; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

    2011-01-01

    SUMMARY The plant SOS2 family of protein kinases and their interacting activators, the SOS3 family of calcium-binding proteins, function together in decoding calcium signals elicited by different environmental stimuli. SOS2 is activated by Ca-SOS3 and subsequently phosphorylates the ion transporter SOS1 to bring about cellular ion homeostasis under salt stress. In addition to possessing the kinase activity, members of the SOS2 family of protein kinases can bind to protein phosphatase 2Cs. The crystal structure of the binary complex of Ca-SOS3 with the C-terminal regulatory moiety of SOS2 resolves central questions regarding the dual function of SOS2 as a kinase and a phosphatase-binding protein. A comparison with the structure of unbound SOS3 reveals the basis of the molecular function of this family of kinases and their interacting calcium sensors. Furthermore, our study suggests that the structure of the phosphatase-interaction domain of SOS2 defines a scaffold module conserved from yeast to human. PMID:17499048

  8. Crystal structures of C4-dicarboxylate ligand complexes with sensor domains of histidine kinases DcuS and DctB.

    PubMed

    Cheung, Jonah; Hendrickson, Wayne A

    2008-10-31

    Two-component signaling systems allow bacteria to adapt to changing environments. Typically, a chemical or other stimulus is detected by the periplasmic sensor domain of a transmembrane histidine kinase sensor, which in turn relays a signal through a phosphotransfer cascade to the cognate cytoplasmic response regulator. Such systems lead ultimately to changes in gene expression or cell motility. Mechanisms of ligand binding and signal transduction through the cell membrane in histidine kinases are not fully understood. In an effort to further understand such processes, we have solved the crystal structures of the periplasmic sensor domains of Escherichia coli DcuS and of Vibrio cholerae DctB in complex with the respective cognate ligands, malate and succinate. Both proteins are involved in the regulation of the transport and metabolism of C(4)-dicarboxylates, but they are not highly related by sequence similarity. Our work reveals that despite disparate sizes, both structures contain a similar characteristic alpha/beta PDC (PhoQ-DcuS-CitA) sensor-domain fold and display similar modes of ligand binding, suggesting similar mechanisms of function.

  9. The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris.

    PubMed

    An, Shi-Qi; Allan, John H; McCarthy, Yvonne; Febrer, Melanie; Dow, J Maxwell; Ryan, Robert P

    2014-05-01

    A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4 μM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle.

  10. The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli.

    PubMed

    Pappalardo, Lucia; Janausch, Ingo G; Vijayan, Vinesh; Zientz, Eva; Junker, Jochen; Peti, Wolfgang; Zweckstetter, Markus; Unden, Gottfried; Griesinger, Christian

    2003-10-03

    The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.

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

    PubMed Central

    Kilmury, Sara L. N.

    2016-01-01

    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

  12. A Novel Sensor Kinase-Response Regulator Hybrid Controls Biofilm Formation and Type VI Secretion System Activity in Burkholderia cenocepacia▿

    PubMed Central

    Aubert, Daniel F.; Flannagan, Ronald S.; Valvano, Miguel A.

    2008-01-01

    Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS. PMID:18316384

  13. Sensing by the membrane-bound sensor kinase DcuS: exogenous versus endogenous sensing of C(4)-dicarboxylates in bacteria.

    PubMed

    Scheu, Patrick D; Kim, Ok Bin; Griesinger, Christian; Unden, Gottfried

    2010-09-01

    Bacteria are able to grow at the expense of both common (succinate, L-malate, fumarate and aspartate) and uncommon (L-tartrate and D-malate) C(4)-dicarboxylates, which are components of central metabolism. Two types of sensors/regulators responding to the C(4)-dicarboxylates function in Escherichia coli, Bacillus, Lactobacillus and related bacteria. The first type represents membrane-integral two-component systems, while the second includes cytoplasmic LysR-type transcriptional regulators. The difference in location and substrate specificity allows the exogenous induction of metabolic genes by common C(4)-dicarboxylates, and endogenous induction by uncommon C(4)-dicarboxylates. The two-component sensors, DcuS and CitA, are composed of an extracellular Per-Arnt-Sim (PAS) domain, two transmembrane helices, a cytoplasmic PAS and the kinase domain. The structures of the extracellular PAS domains of DcuS and CitA have been determined in the ligand-bound and the apo form. Binding of the ligand results in closing and compaction of the binding site, and the structural change gives rise to piston-type movement of the adjacent membrane-spanning helix-2, and signal transmission to the cytoplasmic side. For DcuS, a membrane-embedded construct has been developed that suggests (by experimentation and modeling) that plasticity of the cytoplasmic PAS domain is central to signal transduction from the membrane to the kinase. Sensor kinase DcuS of E. coli requires the C(4)-dicarboxylate transporters DctA or DcuB as co-sensors for function under aerobic and anaerobic conditions, respectively. DcuB contains a regulatory site that controls the function of DcuS and is independent from the transport region. Therefore, DcuS senses C(4)-dicarboxylates in two independent modes, responding to the effector concentration and the metabolic flux of extracellular C(4)-dicarboxylates.

  14. Ultrafast ligand dynamics in the heme-based GAF sensor domains of the histidine kinases DosS and DosT from Mycobacterium tuberculosis†

    PubMed Central

    Vos, Marten H.; Bouzhir-Sima, Latifa; Lambry, Jean-Christophe; Luo, Hao; Eaton-Rye, Julian J.; Ioanoviciu, Alexandra; Ortiz de Montellano, Paul R.; Liebl, Ursula

    2011-01-01

    The transcriptional regulator DosR from M. tuberculosis plays a crucial role in the virulence to dormancy transition of the pathogen. DosR can be activated by DosT and DosS, two histidine kinases with heme-containing sensor GAF domains, capable of diatomic ligand binding, To investigate the initial processes occurring upon ligand dissociation, we performed ultrafast time-resolved absorption spectroscopy of the isolated sensor domains ligated with O2, NO and CO. The results reveal a relatively closed heme pocket for both proteins. For DosT the yield of O2 escape from the heme pocket on the picoseconds timescale upon photodissociation was found to be very low (1.5%), similar to other heme-based oxygen sensor proteins, implying that this sensor acts as an effective O2 trap. Remarkably, this yield is an order of magnitude higher in DosS (18%). For CO, by contrast, the fraction of CO rebinding within the heme pocket is higher in DosS. Experiments with mutant DosT sensor domains and molecular dynamics simulations indicate an important role in ligand discrimination of the distal tyrosine, present in both proteins, which forms a hydrogen bond with heme-bound O2. We conclude that despite their similarity, DosT and DosS display ligand-specific different primary dynamics during the initial phases of intra-protein signaling. The distal tyrosine, present in both proteins, plays an important role in these processes. PMID:22142262

  15. 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.

  16. Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

    PubMed Central

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

    2015-01-01

    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

  17. 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

  18. The sensor kinase CitA (DpiB) of Escherichia coli functions as a high-affinity citrate receptor.

    PubMed

    Kaspar, Sibylle; Bott, Michael

    2002-04-01

    For the CitA-CitB (DpiB-DpiA) two-component signal transduction system from Escherichia coli, three diverse functions have been reported: induction of the citrate fermentation genes citCDEFXGT, repression of the regulator gene appY, and destabilization of the inheritance of iteron-containing plasmids such as pSC101. This poses the question of the principal biological role of this system. Here it is shown that the periplasmic domain of the E. coli sensor kinase CitA functions as a high-affinity citrate receptor. Two CitA derivatives were purified by affinity chromatography and subjected to binding studies using isothermal titration calorimetry (ITC). One of them, termed CitA215MBP, comprised the N-terminal part of CitA (amino acid residues 1-215), including the two transmembrane helices, and was fused to the amino terminus of the E. coli maltose-binding protein lacking its signal peptide. The second CitA derivative, designated CitAP(Ec), encompassed only the periplasmic domain (amino acid residues 38-177). CitA215MBP bound citrate at 25 degrees C with a K(d) of 0.3 microM and a binding stoichiometry of up to 0.9 in 50 mM sodium phosphate buffer, pH 7. Binding was driven by the enthalpy change (Delta H of -95.7 kJ mol(-1)), whereas the entropy change was not favorable for binding ( T Delta S of -58.6 kJ mol(-1)). ITC experiments with CitAP(Ec) yielded similar K(d) values for citrate (0.15-1.0 microM). Besides citrate, also isocitrate ( K(d) approximately tricarballylate ( K(d) approximately t not malate were bound by CitAP(Ec). The results favor the assumption that the primary biological function of the CitA-CitB system is the regulation of the citrate fermentation genes.

  19. 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.

  20. Cloning, expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species.

    PubMed

    Classen, Elisa; Groth, Georg

    2012-03-01

    Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63-80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.

  1. Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor.

    PubMed

    Samanta, Dipanjan; Widom, Joanne; Borbat, Peter P; Freed, Jack H; Crane, Brian R

    2016-12-09

    Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.

  2. Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation.

    PubMed

    Ferris, Hedda U; Coles, Murray; Lupas, Andrei N; Hartmann, Marcus D

    2014-06-01

    Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.

  3. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  4. HAMP Domain Rotation and Tilting Movements Associated with Signal Transduction in the PhoQ Sensor Kinase

    PubMed Central

    Matamouros, Susana; Hager, Kyle R.

    2015-01-01

    ABSTRACT HAMP domains are α-helical coiled coils that often transduce signals from extracytoplasmic sensing domains to cytoplasmic domains. Limited structural information has resulted in hypotheses that specific HAMP helix movement changes downstream enzymatic activity. These hypotheses were tested by mutagenesis and cysteine cross-linking analysis of the PhoQ histidine kinase, essential for resistance to antimicrobial peptides in a variety of enteric pathogens. These results support a mechanistic model in which periplasmic signals which induce an activation state generate a rotational movement accompanied by a tilt in α-helix 1 which activates kinase activity. Biochemical data and a high-confidence model of the PhoQ cytoplasmic domain indicate a possible physical interaction of the HAMP domain with the catalytic domain as necessary for kinase repression. These results support a model of PhoQ activation in which changes in the periplasmic domain lead to conformational movements in the HAMP domain helices which disrupt interaction between the HAMP and the catalytic domains, thus promoting increased kinase activity. PMID:26015499

  5. β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase

    PubMed Central

    Ali, Nada; Ling, Naomi; Krishnamurthy, Srinath; Oakhill, Jonathan S.; Scott, John W.; Stapleton, David I.; Kemp, Bruce E.; Anand, Ganesh Srinivasan; Gooley, Paul R.

    2016-01-01

    The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high. PMID:28000716

  6. 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

  7. Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA.

    PubMed

    Gerharz, Tanja; Reinelt, Stefan; Kaspar, Sibylle; Scapozza, Leonardo; Bott, Michael

    2003-05-20

    The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.

  8. Citrate sensing by the C4-dicarboxylate/citrate sensor kinase DcuS of Escherichia coli: binding site and conversion of DcuS to a C4-dicarboxylate- or citrate-specific sensor.

    PubMed

    Krämer, J; Fischer, J D; Zientz, E; Vijayan, V; Griesinger, C; Lupas, A; Unden, G

    2007-06-01

    The histidine protein kinase DcuS of Escherichia coli senses C(4)-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C(4)-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C(4)-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C(4)-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C(4)-dicarboxylate-specific sensor (DcuS(DC)) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS(Cit)). DcuS(DC) and DcuS(Cit) had complementary effector specificities and responded either to C(4)-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C(4)-dicarboxylates) via the C(4)-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C(4)-dicarboxylates; sites C1 and H are required for other essential purposes.

  9. The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase.

    PubMed

    Ortiz de Orué Lucana, Darío; Bogel, Gabriele; Zou, Peijian; Groves, Matthew R

    2009-03-06

    HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 A) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.

  10. Role of the GacS Sensor Kinase in the Regulation of Volatile Production by Plant Growth-Promoting Pseudomonas fluorescens SBW25

    PubMed Central

    Cheng, Xu; Cordovez, Viviane; Etalo, Desalegn W.; van der Voort, Menno; Raaijmakers, Jos M.

    2016-01-01

    In plant-associated Pseudomonas species, the production of several secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system (the Gac-system). Here, we investigated if a mutation in the GacS sensor kinase affects the production of volatile organic compounds (VOCs) in P. fluorescens SBW25 (Pf.SBW25) and how this impacts on VOCs-mediated growth promotion and induced systemic resistance of Arabidopsis and tobacco. A total of 205 VOCs were detected by Gas Chromatography Mass Spectrometry for Pf. SBW25 and the gacS-mutant grown on two different media for 3 and 6 days. Discriminant function analysis followed by hierarchical clustering revealed 24 VOCs that were significantly different in their abundance between Pf.SBW25 and the gacS-mutant, which included three acyclic alkenes (3-nonene, 4-undecyne, 1-undecene). These alkenes were significantly reduced by the gacS mutation independently of the growth media and of the incubation time. For Arabidopsis, both Pf.SBW25 and the gacS-mutant enhanced, via VOCs, root and shoot biomass, induced systemic resistance against leaf infections by P. syringae and rhizosphere acidification to the same extent. For tobacco, however, VOCs-mediated effects on shoot and root growth were significantly different between Pf.SBW25 and the gacS-mutant. While Pf.SBW25 inhibited tobacco root growth, the gacS-mutant enhanced root biomass and lateral root formation relative to the non-treated control plants. Collectively these results indicate that the sensor kinase GacS is involved in the regulation of VOCs production in Pf.SBW25, affecting plant growth in a plant species-dependent manner. PMID:27917180

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

    PubMed

    Monzel, Christian; Unden, Gottfried

    2015-09-01

    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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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...

  17. The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain.

    PubMed

    Reinelt, Stefan; Hofmann, Eckhard; Gerharz, Tanja; Bott, Michael; Madden, Dean R

    2003-10-03

    The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.

  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 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

  19. 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

  20. Putative regulatory mechanism of prothoracicotropic hormone (PTTH) secretion in the American cockroach, Periplaneta americana as inferred from co-localization of Rab8, PTTH, and protein kinase C in neurosecretory cells.

    PubMed

    Hiragaki, Susumu; Uno, Tomohide; Takeda, Makio

    2009-03-01

    Small GTPases of the Rab family act as essential regulators of vesicle transport pathways, including the exocytosis of neurohormones. These processes are not well-understood in insects. To address the physiological function of Rab proteins and their phosphorylation in insect neurosecretion, Rab8-like, prothoracicotropic hormone (PTTH)-like, and protein kinase C (PKC)-like immunohistochemical reactivities (-ir) were investigated in the brain of the American cockroach, Periplaneta americana. All the antibodies tested reacted with neurons in the pars intercerebralis, corpora cardiaca, and nervi corporis allati I. Double-labeling experiments demonstrated that all PTTH-ir were colocalized with Rab8-ir and PKC-ir in the pars intercerebralis, although exclusive reactivity was present to antisera against Rab8 or PKC. These findings support the notion that Rab8-like antigen is phosphorylated by PKC, and that this phosphorylation is involved in the axonal transport and secretion of PTTH in this species.

  1. The threshold level of the sensor histidine kinase KinA governs entry into sporulation in Bacillus subtilis.

    PubMed

    Eswaramoorthy, Prahathees; Duan, Daniel; Dinh, Jeffrey; Dravis, Ashlee; Devi, Seram Nganbiton; Fujita, Masaya

    2010-08-01

    Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a sigma(H)-dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).

  2. 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

  3. Bioelectrocatalytic sensor for triglycerides in human skin sebum based on enzymatic cascade reaction of lipase, glycerol kinase and glycerophosphate oxidase.

    PubMed

    Jeong, Chi Yong; Han, Yong Duk; Yoon, Jae Ho; Yoon, Hyun C

    2014-04-10

    We report the development of an electrochemical biosensor for the quantification of triglycerides in human skin sebum, based on a multienzyme cascade reaction. The presence of excessive triglycerides in human sebum is one of the leading causes of various skin ailments. However, to the best of our knowledge, no bioelectrocatalytic approach for the quantification of sebum triglycerides has been made. In order to develop triglyceride biosensor, we fabricated a multienzyme-associated electrode incorporating lipase, glycerol kinase, and glycerophosphate oxidase. Enzymes were deposited by electrostatic force and further stabilized via crosslinking between enzymes and polymer matrices. The enzyme-modified biosensing electrode maintained its bioelectrocatalytic activity for five days. An additional constraint was the limited solubility of sebum triglycerides in aqueous electrolytes, impeding the analysis. To address this issue, triglyceride samples were prepared in the form of micelles, enabling efficient sample preparation for biosensor signaling. Calibration tests revealed that the designed assay had a detection range of 15-200mg/dL of micellar triglyceride, which covered the required determination range. The developed biosensing approach was successfully used to determine triglyceride concentrations in real sebum samples of unknown triglyceride content.

  4. Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae.

    PubMed

    Yamamoto, Shouji; Mitobe, Jiro; Ishikawa, Takahiko; Wai, Sun Nyunt; Ohnishi, Makoto; Watanabe, Haruo; Izumiya, Hidemasa

    2014-01-01

    In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed 'TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR.

  5. 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.

  6. Visualizing Evolutionary Relationships of Multidomain Proteins: An Example from Receiver (REC) Domains of Sensor Histidine Kinases in the Candidatus Maribeggiatoa str. Orange Guaymas Draft Genome

    PubMed Central

    MacGregor, Barbara J.

    2016-01-01

    For multidomain proteins, evolutionary changes may occur at the domain as well as the whole-protein level. An example is presented here, with suggestions for how such complicated relationships might be visualized. Earlier analysis of the Candidatus Maribeggiatoa str. Orange Guaymas (BOGUAY; Gammaproteobacteria) single-filament draft genome found evidence of gene exchange with the phylogenetically distant Cyanobacteria, particularly for sensory and signal transduction proteins. Because these are modular proteins, known to undergo frequent duplication, domain swapping, and horizontal gene transfer, a single domain was chosen for analysis. Recognition (REC) domains are short (~125 amino acids) and well conserved, simplifying sequence alignments and phylogenetic calculations. Over 100 of these were identified in the BOGUAY genome and found to have a wide range of inferred phylogenetic relationships. Two sets were chosen here for detailed study. One set of four BOGUAY ORFs has closest relatives among other Beggiatoaceae and Cyanobacteria. A second set of four has REC domains with more mixed affiliations, including other Beggiatoaceae, several sulfate-reducing Deltaproteobacteria and Firmicutes, magnetotactic Nitrospirae, one Shewanella and one Ferrimonas strain (both Gammaproteobacteria), and numerous Vibrio vulnificus and V. navarrensis strains (also Gammaproteobacteria). For an overview of the possible origins of the whole proteins and the surrounding genomic regions, color-coded BLASTP results were produced and displayed against cartoons showing protein domain structure of predicted genes. This is suggested as a visualization method for investigation of possible horizontally transferred regions, giving more detail than scans of DNA composition and codon usage but much faster than carrying out full phylogenetic analyses for multiple proteins. As expected, most of the predicted sensor histidine kinases investigated have two or more segments with distinct BLASTP

  7. In Vivo Evolution to Colistin Resistance by PmrB Sensor Kinase Mutation in KPC-Producing Klebsiella pneumoniae Is Associated with Low-Dosage Colistin Treatment

    PubMed Central

    Cannatelli, Antonio; Di Pilato, Vincenzo; Giani, Tommaso; Arena, Fabio; Ambretti, Simone; Gaibani, Paolo; D'Andrea, Marco Maria

    2014-01-01

    Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrBL82R in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrBL82R mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment. PMID:24841267

  8. Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system.

    PubMed Central

    Brian, P; Riggle, P J; Santos, R A; Champness, W C

    1996-01-01

    Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. The S. coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system. All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase. A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator. In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin. Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene. We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor. PMID:8655502

  9. The Wall-associated Kinase gene family in rice genomes.

    PubMed

    de Oliveira, Luiz Felipe Valter; Christoff, Ana Paula; de Lima, Júlio Cesar; de Ross, Bruno Comparsi Feijó; Sachetto-Martins, Gilberto; Margis-Pinheiro, Marcia; Margis, Rogerio

    2014-12-01

    The environment is a dynamic system in which life forms adapt. Wall-Associated Kinases (WAK) are a subfamily of receptor-like kinases associated with the cell wall. These genes have been suggested as sensors of the extracellular environment and triggers of intracellular signals. They belong to the ePK superfamily with or without a conserved arginine before the catalytic subdomain VIB, which characterizes RD and non-RD WAKs. WAK is a large subfamily in rice. We performed an extensive comparison of WAK genes from A. thaliana (AtWAK), O. sativa japonica and indica subspecies (OsWAK). Phylogenetic studies and WAK domain characterization allowed for the identification of two distinct groups of WAK genes in Arabidopsis and rice. One group corresponds to a cluster containing only OsWAKs that most likely expanded after the monocot-dicot separation, which evolved into a non-RD kinase class. The other group comprises classical RD-kinases with both AtWAK and OsWAK representatives. Clusterization analysis using extracellular and kinase domains demonstrated putative functional redundancy for some genes, but also highlighted genes that could recognize similar extracellular stimuli and activate different cascades. The gene expression pattern of WAKs in response to cold suggests differences in the regulation of the OsWAK genes in the indica and japonica subspecies. Our results also confirm the hypothesis of functional diversification between A. thaliana and O. sativa WAK genes. Furthermore, we propose that plant WAKs constitute two evolutionarily related but independent subfamilies: WAK-RD and WAK-nonRD. Recognition of this structural division will further provide insights to understanding WAK functions and regulations.

  10. Translating neuronal activity at the synapse: presynaptic calcium sensors in short-term plasticity

    PubMed Central

    de Jong, Arthur P. H.; Fioravante, Diasynou

    2014-01-01

    The complex manner in which patterns of presynaptic neural activity are translated into short-term plasticity (STP) suggests the existence of multiple presynaptic calcium (Ca2+) sensors, which regulate the amplitude and time-course of STP and are the focus of this review. We describe two canonical Ca2+-binding protein domains (C2 domains and EF-hands) and define criteria that need to be met for a protein to qualify as a Ca2+ sensor mediating STP. With these criteria in mind, we discuss various forms of STP and identify established and putative Ca2+ sensors. We find that despite the multitude of proposed sensors, only three are well established in STP: Munc13, protein kinase C (PKC) and synaptotagmin-7. For putative sensors, we pinpoint open questions and potential pitfalls. Finally, we discuss how the molecular properties and modes of action of Ca2+ sensors can explain their differential involvement in STP and shape net synaptic output. PMID:25400547

  11. A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

    PubMed Central

    Charpenteau, Martine; Jaworski, Krzysztof; Ramirez, Bertha C; Tretyn, Andrzej; Ranjeva, Raoul; Ranty, Benoît

    2004-01-01

    Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways. PMID:14720124

  12. 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.

  13. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-03

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

  14. 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.

  15. Phosphorylation of calcineurin B-like (CBL) calcium sensor proteins by their CBL-interacting protein kinases (CIPKs) is required for full activity of CBL-CIPK complexes toward their target proteins.

    PubMed

    Hashimoto, Kenji; Eckert, Christian; Anschütz, Uta; Scholz, Martin; Held, Katrin; Waadt, Rainer; Reyer, Antonella; Hippler, Michael; Becker, Dirk; Kudla, Jörg

    2012-03-09

    Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

  16. Domain Analysis of ArcS, the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System, Reveals Functional Differentiation of Its Two Receiver Domains

    PubMed Central

    Bubendorfer, Sebastian

    2013-01-01

    In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli. PMID:23161031

  17. 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

  18. Gene ercA, Encoding a Putative Iron-Containing Alcohol Dehydrogenase, Is Involved in Regulation of Ethanol Utilization in Pseudomonas aeruginosa

    PubMed Central

    Hempel, Niels; Görisch, Helmut

    2013-01-01

    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. PMID:23813731

  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. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  1. Inverse modulation of the energy sensor Snf1-related protein kinase 1 on hypoxia adaptation and salt stress tolerance in Arabidopsis thaliana.

    PubMed

    Im, Jong Hee; Cho, Young-Hee; Kim, Geun-Don; Kang, Geun-Ho; Hong, Jung-Woo; Yoo, Sang-Dong

    2014-10-01

    Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater.

  2. 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.

  3. 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.

  4. In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS.

    PubMed

    Sun, Fei; Li, Chunling; Jeong, Dowon; Sohn, Changmo; He, Chuan; Bae, Taeok

    2010-04-01

    Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN(6)GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.

  5. The Pseudomonas putida HskA hybrid sensor kinase responds to redox signals and contributes to the adaptation of the electron transport chain composition in response to oxygen availability.

    PubMed

    Sevilla, Emma; Alvarez-Ortega, Carolina; Krell, Tino; Rojo, Fernando

    2013-12-01

    Pseudomonas putida has a branched aerobic electron transport that includes five terminal oxidases, each of which has different properties. The relative expression of each oxidase is carefully regulated to assemble the most suitable electron transport chain for the prevailing conditions. The HskA hybrid sensor kinase participates in this control, but the signals to which HskA responds were unknown. Here, the influence of HskA on the mRNA abundance of genes coding for all terminal oxidases and for the bc1 complex was analysed in cells growing under controlled aerobic, semiaerobic or microaerobic conditions. The results indicate that the influence of HskA on the expression of each terminal oxidase and the bc1 complex varies depending on oxygen availability. This effect was more pronounced under aerobic or semiaerobic conditions, but decreased under microaerobic conditions. The expression of hskA was regulated by oxygen availability. We show that HskA autophosphorylation is inhibited by ubiquinone but not by ubiquinol, its reduced derivative. This suggests that HskA could sense the oxidation state of the respiratory ubiquinones, which may be a key factor in HskA activity. Inactivation of hskA reduced growth rate and oxygen consumption, stressing the importance of HskA for the assembly of an efficient electron transport chain.

  6. 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

  7. 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

  8. 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.

  9. 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

    2005-03-07

    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.

  10. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  11. 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

  12. Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

    PubMed

    Smith, Johanna A; Wang, Feng-Xiang; Zhang, Hui; Wu, Kou-Juey; Williams, Kevin Jon; Daniel, René

    2008-01-22

    Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

  13. 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…

  14. 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.

  15. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  16. 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

  17. The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

    PubMed Central

    Saunders, Neil F. W.

    2008-01-01

    The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au. PMID:18477637

  18. Molecular and structural insight into plasmodium falciparum RIO2 kinase.

    PubMed

    Chouhan, Devendra K; Sharon, Ashoke; Bal, Chandralata

    2013-02-01

    Among approximately 65 kinases of the malarial genome, RIO2 (right open reading frame) kinase belonging to the atypical class of kinase is unique because along with a kinase domain, it has a highly conserved N-terminal winged helix (wHTH) domain. The wHTH domain resembles the wing like domain found in DNA binding proteins and is situated near to the kinase domain. Ligand binding to this domain may reposition the kinase domain leading to inhibition of enzyme function and could be utilized as a novel allosteric site to design inhibitor. In the present study, we have generated a model of RIO2 kinase from Plasmodium falciparum utilizing multiple modeling, simulation approach. A novel putative DNA-binding site is identified for the first time in PfRIO2 kinase to understand the DNA binding events involving wHTH domain and flexible loop. Induced fit DNA docking followed by minimization, molecular dynamics simulation, energetic scoring and binding mode studies are used to reveal the structural basis of PfRIO2-ATP-DNA complex. Ser105 as a potential site of phosphorylation is revealed through the structural studies of ATP binding in PfRIO2. Overall the present study discloses the structural facets of unknown PfRIO2 complex and opens an avenue toward exploration of novel drug target.

  19. 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

  20. Alternative splicing produces two transcripts encoding putative female-biased odorant receptors in the navel orangeworm, Amyelois transitella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect odorant receptors are key sensors of environmental odors, and members of the lepidopteran pheromone receptor subfamily are thought to play important roles in mate finding, and oviposition site location. Much research has been done to identify putative pheromone receptors in lepidopteran male...

  1. 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.

  2. [MAST2-like protein kinase from grape vine Vitis vinifera: cloning of catalytic domain cDNA].

    PubMed

    Briantseva, S A; Gavriushina, E S; Emets, A I; Karpov, P A; Blium, Ia B; Drygin, Iu F; Nadezhdina, E S

    2010-01-01

    The aim of our work is the identification of protein kinases phosphorylating microtubule proteins in plant cells. Using bioinformatic approach, we found genes of putative homologues of microtubule-associated mammalian protein kinase MAST2 in higher plant genomes. The gene of closest MAST2 homologue, putative protein, named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI), was found in grape Vitis vinifera. We report here the cloning of cDNA of GMLK (A7NTE9) from Pinot Noir grape vine leaves.

  3. 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.

  4. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  5. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  6. Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

    PubMed

    Schlattner, Uwe; Klaus, Anna; Ramirez Rios, Sacnicte; Guzun, Rita; Kay, Laurence; Tokarska-Schlattner, Malgorzata

    2016-08-01

    There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus

  7. 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°.

  8. Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency

    PubMed Central

    Han, Ji-Young; Kang, Min-Ji; Kim, Kyung-Hee; Han, Pyung-Lim; Kim, Hyun-Seok; Ha, Ji-Young; Son, Jin H.

    2015-01-01

    The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser1412) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease. PMID:25716315

  9. 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

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

    PubMed

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

    2017-04-01

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

  11. 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

  12. Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti.

    PubMed

    Meier, Veronika M; Muschler, Paul; Scharf, Birgit E

    2007-03-01

    The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.

  13. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  14. Molecular cloning of plant transcripts encoding protein kinase homologs.

    PubMed Central

    Lawton, M A; Yamamoto, R T; Hanks, S K; Lamb, C J

    1989-01-01

    Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences. Images PMID:2541432

  15. 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.

  16. Modulation of Neuronal Survival Factor MEF2 by Kinases in Parkinson’s Disease

    PubMed Central

    Yin, Yue; She, Hua; Li, Wenming; Yang, Qian; Guo, Shuzhong; Mao, Zixu

    2012-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disorder due to selective death of neurons in the substantia nigra pars compacta. The cause of cell death remains largely unknown. Myocyte enhancer factor 2 (MEF2) is a group of transcriptional factors required to regulate neuronal development, synaptic plasticity, as well as survival. Recent studies show that MEF2 functions are regulated in multiple subcellular organelles and suggest that dysregulation of MEF2 plays essential roles in the pathogenesis of PD. Many kinases associated with transcription, translation, protein misfolding, autophagy, and cellular energy homeostasis are involved in the neurodegenerative process. Following the first demonstration that mitogen-activated protein kinase p38 (p38 MAPK) directly phosphorylates and activates MEF2 to promote neuronal survival, several other kinase regulators of MEF2s have been identified. These include protein kinase A and extracellular signal regulated kinase 5 as positive MEF2 regulators, and cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β as negative regulators in response to diverse toxic signals relevant to PD. It is clear that MEF2 has emerged as a key point where survival and death signals converge to exert their regulatory effects, and dysregulation of MEF2 function in multiple subcellular organelles may underlie PD pathogenesis. Moreover, several other kinases such as leucine-rich repeat kinase 2 and PTEN-induced putative kinase 1 (PINK1) are of particular interest due to their potential interaction with MEF2. PMID:22661957

  17. 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

  18. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  19. Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases.

    PubMed

    Erni, B; Siebold, C; Christen, S; Srinivas, A; Oberholzer, A; Baumann, U

    2006-04-01

    Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nepsilon2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA(+) family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family.

  20. 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.

  1. 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.

  2. 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.

  3. Putative Bronchopulmonary Flagellated Protozoa in Immunosuppressed Patients

    PubMed Central

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Çelik, Pınar; 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. PMID:24804259

  4. The Biogeography of Putative Microbial Antibiotic Production.

    PubMed

    Morlon, Hélène; O'Connor, Timothy K; 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.

  5. 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

  6. PUTATIVE ADVERSE OUTCOME PATHWAY FOR INHIBITON ...

    EPA Pesticide Factsheets

    The adverse outcome pathway (AOP) provides a framework for organizing knowledge to define links between a molecular initiating event (MIE) and an adverse outcome (AO) occurring at a higher level of biological organization, such as the individual or population. The AOP framework proceeds from a general (e.g., not chemical specific) molecular mode of action, designated as a MIE, through stepwise changes in biological status, defined as key events (KEs), to a final AO that can be used in risk assessment. Because aromatase-inhibiting pharmaceuticals are widely used to treat breast cancer patients, we explored the unintended consequences that might occur in fish exposed to these chemicals through wastewater discharge into the aquatic environment. Unlike mammals, fish have two isoforms of aromatase, one that predominates in the ovary (cyp19a1a) and a second (cyp19a1b) that prevails in the brain. Aromatase activity in fish brain can be 100 to 1000 times that in mammals and is associated with reproduction. We have developed a putative AOP for inhibition of brain aromatase in fish leading to reproductive dysfunction based on review of relevant literature and reproductive experiments with the marine fish cunner (Tautogolabrus adspersus) exposed to aromatase-inhibiting pharmaceuticals in the laboratory. The first KE in this AOP is a decrease in brain aromatase activity due to exposure to an aromatase inhibitor. KEs then progress through subsequent steps including decreas

  7. STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis

    PubMed Central

    Chevalier, David; Batoux, Martine; Fulton, Lynette; Pfister, Karen; Yadav, Ram Kishor; Schellenberg, Maja; Schneitz, Kay

    2005-01-01

    An open question remains as to what coordinates cell behavior during organogenesis, permitting organs to reach their appropriate size and shape. The Arabidopsis gene STRUBBELIG (SUB) defines a receptor-mediated signaling pathway in plants. SUB encodes a putative leucine-rich repeat transmembrane receptor-like kinase. The mutant sub phenotype suggests that SUB affects the formation and shape of several organs by influencing cell morphogenesis, the orientation of the division plane, and cell proliferation. Mutational analysis suggests that the kinase domain is important for SUB function. Biochemical assays using bacterially expressed fusion proteins indicate that the SUB kinase domain lacks enzymatic phosphotransfer activity. Furthermore, transgenes encoding WT and different mutant variants of SUB were tested for their ability to rescue the mutant sub phenotype. These genetic data also indicate that SUB carries a catalytically inactive kinase domain. The SUB receptor-like kinase may therefore signal in an atypical fashion. PMID:15951420

  8. 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.

  9. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  10. Orphan kinases turn eccentric

    PubMed Central

    Mikolcevic, Petra; Rainer, Johannes; Geley, Stephan

    2012-01-01

    PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIREs are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed “PCTAIRE” sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIREs and discuss their established functions. PMID:22895054

  11. 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

  12. 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...

  13. 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.

  14. Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 from Methanopyrus kandleri.

    PubMed

    Schacherl, Magdalena; Waltersperger, Sandro; Baumann, Ulrich

    2013-12-01

    Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such as Methanopyrus kandleri contain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 from M. kandleri in apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified.

  15. PAK family kinases

    PubMed Central

    Zhao, Zhuo-shen; Manser, Ed

    2012-01-01

    The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1–6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4–6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies. PMID:23162738

  16. Kinase Inhibitors from Marine Sponges

    PubMed Central

    Skropeta, Danielle; Pastro, Natalie; Zivanovic, Ana

    2011-01-01

    Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included. PMID:22073013

  17. 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.

  18. Smart sensors

    NASA Astrophysics Data System (ADS)

    Corsi, Carlo

    1991-09-01

    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. In a broad sense, they include any sensor system 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 performances. Thus, sophisticated signal processing operations will be 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 has achieved higher goals by a new and revolutionary sensor concept which introduces inside the sensor some of the basic functions of living eyes, such as dynamic stare, dishomogeneity 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 system. This paper concerns the processing techniques limited to 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 a smart pattern correlation thresholding.

  19. Secondary kinase reactions catalyzed by yeast pyruvate kinase.

    PubMed

    Leblond, D J; Robinson, J L

    1976-06-07

    1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.

  20. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

    PubMed Central

    Frey, Stefan; Reschka, Eva J.; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain. PMID:26418262

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Cyclin-dependent kinases

    PubMed Central

    2014-01-01

    Summary 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

  13. 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

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

    PubMed Central

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

    2016-01-01

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo. After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  15. Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.

    PubMed

    Cabezas, Alicia; Costas, María Jesús; Pinto, Rosa María; Couto, Ana; Cameselle, José Carlos

    2005-12-30

    Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.

  16. 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.

  17. 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

  18. Aptamer Sensors

    PubMed Central

    Marrazza, Giovanna

    2017-01-01

    In the last years, great progress has been accomplished in the development of aptamer sensors with different transducers. In order to improve the sensitivity of these biosensors, several methodologies have been employed. In this Special Issue, the state of art and the future trends in the field of aptamer sensors have been explored. PMID:28054983

  19. 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.

  20. A high-throughput radiometric kinase assay

    PubMed Central

    Duong-Ly, Krisna C.; Peterson, Jeffrey R.

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  1. 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.

  2. Suborganelle sensing of mitochondrial cAMP-dependent protein kinase activity.

    PubMed

    Agnes, Richard S; Jernigan, Finith; Shell, Jennifer R; Sharma, Vyas; Lawrence, David S

    2010-05-05

    A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix/intermembrane space/outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.

  3. 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.

  4. Aurora Kinases Throughout Plant Development.

    PubMed

    Weimer, Annika K; Demidov, Dmitri; Lermontova, Inna; Beeckman, Tom; Van Damme, Daniël

    2016-01-01

    Aurora kinases are evolutionarily conserved key mitotic determinants in all eukaryotes. Yeasts contain a single Aurora kinase, whereas multicellular eukaryotes have at least two functionally diverged members. The involvement of Aurora kinases in human cancers has provided an in-depth mechanistic understanding of their roles throughout cell division in animal and yeast models. By contrast, understanding Aurora kinase function in plants is only starting to emerge. Nevertheless, genetic, cell biological, and biochemical approaches have revealed functional diversification between the plant Aurora kinases and suggest a role in formative (asymmetric) divisions, chromatin modification, and genome stability. This review provides an overview of the accumulated knowledge on the function of plant Aurora kinases as well as some major challenges for the future.

  5. 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.

  6. PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

    PubMed

    Prieto-Echagüe, Victoria; Chan, Perry M; Craddock, Barbara P; Manser, Edward; Miller, W Todd

    2011-04-26

    Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

  7. Bisubstrate fluorescent probes and biosensors in binding assays for HTS of protein kinase inhibitors.

    PubMed

    Uri, Asko; Lust, Marje; Vaasa, Angela; Lavogina, Darja; Viht, Kaido; Enkvist, Erki

    2010-03-01

    Conjugates of adenosine mimics and d-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.

  8. Sensor technology

    NASA Technical Reports Server (NTRS)

    Sokoloski, Martin M.

    1988-01-01

    The objective is to provide necessary expertise and technology to advance space remote sensing of terrestrial, planetary, and galactic phenomena through the use of electromagnetic and electro-optic properties of gas, liquid, and solid state materials technology. The Sensor Technology Program is divided into two subprograms: a base research and development part and a Civil Space Technology Initiative (CSTI) part. The base research and development consists of research on artificially grown materials such as quantum well and superlattice structure with the potential for new and efficient means for detecting electromagnetic phenomena. Research is also being done on materials and concepts for detector components and devices for measuring high energy phenomena such as UV, X-, and gamma rays that are required observables in astrophysis and solar physics missions. The CSTI program is more mission driven and is balanced among four major disciplines: detector sensors; submillimeter wave sensors; LIDAR/DIAL sensors; and cooler technology.

  9. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  10. Understanding the Polo Kinase machine.

    PubMed

    Archambault, V; Lépine, G; Kachaner, D

    2015-09-10

    The Polo Kinase is a central regulator of cell division required for several events of mitosis and cytokinesis. In addition to a kinase domain (KD), Polo-like kinases (Plks) comprise a Polo-Box domain (PBD), which mediates protein interactions with targets and regulators of Plks. In all organisms that contain Plks, one Plk family member fulfills several essential functions in the regulation of cell division, and here we refer to this conserved protein as Polo Kinase (Plk1 in humans). The PBD and the KD are capable of both cooperation and mutual inhibition in their functions. Crystal structures of the PBD, the KD and, recently, a PBD-KD complex have helped understanding the inner workings of the Polo Kinase. In parallel, an impressive array of molecular mechanisms has been found to mediate the regulation of the protein. Moreover, the targeting of Polo Kinase in the development of anti-cancer drugs has yielded several molecules with which to chemically modulate Polo Kinase to study its biological functions. Here we review our current understanding of the protein function and regulation of Polo Kinase as a fascinating molecular device in control of cell division.

  11. 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

  12. Spectral Evidence of Aqueous Activity in Two Putative Martian Paleolakes

    NASA Technical Reports Server (NTRS)

    Roush, Ted L.; Marzo, Giuseppe A.; Fonti, Sergio; Orofino, Vincenzo; Blanco, Armando

    2010-01-01

    CRISM observations of putative paleolakes in Cankuzo and Luqa craters exhibit spectral features consistent with the activity of water. The spatial distributions suggest different formation scenarios for each site. In Cankuzo the distribution suggests postimpact alteration whereas in Luqa there are hints of possible formation of a layer of phyllosilicate materials.

  13. Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.

    PubMed

    de María, Nuria; Guevara, Angeles; Serra, M Teresa; García-Luque, Isabel; González-Sama, Alfonso; García de Lacoba, Mario; de Felipe, M Rosario; Fernández-Pascual, Mercedes

    2007-08-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed.

  14. 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.

  15. 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

  16. 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,...

  17. P21 activated kinases

    PubMed Central

    Rane, Chetan K; Minden, Audrey

    2014-01-01

    The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer. PMID:24658305

  18. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  19. Thymidine kinase mutants obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Loeb, L A

    1993-05-01

    Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

  20. 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.

  1. 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

  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. 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

  4. 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.

  5. 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

  6. Sensor domains of two-component regulatory systems.

    PubMed

    Cheung, Jonah; Hendrickson, Wayne A

    2010-04-01

    Two-component systems regulate crucial cellular processes in microorganisms, and each comprises a homodimeric histidine kinase receptor and a cytoplasmic response regulator. Histidine kinases, often membrane associated, detect environmental input at sensor domains and propagate resulting signals to catalytic cytoplasmic transmitter domains. Recent studies on the great diversity of sensor domains reveal patterns of domain organization and biochemical properties that provide insight into mechanisms of signaling. Despite the enormous sequence variability found within sensor input domains, they fall into a relatively small number of discrete structural classes. Subtle rearrangements along a structurally labile dimer interface, in the form of possible sliding or rotational motions, are propagated from the sensor domain to the transmitter domain to modulate activity of the receptor.

  7. Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora subsp. carotovora that encodes a two-component sensor-regulator protein.

    PubMed

    Frederick, R D; Chiu, J; Bennetzen, J L; Handa, A K

    1997-04-01

    A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.

  8. 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.

  9. Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

    PubMed Central

    de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

    2007-01-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

  10. 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.

  11. 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.

  12. Gas sensor

    SciTech Connect

    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.

  13. 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.

  14. 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.

  15. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  16. Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii.

    PubMed

    Daher, Wassim; Morlon-Guyot, Juliette; Sheiner, Lilach; Lentini, Gaëlle; Berry, Laurence; Tawk, Lina; Dubremetz, Jean-François; Wengelnik, Kai; Striepen, Boris; Lebrun, Maryse

    2015-04-01

    Phosphoinositides regulate numerous cellular processes by recruiting cytosolic effector proteins and acting as membrane signalling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3 kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiotic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P-binding proteins and identified in total six PX and FYVE domain-containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2 . Although depletion of putative PI(3)P-binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2 . Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species that lack an apicoplast, supporting a direct role of PI(3,5)P2 in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.

  17. Benzimidazole derivatives as kinase inhibitors.

    PubMed

    Garuti, Laura; Roberti, Marinella; Bottegoni, Giovanni

    2014-01-01

    Benzimidazole is a common kinase inhibitor scaffold and benzimidazole-based compounds interact with enzymes by multiple binding modes. In some cases, the benzimidazole acts as part of the hinge-binding motif, in others it has a scaffolding role without evidence for direct hinge binding. Several of these compounds are ATP-competitive inhibitors and show high selectivity by exploiting unique structural properties that distinguish one kinase from the majority of other kinases. However, the high specificity for a single target is not always sufficient. Thus another approach, called multi-target therapy, has been developed over the last few years. The simultaneous inhibition of various kinases may be useful because the disease is attacked at several relevant targets. Moreover, if a kinase becomes drug-resistant, a multitargeted drug can act on the other kinases. Some benzimidazole derivatives are multi-target inhibitors. In this article benzimidazole inhibitors are reported with their mechanisms of action, structure-activity relationship (SAR) and biological properties.

  18. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  19. TNF and MAP kinase signaling pathways

    PubMed Central

    Sabio, Guadalupe; Davis, Roger J.

    2014-01-01

    The binding of tumor necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα. PMID:24647229

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

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  1. An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease.

    PubMed

    Cavallini, Annalisa; Brewerton, Suzanne; Bell, Amanda; Sargent, Samantha; Glover, Sarah; Hardy, Clare; Moore, Roger; Calley, John; Ramachandran, Devaki; Poidinger, Michael; Karran, Eric; Davies, Peter; Hutton, Michael; Szekeres, Philip; Bose, Suchira

    2013-08-09

    Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

  2. A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK6.

    PubMed

    Gutiérrez-Beltrán, Emilio; Personat, José María; de la Torre, Fernando; Del Pozo, Olga

    2017-01-01

    Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca(2+) signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named "SlRd2". SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2 treatment. Finally, transient expression of SlCipk6 in transgenic N benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation.

  3. Pressure sensor

    DOEpatents

    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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Yeast Pho85 kinase is required for proper gene expression during the diauxic shift.

    PubMed

    Nishizawa, Masafumi; Katou, Yuki; Shirahige, Katsuhiko; Toh-e, Akio

    2004-08-01

    The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.

  10. The "kynurenate test", a biochemical assay for putative cognition enhancers.

    PubMed

    Pittaluga, A; Vaccari, D; Raiteri, M

    1997-10-01

    Some putative cognition enhancers (oxiracetam, aniracetam and D-cycloserine) were previously shown to prevent the kynurenic acid antagonism of the N-methyl-D-aspartate (NMDA)-evoked norepinephrine (NE) release in rat hippocampal slices. This functional in vitro assay was further characterized in the present work. D-Serine, a glutamate coagonist at the NMDA receptor glycine site, concentration-dependently (EC50 approximately 0.1 microM) prevented the kynurenate (100 microM) block of the NMDA (100 microM)-evoked [3H]NE release. L-Serine was ineffective up to 10 microM. The gamma-aminobutyric acidB (GABA[B]) receptor antagonist CGP 36742, reported to improve cognitive performance, potently prevented the kynurenate antagonism. The activity of CGP 36742 (1 microM) appeared to be unaffected by 10 microM (-)-baclofen, a GABA(B) receptor agonist; furthermore, CGP 52432, a GABA(B) antagonist more potent than CGP 36742, but reportedly devoid of nootropic properties, was inactive in the "kynurenate test." The novel putative cognition enhancer CR2249, but not its enantiomer CR2361, also potently prevented the kynurenate antagonism. In contrast, linopirdine, nicotine and tacrine were inactive. In rat hippocampal synaptosomes glycine and D-cycloserine enhanced the NMDA-evoked [3H]NE release, whereas oxiracetam and CR2249 did not. These four compounds were all similarly effective in preventing kynurenate antagonism, both in slices and in synaptosomes. The NMDA potentiation caused by glycine (0.1-100 microM) was not affected by 100 microM oxiracetam, which suggested that drugs active in the "kynurenate test" may bind to sites different from the glycine site of the NMDA receptor. To conclude, the "kynurenate test" is an in vitro assay useful in the identification and characterization of putative cognition enhancers acting via NMDA receptors.

  11. The sensory histidine kinases TorS and EvgS tend to form clusters in Escherichia coli cells.

    PubMed

    Sommer, Erik; Koler, Moriah; Frank, Vered; Sourjik, Victor; Vaknin, Ady

    2013-01-01

    Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis.

  12. An ORF from Bacillus licheniformis encodes a putative DNA repressor.

    PubMed

    Naval, J; Aguilar, D; Serra, X; Pérez-Pons, J A; Piñol, J; Lloberas, J; Querol, E

    2000-01-01

    The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported. It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome. Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins. A maxicells assay shows that the encoded polypeptide is expressed. A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

  13. Molecular genetics: DNA analysis of a putative dog clone.

    PubMed

    Parker, Heidi G; Kruglyak, Leonid; Ostrander, Elaine A

    2006-03-09

    In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..

  14. Discovering the first tyrosine kinase.

    PubMed

    Hunter, Tony

    2015-06-30

    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.

  15. 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

  16. Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

    PubMed Central

    Guo, Chang-An; Danai, Laura V.; Yawe, Joseph C.; Gujja, Sharvari; Edwards, Yvonne J. K.

    2016-01-01

    The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate. PMID:27044870

  17. Tyrosine Kinase Inhibitors in Lung Cancer

    PubMed Central

    Thomas, Anish; Rajan, Arun; Giaccone, Giuseppe

    2012-01-01

    SYNOPSIS ‘Driver mutations’ are essential for carcinogenesis as well as tumor progression as they confer a selective growth advantage to cancer cells. Identification of driver mutations in growth related protein kinases, especially tyrosine kinases have led to clinical development of an array of tyrosine kinase inhibitors in various malignancies, including lung cancer. Inhibition of epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinases have proven to be of meaningful clinical benefit, while inhibition of several other tyrosine kinases have been of limited clinical benefit, thus far. An improved understanding of tyrosine kinase biology has also led to faster drug development, identification of resistance mechanisms and ways to overcome resistance. In this review, we discuss the clinical data supporting the use and practical aspects of management of patients on epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors. PMID:22520981

  18. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells.

    PubMed Central

    Corey, S; Eguinoa, A; Puyana-Theall, K; Bolen, J B; Cantley, L; Mollinedo, F; Jackson, T R; Hawkins, P T; Stephens, L R

    1993-01-01

    The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3. Images PMID:8392933

  19. Choristoneura fumiferana Granulovirus pk-1: a baculoviral protein kinase.

    PubMed

    Giannopoulos, Paresa N; Nassoury, Nasha; Lamontagne, Lucie; Guertin, Claude; Rashidan, Kianoush Khajeh

    2005-07-31

    Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk- 1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80 % amino acid identity.

  20. A lipid-regulated docking site on vinculin for protein kinase C.

    PubMed

    Ziegler, Wolfgang H; Tigges, Ulrich; Zieseniss, Anke; Jockusch, Brigitte M

    2002-03-01

    During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.

  1. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  2. Characterization of a Putative Ancestor of Coxsackievirus B5 ▿

    PubMed Central

    Gullberg, Maria; Tolf, Conny; Jonsson, Nina; Mulders, Mick N.; Savolainen-Kopra, Carita; Hovi, Tapani; Van Ranst, Marc; Lemey, Philippe; Hafenstein, Susan; Lindberg, A. Michael

    2010-01-01

    Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines. PMID:20631132

  3. 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.

  4. 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.

  5. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  6. [Kinase inhibitors against hematological malignancies].

    PubMed

    Tojo, Arinobu

    2014-06-01

    Dysregulation of protein phosphorylation, especially on tyrosine residues, plays a crucial role in development and progression of hematological malignancies. Since remarkable success in imatinib therapy of CML and Ph+ALL, extensive efforts have made to explore candidate molecular targets and next breakthrough drugs. Now that next generation ABL kinase inhibitors are available for CML, the therapeutic algorithm has been revolutionized. As for AML and lymphoid malignancies, many kinase inhibitors targeting FLT3, BTK and aurora-A are on early and late clinical trials, and a number of promising drugs including ibrutinib are picked up for further evaluation.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    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. PMID:26742849

  12. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  13. 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.

  14. Differential accumulation of transcripts encoding protein kinase homologs in greening pea seedlings.

    PubMed Central

    Lin, X; Feng, X H; Watson, J C

    1991-01-01

    Degenerate oligonucleotides, corresponding to conserved regions within the catalytic domain of known protein-serine/threonine kinases, were used as primers for the polymerase chain reaction to amplify cDNA synthesized from poly(A)+ RNA purified from the apical buds of 7-day-old pea seedlings. Five partial cDNAs were obtained and designated PsPK1 through PsPK5 (for Pisum sativum protein kinase) in order of decreasing length. The deduced amino acid sequences show that each member of the PsPK series is different in length, and, although their sequences are quite similar overall, each has a unique sequence. Moreover, each member of the PsPK series has structural features typical of members of the protein-serine/threonine kinase family of protein kinases. All are equally similar to cyclic nucleotide-dependent protein kinase and protein kinase C, suggesting that the pea homologs may be involved in signal transduction. DNA gel blots show that each PsPK cDNA is likely to be encoded by a single gene within the pea genome. RNA blot analyses show that the PsPK transcripts accumulate differentially during greening of etiolated seedlings. PsPK3 and PsPK5 transcripts show a large and rapid decline during deetiolation. In contrast, the level of PsPK4 RNA increases steadily during deetiolation whereas PsPK1 and PsPK2 transcripts show little change during the greening period. Thus light regulates changes in the levels of transcripts encoding putative protein kinases in plants. Images PMID:1714582

  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, 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.

  18. 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.

  19. 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.

  20. The putative role of MALDI-MSI in the study of Membranous Nephropathy.

    PubMed

    Smith, Andrew; L'Imperio, Vincenzo; Ajello, Elena; Ferrario, Franco; Mosele, Niccolò; Stella, Martina; Galli, Manuel; Chinello, Clizia; Pieruzzi, Federico; Spasovski, Goce; Pagni, Fabio; Magni, Fulvio

    2016-11-24

    Membranous Nephropathy (MN) is an immunocomplex mediated renal disease that represents one of the most frequent glomerulopathies worldwide. This glomerular disease can manifest as primary (idiopathic) or secondary and this distinction is crucial when choosing the most appropriate course of treatment. In secondary cases, the best strategy involves treating the underlying disease, whereas in primary forms, the identification of confirmatory markers of the idiopathic etiology underlining the process is requested by clinicians. Among those currently reported, the positivity to circulating antigens (PLA2R, IgG4 and THSD7A) was demonstrated in approximately 75% of iMN patients, while approximately 1 in 4 patients with iMN still lack a putative diagnostic marker. Ultimately, the discovery of biomarkers to help further stratify these two different forms of glomerulopathy seems mandatory. Here, MALDI-MSI was applied to FFPE renal biopsies from histologically diagnosed primary and secondary MN patients (n=20) in order to detect alterations in their tissue proteome. MALDI-MSI was able to generate molecular signatures of primary and secondary MN, with one particular signal (m/z 1459), identified as Serine/threonine-protein kinase MRCK gamma, being over-expressed in the glomeruli of primary MN patients with respect to secondary MN. Furthermore, a number of signals that could differentiate the different forms of iMN that were positive to PLA2R or IgG4 were detected, as well as a further set of signals (m/z 1094, 1116, 1381 and 1459) that could distinguish these patients from those who were negative to both. These signals could potentially represent future targets for the further stratification of iMN patients.

  1. Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

    PubMed Central

    Saunders, Neil FW; Brinkworth, Ross I; Huber, Thomas; Kemp, Bruce E; Kobe, Bostjan

    2008-01-01

    Background We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. Results Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. Conclusion New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at . PMID:18501020

  2. Expressional Analysis and Role of Calcium Regulated Kinases in Abiotic Stress Signaling

    PubMed Central

    Das, Ritika; Pandey, Girdhar K

    2010-01-01

    Perception of stimuli and activation of a signaling cascade is an intrinsic characteristic feature of all living organisms. Till date, several signaling pathways have been elucidated that are involved in multiple facets of growth and development of an organism. Exposure to unfavorable stimuli or stress condition activates different signaling cascades in both plants and animal. Being sessile, plants cannot move away from an unfavorable condition, and hence activate the molecular machinery to cope up or adjust against that particular stress condition. In plants, role of calcium as second messenger has been studied in detail in both abiotic and biotic stress signaling. Several calcium sensor proteins such as calmodulin (CaM), calcium dependent protein kinases (CDPK) and calcinuerin B-like (CBL) were discovered to play a crucial role in abiotic stress signaling in plants. Unlike CDPK, CBL and CaM are calcium-binding proteins, which do not have any protein kinase enzyme activity and interact with a target protein kinase termed as CBL-interacting protein kinase (CIPK) and CaM kinases respectively. Genome sequence analysis of Arabidopsis and rice has led to the identification of multigene familes of these calcium signaling protein kinases. Individual and global gene expression analysis of these protein kinase family members has been analyzed under several developmental and different abiotic stress conditions. In this review, we are trying to overview and emphasize the expressional analysis of calcium signaling protein kinases under different abiotic stress and developmental stages, and linking the expression to possible function for these kinases. PMID:20808518

  3. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation.

    PubMed

    Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R; Correa, Fernando; Gardner, Kevin H

    2014-12-16

    Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.

  4. Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

    DOE PAGES

    Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R.; ...

    2014-12-02

    Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. In this paper, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light–oxygen–voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain.more » The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. Finally, we suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.« less

  5. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... shape, leading to a reduction of mevalonate kinase enzyme activity. Despite this shortage (deficiency) of mevalonate kinase activity, ... who have less than 1 percent of normal enzyme activity usually develop MVA. Learn more about the gene ...

  6. 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.

  7. 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.

  8. Kinase signalling in Huntington's disease.

    PubMed

    Bowles, Kathryn R; Jones, Lesley

    2014-01-01

    Alterations in numerous signal transduction pathways and aberrant activity of specific kinases have been identified in multiple cell and mouse models of Huntington's disease (HD), as well as in human HD brain. The balance and integration of a network of kinase signalling pathways is paramount for the regulation of a wide range of cellular and physiological processes, such as proliferation, differentiation, inflammation, neuronal plasticity and apoptosis. Unbalanced activity within these pathways provides a potential mechanism for many of the pathological phenotypes associated with HD, such as transcriptional dysregulation, inflammation and ultimately neurodegeneration. The characterisation of aberrant kinase signalling regulation in HD has been inconsistent and may be a result of failure to consider integration between multiple signalling pathways, as well as alterations that may occur over time with both age and disease progression. Collating the information about the effect of mHTT on signalling pathways demonstrates that it has wide ranging effects on multiple pro- and anti-apoptotic kinases, resulting in the dysregulation of numerous complex interactions within a dynamic network.

  9. Case report: pyruvate kinase deficiency.

    PubMed

    Rothman, J M

    1995-09-01

    Pyruvate kinase deficiency is a rare cause of congenital hemolytic anemia. Despite a paucity of reports, splenectomy resulted in successful outcomes for two siblings with this disorder. The sisters were diagnosed at birth with profound jaundice and congenital nonspherocytic hemolytic anemia.

  10. Structural plasticity and catalysis regulation of a thermosensor histidine kinase

    PubMed Central

    Albanesi, Daniela; Martín, Mariana; Trajtenberg, Felipe; Mansilla, María C.; Haouz, Ahmed; Alzari, Pedro M.; de Mendoza, Diego; Buschiazzo, Alejandro

    2009-01-01

    Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases. PMID:19805278

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

    DOE PAGES

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; ...

    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

  12. 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.

  13. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  14. Mass Sensor

    SciTech Connect

    Adams, B.E.

    2001-01-18

    The purpose of this CRADA was to use Honeywell's experience in low temperature cofire ceramics and traditional ceramics to assemble a relatively low-cost, mass-producible miniature mass analyzer. The specific design, given to us by Mass Sensors, LLC, was used to test for helium. The direct benefit for the participant was to have a prototype unit assembled for the purpose of proof of concept and the ability to secure venture capital investors. From that, the company would begin producing their own product for sale. The consumer/taxpayer benefits come from the wide variety of industries that can utilize this technology to improve quality of life. Medical industry can use this technology to improve diagnostic ability; manufacturing industry can use it for improved air, water, and soil monitoring to minimize pollution; and the law enforcement community can use this technology for identification of substances. These are just a few examples of the benefit of this technology. The benefits to DOE were in the area of process improvement for cofire and ceramic materials. From this project we demonstrated nonlinear thickfilm fine lines and spaces that were 5-mil wide with 5-mil spaces; determined height-to diameter-ratios for punched and filled via holes; demonstrated the ability to punch and fill 5-mil microvias; developed and demonstrated the capability to laser cut difficult geometries in 40-mil ceramic; developed and demonstrated coupling LTCC with standard alumina and achieving hermetic seals; developed and demonstrated three-dimensional electronic packaging concepts; and demonstrated printing variable resistors within 1% of the nominal value and within a tightly defined ratio. The capability of this device makes it invaluable for many industries. The device could be used to monitor air samples around manufacturing plants. It also could be used for monitoring automobile exhaust, for doing blood gas analysis, for sampling gases being emitted by volcanoes, for studying

  15. Variation in Arabidopsis flooding responses identifies numerous putative "tolerance genes".

    PubMed

    Vashisht, Divya; van Veen, Hans; Akman, Melis; Sasidharan, Rashmi

    2016-11-01

    Plant survival in flooded environments requires a combinatory response to multiple stress conditions such as limited light availability, reduced gas exchange and nutrient uptake. The ability to fine-tune the molecular response at the transcriptional and/or post-transcriptional level that can eventually lead to metabolic and anatomical adjustments are the underlying requirements to confer tolerance. Previously, we compared the transcriptomic adjustment of submergence tolerant, intolerant accessions and identified a core conserved and genotype-specific response to flooding stress, identifying numerous 'putative' tolerance genes. Here, we performed genome wide association analyses on 81 natural Arabidopsis accessions that identified 30 additional SNP markers associated with flooding tolerance. We argue that, given the many genes associated with flooding tolerance in Arabidopsis, improving resistance to submergence requires numerous genetic changes.

  16. Akt1 as a putative regulator of Hox genes.

    PubMed

    Kong, Kyoung-Ah; Yoon, Heejei; Kim, Myoung Hee

    2013-01-25

    In mammals, precise spatiotemporal expressions of Hox genes control the main body axis during embryogenesis. However, the mechanism by which Hox genes are regulated is poorly understood. To discover the putative regulator of Hox genes, in silico analyses were performed using GEO profiles, and Akt1 emerged as a candidate regulator of Hox genes in E13.5 MEFs. The results of the RT-PCR showed that 5' Hoxc genes, including ncRNA were upregulated in Akt1 null MEF. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing showed that the CpG island of a 5' Hoxc gene was hypomethylated in Akt1 null cells. These results indicate that Hox expression could be controlled by the function of Akt1 through epigenetic modification such as DNA methylation.

  17. Motor abnormalities as a putative endophenotype for Autism Spectrum Disorders

    PubMed Central

    Esposito, Gianluca; Paşca, Sergiu P.

    2013-01-01

    Autism Spectrum Disorders (ASDs) represent a complex group of behaviorally defined conditions with core deficits in social communication and the presence of repetitive and restrictive behaviors. To date, neuropathological studies have failed to identify pathognomonic cellular features for ASDs and there remains a fundamental disconnection between the complex clinical aspects of ASDs and the underlying neurobiology. Although not listed among the core diagnostic domains of impairment in ASDs, motor abnormalities have been consistently reported across the spectrum. In this perspective article, we summarize the evidence that supports the use of motor abnormalities as a putative endophenotype for ASDs. We argue that because these motor abnormalities do not directly depend on social or linguistic development, they may serve as an early disease indicator. Furthermore, we propose that stratifying patients based on motor development could be useful not only as an outcome predictor and in identifying more specific treatments for different ASDs categories, but also in exposing neurobiological mechanisms. PMID:23781177

  18. Catalysis-based total synthesis of putative mandelalide A.

    PubMed

    Willwacher, Jens; Fürstner, Alois

    2014-04-14

    A concise synthesis of the putative structure assigned to the highly cytotoxic marine macrolide mandelalide A (1) is disclosed. Specifically, an iridium-catalyzed two-directional Krische allylation and a cobalt-catalyzed carbonylative epoxide opening served as convenient entry points for the preparation of the major building blocks. The final stages feature the first implementation of terminal-acetylene metathesis into natural product synthesis, which is remarkable as this class of substrates was beyond reach until very recently; key to success was the use of the highly selective molybdenum alkylidyne complex 42 as the catalyst. Although the constitution and stereochemistry of the synthetic samples are unambiguous, the spectra of 1 as well as of 11-epi-1 deviate from those of the natural product, which implies a subtle but deep-seated error in the original structure assignment.

  19. Quartet analysis of putative horizontal gene transfer in Crenarchaeota.

    PubMed

    Ching, Travers H; Yoza, Brandon A; Li, Qing X

    2014-02-01

    Horizontal gene transfers (HGT) between four Crenarchaeota species (Metallosphaera cuprina Ar-4T, Acidianus hospitalis W1T, Vulcanisaeta moutnovskia 768-28T, and Pyrobaculum islandicum DSM 4184T) were investigated with quartet analysis. Strong support was found for individual genes that disagree with the phylogeny of the majority, implying genomic mosaicism. One such gene, a ferredoxin-related gene, was investigated further and incorporated into a larger phylogeny, which provided evidence for HGT of this gene from the Vulcanisaeta lineage to the Acidianus lineage. This is the first application of quartet analysis of HGT for the phylum Crenarchaeota. The results have shown that quartet analysis is a powerful technique to screen homologous sequences for putative HGTs and is useful in visually describing genomic mosaicism and HGT within four taxa.

  20. Novel putative mechanisms to link circadian clocks to healthy aging.

    PubMed

    Popa-Wagner, Aurel; Catalin, Bogdan; Buga, Ana-Maria

    2015-08-01

    The circadian clock coordinates the internal physiology to increase the homeostatic capacity thereby providing both a survival advantage to the system and an optimization of energy budgeting. Multiple-oscillator circadian mechanisms are likely to play a role in regulating human health and may contribute to the aging process. Our aim is to give an overview of how the central clock in the hypothalamus and peripheral clocks relate to aging and metabolic disorders, including hyperlipidemia and hyperglycemia. In particular, we unravel novel putative mechanisms to link circadian clocks to healthy aging. This review may lead to the design of large-scale interventions to help people stay healthy as they age by adjusting daily activities, such as feeding behavior, and or adaptation to age-related changes in individual circadian rhythms.

  1. Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda)

    PubMed Central

    Schön, Isa; Pinto, Ricardo L.; Halse, Stuart; Smith, Alison J.; Martens, Koen; Birky, C. William

    2012-01-01

    Background Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. Methodology/Principal Findings We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. Conclusions/Significance Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated. PMID:22802945

  2. 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

  3. Characterization of two new putative adhesins of Leptospira interrogans.

    PubMed

    Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

    2017-01-01

    We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

  4. Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1.

    PubMed

    Burch, Lindsay R; Scott, Mary; Pohler, Elizabeth; Meek, David; Hupp, Ted

    2004-03-12

    Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.

  5. 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.

  6. A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila.

    PubMed Central

    Therrien, M; Morrison, D K; Wong, A M; Rubin, G M

    2000-01-01

    kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed. PMID:11063697

  7. The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

    PubMed Central

    Awad, Rida; Marion, Sévajol; Isabel, Ayala; Anne, Chouquet; Philippe, Frachet; Pierre, Gans; Jean-Baptiste, Reiser; Jean-Philippe, Kleman

    2015-01-01

    Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling. PMID:25737835

  8. 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

  9. The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

    PubMed Central

    Lorenz, W; Inglese, J; Palczewski, K; Onorato, J J; Caron, M G; Lefkowitz, R J

    1991-01-01

    Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors. Images PMID:1656454

  10. 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

  11. G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

    PubMed

    Chakraborty, Prabir Kumar; Zhang, Yushan; Coomes, Alexandra S; Kim, Wan-Ju; Stupay, Rachel; Lynch, Lauren D; Atkinson, Tamieka; Kim, Jae I; Nie, Zhongzhen; Daaka, Yehia

    2014-07-01

    G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

  12. 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.

  13. Aurora kinases: novel therapy targets in cancers.

    PubMed

    Tang, Anqun; Gao, Keyu; Chu, Laili; Zhang, Rui; Yang, Jing; Zheng, Junnian

    2017-01-29

    Aurora kinases, a family of serine/threonine kinases, consisting of Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC), are essential kinases for cell division via regulating mitosis especially the process of chromosomal segregation. Besides regulating mitosis, Aurora kinases have been implicated in regulating meiosis. The deletion of Aurora kinases could lead to failure of cell division and impair the embryonic development. Overexpression or gene amplification of Aurora kinases has been clarified in a number of cancers. And a growing number of studies have demonstrated that inhibition of Aurora kinases could potentiate the effect of chemotherapies. For the past decades, a series of Aurora kinases inhibitors (AKIs) developed effectively repress the progression and growth of many cancers both in vivo and in vitro, suggesting that Aurora kinases could be a novel therapeutic target. In this review, we'll first briefly present the structure, localization and physiological functions of Aurora kinases in mitosis, then describe the oncogenic role of Aurora kinases in tumorigenesis, we shall finally discuss the outcomes of AKIs combination with conventional therapy.

  14. Receptor tyrosine kinases in carcinogenesis.

    PubMed

    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  15. Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases.

    PubMed

    Murungi, Edwin K; Kariithi, Henry M

    2017-03-21

    The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

  16. Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

    PubMed Central

    Murungi, Edwin K.; Kariithi, Henry M.

    2017-01-01

    The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host. PMID:28335576

  17. Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

    PubMed

    Vijapurkar, Ulka; Kim, Myong-Soo; Koland, John G

    2003-04-01

    ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here. To this end, cDNAs encoding the wild-type ErbB3 protein (ErbB3-WT) and ErbB3 proteins with amino acid substitutions in either the Shc-binding site (ErbB3-Y1325F), the six putative PI 3-kinase-binding sites (ErbB3-6F), or both (ErbB3-7F) were generated and expressed in NIH-3T3 cells to form functional ErbB2/ErbB3 heregulin coreceptors. While the coreceptor incorporating ErbB3-WT activated both the MAPK and the PI 3-kinase signaling pathways, those incorporating ErbB3-Y1325F or ErbB3-6F activated either PI 3-kinase or MAPK, respectively. The ErbB2/ErbB3-7F coreceptor activated neither. Elimination of either signaling pathway lowered basal and eliminated heregulin-dependent expression of cyclin D1, which was in each case accompanied by an attenuated mitogenic response. Selective elimination of the PI 3-kinase pathway severely impaired the ability of heregulin to transform cells expressing the coreceptor, whereas attenuation of the MAPK pathway had a lesser effect. Thus, while both pathways contributed in a roughly additive manner to the mitogenic response elicited by the activated ErbB2/ErbB3 coreceptor, the PI 3-kinase pathway predominated in the induction of cellular transformation.

  18. 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.

  19. Carbon Dioxide Sensor Technology.

    DTIC Science & Technology

    1983-04-01

    identify the promising sensor concepts is provided in this section along with the basic conclusions that were reached regarding avail- able sensor techniques ...63 Estimated Operating Characteristics ... .......... 65 Conclusions and Recommendations (SAW) ... ......... 68 Calorimetric Technique ...Desired Sensor Properties. .. .. ... .. ... .. ..... 8 Table 2A. Candidate Sensor Techniques . .. .. ... .. ... .. ... 14 Selected for Further Analysis

  20. 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.

  1. Wireless ferroelectric resonating sensor.

    PubMed

    Viikari, Ville; Seppa, Heikki; Mattila, Tomi; Alastalo, Ari

    2010-04-01

    This paper presents a passive wireless resonating sensor that is based on a ferroelectric varactor. The sensor replies with its data at an intermodulation frequency when a reader device illuminates it at 2 closely located frequencies. The paper derives a theoretical equation for the response of such a sensor, verifies the theory by simulations, and demonstrates a temperature sensor based on a ferroelectric varactor.

  2. 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

  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. Nucleotide selectivity of antibiotic kinases.

    PubMed

    Shakya, Tushar; Wright, Gerard D

    2010-05-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

  5. Nanotube Sensors

    NASA Technical Reports Server (NTRS)

    McEuen, Paul L.

    2002-01-01

    Under this project, we explored the feasibility of utilizing carbon nanotubes in sensing applications. The grant primarily supported a graduate student, who worked on a number of aspects of the electrical properties of carbon nanotubes in collaboration with other researchers in my group. The two major research accomplishments are described below. The first accomplishment is the demonstration that solution carbon nanotube transistors functioned well in an electrolyte environment. This was important for two reasons. First, it allowed us to explore the ultimate limits of nanotube electronic performance by using the electrolyte as a highly effective gate, with a dielectric constant of approximately 80 and an effective insulator thickness of approximately 1 nm. Second, it showed that nanotubes function well under biologically relevant conditions (salty water) and therefore offer great promise as biological sensors. The second accomplishment was the demonstration that a voltage pulse applied to an AFM tip could be used to electrically cut carbon nanotubes. We also showed that a carefully applied pulse could also 'nick' a nanotube, creating a tunnel barrier without completely breaking the tube. Nicking was employed to make, for example, a quantum dot within a nanotube.

  6. 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.

  7. 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

  8. 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.

  9. 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...

  10. A human NDP-kinase B specifically binds single-stranded poly-pyrimidine sequences.

    PubMed Central

    Hildebrandt, M; Lacombe, M L; Mesnildrey, S; Véron, M

    1995-01-01

    Recently, a DNA binding protein 'PUF' was purified that binds to a poly-pyrimidine rich element in the human c-myc promoter. Cloning of the corresponding gene surprisingly identified this putative transcription factor as isoform B of the enzyme nucleoside diphosphate kinase (NDPK-B) [Postel et al. (1993) Science, 261, 478-480], the product of the potential metastasis suppressor gene nm23-H2. Using different recombinant NDP kinases, we demonstrate by electrophoretic mobility shift analysis (EMSA) that the NDP kinase DNA binding properties are predominantly observed with human isoform B. Unlike typical DNA binding proteins that are involved in transcriptional regulation, binding occurs to single-stranded DNA rather than to a double-stranded oligonucleotide. As a consequence, complexes of single-stranded DNA and NDPK-B are generated from double-stranded oligonucleotide hybrids in an ATP independent manner. In addition to the c-myc element, NDPK-B is binding in vitro to a variety of poly-pyrimidine rich sequences including dC or dT homo-oligomers, (CT)n dinucleotide repeats, the initiator region of the Adenovirus major late promoter and even poly-pyrimidine rich RNAs. The possible consequences of these findings in understanding the multiple roles of NDP kinase are discussed. Images PMID:7479028

  11. 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.

  12. A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

    PubMed

    Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

    2017-03-01

    Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface.

  13. High-content screen using zebrafish (Danio rerio) embryos identifies a novel kinase activator and inhibitor.

    PubMed

    Geldenhuys, Werner J; Bergeron, Sadie A; Mullins, Jackie E; Aljammal, Rowaa; Gaasch, Briah L; Chen, Wei-Chi; Yun, June; Hazlehurst, Lori A

    2017-02-28

    In this report we utilized zebrafish (Danio rerio) embryos in a phenotypical high-content screen (HCS) to identify novel leads in a cancer drug discovery program. We initially validated our HCS model using the flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum (ER) enzyme, endoplasmic reticulum oxidoreductase (ERO1) inhibitor EN460. EN460 showed a dose response effect on the embryos with a dose of 10μM being significantly lethal during early embryonic development. The HCS campaign which employed a small library identified a promising lead compound, a naphthyl-benzoic acid derivative coined compound 1 which had significant dosage and temporally dependent effects on notochord and muscle development in zebrafish embryos. Screening a 369 kinase member panel we show that compound 1 is a PIM3 kinase inhibitor (IC50=4.078μM) and surprisingly a DAPK1 kinase agonist/activator (EC50=39.525μM). To our knowledge this is the first example of a small molecule activating DAPK1 kinase. We provide a putative model for increased phosphate transfer in the ATP binding domain when compound 1 is virtually docked with DAPK1. Our data indicate that observable phenotypical changes can be used in future zebrafish screens to identify compounds acting via similar molecular signaling pathways.

  14. 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.

  15. 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.

  16. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

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

    2013-01-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  17. 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

  18. Small molecule adenosine 5'-monophosphate activated protein kinase (AMPK) modulators and human diseases.

    PubMed

    Rana, Sandeep; Blowers, Elizabeth C; Natarajan, Amarnath

    2015-01-08

    Adenosine 5'-monophosphate activated protein kinase (AMPK) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy homeostasis. AMPK is a serine/threonine kinase that is activated by upstream kinases LKB1, CaMKKβ, and Tak1, among others. AMPK exists as αβγ trimeric complexes that are allosterically regulated by AMP, ADP, and ATP. Dysregulation of AMPK has been implicated in a number of metabolic diseases including type 2 diabetes mellitus and obesity. Recent studies have associated roles of AMPK with the development of cancer and neurological disorders, making it a potential therapeutic target to treat human diseases. This review focuses on the structure and function of AMPK, its role in human diseases, and its direct substrates and provides a brief synopsis of key AMPK modulators and their relevance in human diseases.

  19. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Putative transmembrane transporter modulates higher-level aggression in Drosophila.

    PubMed

    Chowdhury, Budhaditya; Chan, Yick-Bun; Kravitz, Edward A

    2017-02-28

    By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

  5. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury.

    PubMed

    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.

  6. Phytophthora infestans specific phosphorylation patterns and new putative control targets.

    PubMed

    Frades, Itziar; Andreasson, Erik

    2016-04-01

    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

  7. Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae.

    PubMed

    Blau, Karin; Portnoi, Maxim; Shagan, Marilou; Kaganovich, Antonina; Rom, Slava; Kafka, Daniel; Chalifa Caspi, Vered; Porgador, Angel; Givon-Lavi, Noga; Gershoni, Jonathan M; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2007-06-15

    Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.

  8. 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.

  9. General pharmacology of the putative cognition enhancer linopirdine.

    PubMed

    Flagmeyer, I; Gebert, I; van der Staay, F J

    1995-04-01

    The putative cognition enhancer linopirdine (3,3-bis(4-pyrindinylmethyl)-1-phenylindolin-2-one, CAS 105431-72-9) is supposed to act by enhancing the release of neurotransmitters, especially acetylcholine. The present study assessed the effects of a single administration of this compound on the central nervous system in eight different rat and mouse models (CNS general pharmacology). In each test performed, linopirdine was administered subcutaneously in doses of 3, 10, and 30 mg/kg. The compound did not affect traction ability and nociceptive responsiveness nor did it induce catalepsy. Linopirdine impaired motor coordination in the balance rod test. The compound showed a distinct proconvulsive action in the pentylenetetrazole threshold dose test and induced in the highest dose tested (30 mg/kg) lethal seizures in some mice. It increased the duration of hexobarbital-induced anaesthesia in mice. Rats treated with linopirdine showed ptosis, salivation, slight sedation, paw beating and slight hypothermia. These results support the hypothesis that linopirdine acts by elevating the release of different neurotransmitters such as acetylcholine and dopamine. The compound has a low potential to produce side effects at pharmacodynamic active doses.

  10. Inhalation of two putative Gulf War toxins by mice.

    PubMed

    Repine, John E; Wilson, Paul; Elkins, Nancy; Klawitter, Jelena; Christians, Uwe; Peters, Ben; Smith, Dwight M

    2016-01-01

    We employed our inhalation methodology to examine whether biomarkers of inflammation and oxidative stress would be produced in mice following inhalation of aerosols containing carbonaceous particles or the vapor of pesticides prevalent during the first Gulf War. Exposure to two putative Gulf War Illness toxins, fine airborne particles and the pesticide malathion, increased biomarkers of inflammation and oxidative stress in Friend virus B (FVB) female mice. Mice inhaling particles 24 h before had increased lung lavage and plasma Leukotriene B4 (LTB4) (a biomarker of inflammation) and PGF2α (a biomarker of oxidative stress) levels, lung lavage protein and lung lavage lactic dehydrogenase (LDH) levels. These changes were a function of particle density and exposure time. Compared to particle inhalation, mice inhaling malathion 24 h before had small increase in plasma LTB4 and PGF2α levels but no increase in lung lavage LTB4, lung lavage protein, lung lavage LDH, and lung lavage alveolar macrophage (AM) levels compared to unexposed control mice. AM from particle-exposed mice contained phagocytosed particles, while AM from malathion-exposed mice showed no abnormalities. Our results indicate that inhaling particles or malathion can alter inflammatory and oxidative biomarkers in mice and raise the possibility that these toxins may have altered inflammation and oxidative stress biomarkers in Gulf War-exposed individuals.

  11. Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens

    PubMed Central

    1987-01-01

    The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma- derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12- O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha- phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2- dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become

  12. 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

  13. Aurora kinase inhibitors as anticancer molecules.

    PubMed

    Katayama, Hiroshi; Sen, Subrata

    2010-01-01

    Aurora kinase family of serine/threonine kinases are important regulators of mitosis that are frequently over expressed in human cancers and have been implicated in oncogenic transformation including development of chromosomal instability in cancer cells. In humans, among the three members of the kinase family, Aurora-A, -B and -C, only Aurora-A and -B are expressed at detectable levels in all somatic cells undergoing mitotic cell division and have been characterized in greater detail for their involvement in cellular pathways relevant to the development of cancer associated phenotypes. Aurora-A and -B are being investigated as potential targets for anticancer therapy. Development of inhibitors against Aurora kinases as anticancer molecules gained attention because of the facts that aberrant expression of these kinases leads to chromosomal instability and derangement of multiple tumor suppressor and oncoprotein regulated pathways. Preclinical studies and early phase I and II clinical trials of multiple Aurora kinase inhibitors as targeted anticancer drugs have provided encouraging results. This article discusses functional involvement of Aurora kinase-A and -B in the regulation of cancer relevant cellular phenotypes together with findings on some of the better characterized Aurora kinase inhibitors in modulating the functional interactions of Aurora kinases. Future possibilities about developing next generation Aurora kinase inhibitors and their clinical utility as anticancer therapeutic drugs are also discussed.

  14. Aurora Kinase inhibitors as Anticancer Molecules

    PubMed Central

    Katayama, Hiroshi; Sen, Subrata

    2015-01-01

    Aurora kinase family of serine/threonine kinases are important regulators of mitosis that are frequently over expressed in human cancers and have been implicated in oncogenic transformation including development of chromosomal instability in cancer cells. In humans, among the three members of the kinase family, Aurora-A, -B and -C, only Aurora-A and -B are expressed in detectable levels in somatic cells undergoing mitotic cell division and have been characterized in greater detail for their involvement in cellular pathways relevant to the development of cancer associated phenotypes. Aurora-A and -B are being investigated as potential targets for anticancer therapy. Development of inhibitors against Aurora kinases as anticancer molecules gained attention because of the facts that aberrant expression of these kinases lead to chromosomal instability and derangement of multiple tumor suppressor and oncoprotein regulated pathways. Pre-clinical studies and early phase I and II clinical trials of multiple Aurora kinase inhibitors as targeted anticancer drugs have provided encouraging results. This article discusses functional involvement of Aurora kinase-A and -B in the regulation of cancer relevant cellular phenotypes together with findings on some of the better characterized Aurora kinase inhibitors in modulating the functional interactions of Aurora kinases. Future possibilities about developing next generation Aurora kinase inhibitors and their clinical utility as anticancer therapeutic drugs are also discussed. PMID:20863917

  15. Shewanella oneidensis MR-1 H-NOX Regulation of a Histidine Kinase by Nitric Oxide

    PubMed Central

    Price, Mark S.; Chao, Lily; Marletta, Michael A.

    2008-01-01

    Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologs of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis, directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor. PMID:17988156

  16. Sensor modules for wireless distributed sensor networks

    SciTech Connect

    Lee, A P; McConaghy, C F; Simon, J N; Benett, W; Jones, L; Trevino, J

    1999-02-22

    A national security need as well as environmental monitoring need exists for networks of sensors. The advantages of a network of sensors over a single sensor are improved range, sensitivity, directionality, and data readability. Depending upon the particular application, sensors can be acoustic, chemical, biological, thermal or inertial. A major desire in these sensor networks is to have the individual sensor and associated electronics small and low enough in power that the battery can also be small and of long life. Smaller, low power sensor nodes can allow more nodes per network. A typical network for security applications is depicted in Figure 1. Here a number of sensor nodes are deployed around a central hub node in a star configuration. In this scenario the hubs communicate with each other and ultimately relay information to a satellite. Future networks might follow this scenario or some other network architecture such as a hopping network where individual nodes communicate directly with each other. The focus of our research has been on development of the small low power nodes and less on the overall network topology. However, some consideration of the network must be given when designing the nodes and some consideration of the nodes must be given when designing the network. An individual sensor node contains not only the sensor but also the sensor interface electronics, analog to digital (A/D) converter, logic, RF communication link, antenna, and the battery. Future nodes will also contain some form of signal processing to allow more sophisticated network architectures. The FY98 goal for this project was to make a sensor node with a physical form factor of a 2 inch x 2 inch x 2 inch cube.

  17. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Inferring protein function from genomic sequence: Giardia lamblia expresses a phosphatidylinositol kinase-related kinase similar to yeast and mammalian TOR.

    PubMed

    Morrison, Hilary G; Zamora, Gus; Campbell, Robert K; Sogin, Mitchell L

    2002-12-01

    Functional assays of genes have historically led to insights about the activities of a protein or protein cascade. However, the rapid expansion of genomic and proteomic information for a variety of diverse taxa is an alternative and powerful means of predicting function by comparing the enzymes and metabolic pathways used by different organisms. As part of the Giardia lamblia genome sequencing project, we routinely survey the complement of predicted proteins and compare those found in this putatively early diverging eukaryote with those of prokaryotes and more recently evolved eukaryotic lineages. Such comparisons reveal the minimal composition of conserved metabolic pathways, suggest which proteins may have been acquired by lateral transfer, and, by their absence, hint at functions lost in the transition from a free-living to a parasitic lifestyle. Here, we describe the use of bioinformatic approaches to investigate the complement and conservation of proteins in Giardia involved in the regulation of translation. We compare an FK506 binding protein homologue and phosphatidylinositol kinase-related kinase present in Giardia to those found in other eukaryotes for which complete genomic sequence data are available. Our investigation of the Giardia genome suggests that PIK-related kinases are of ancient origin and are highly conserved.

  3. 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

  4. 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

  5. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  6. 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…

  7. Spatial gradients in kinase cascade regulation.

    PubMed

    Kazmierczak, B; Lipniacki, T

    2010-11-01

    The spatiotemporal kinetics of proteins and other substrates regulate cell fate and signaling. In this study, we consider a reaction-diffusion model of interaction of membrane receptors with a two-step kinase cascade. The receptors activate the 'up-stream' kinase, which may diffuse over cell volume and activate the 'down-stream' kinase, which is also diffusing. Both kinase species and receptors are inactivated by uniformly distributed phosphatases. The positive feedback, key to the considered dynamics, arises since the up-stream kinase activates the receptors. Such a mutual interaction is characteristic for immune cell receptors. Based on the proposed model, we demonstrated that cell sensitivity (measured as a critical value of phosphatase activity at which cell maybe activated) increases with decreasing motility of receptor-interacting kinases and with increasing polarity of receptors distribution. These two effects are cooperating, the effect of receptors localisation close to one pole of the cell grows with the decreasing kinase diffusion and vanishes in the infinite diffusion limit. As the cell sensitivity increases with decreasing diffusion of receptor-interacting kinase, the overall activity of the down-stream kinase increases with its diffusion. In conclusion, the analysis of the proposed model shows that, for the fixed substrate interaction rates, spatial distribution of the surface receptors together with the motility of intracellular kinases control cell signalling and sensitivity to extracellular signals. The increase of the cell sensitivity can be achieved by (i) localisation of receptors in a small subdomain of the cell membrane, (ii) lowering the motility of receptor-interacting kinase, (iii) increasing the motility of down-stream kinases which distribute the signal over the whole cell.

  8. The phosphoinositide 3-kinase pathway.

    PubMed

    Cantley, Lewis C

    2002-05-31

    Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

  9. 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

  10. 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.

  11. Glycerate kinase of the hyperthermophilic archaeon Thermoproteus tenax: new insights into the phylogenetic distribution and physiological role of members of the three different glycerate kinase classes

    PubMed Central

    Kehrer, Daniel; Ahmed, Hatim; Brinkmann, Henner; Siebers, Bettina

    2007-01-01

    Background The presence of the branched Entner-Doudoroff (ED) pathway in two hyperthermophilic Crenarchaea, the anaerobe Thermoproteus tenax and the aerobe Sulfolobus solfataricus, was suggested. However, so far no enzymatic information of the non-phosphorylative ED branch and especially its key enzyme – glycerate kinase – was available. In the T. tenax genome, a gene homolog with similarity to putative hydroxypyruvate reductase/glycerate dehydrogenase and glycerate kinase was identified. Results The encoding gene was expressed in E. coli in a recombinant form, the gene product purified and the glycerate kinase activity was confirmed by enzymatic studies. The enzyme was active as a monomer and catalyzed the ATP-dependent phosphorylation of D-glycerate forming exclusively 2-phosphoglycerate. The enzyme was specific for glycerate and highest activity was observed with ATP as phosphoryl donor and Mg2+ as divalent cation. ATP could be partially replaced by GTP, CTP, TTP and UTP. The enzyme showed high affinity for D-glycerate (Km 0.02 ± 0.01 mM, Vmax of 5.05 ± 0.52 U/mg protein) as well as ATP (Km of 0.03 ± 0.01 mM, Vmax of 4.41 ± 0.04 U/mg protein), although at higher glycerate concentrations, substrate inhibition was observed. Furthermore, the enzyme was inhibited by its product ADP via competitive inhibition. Data bank searches revealed that archaeal glycerate kinases are members of the MOFRL (multi-organism fragment with rich leucine) family, and homologs are found in all three domains of life. Conclusion A re-evaluation of available genome sequence information as well as biochemical and phylogenetic studies revealed the presence of the branched ED pathway as common route for sugar degradation in Archaea that utilize the ED pathway. Detailed analyses including phylogenetic studies demonstrate the presence of three distinct glycerate kinase classes in extant organisms that share no common origin. The affiliation of characterized glycerate kinases with the

  12. 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.

  13. Tyrosine kinases in inflammatory dermatologic disease

    PubMed Central

    Paniagua, Ricardo T.; Fiorentino, David; Chung, Lorinda; Robinson, William H.

    2010-01-01

    Tyrosine kinases are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific tyrosine kinases have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of tyrosine kinases are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight tyrosine kinase signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development. PMID:20584561

  14. MST kinases in development and disease

    PubMed Central

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease. PMID:26370497

  15. JAK2 and AMP-kinase inhibition in vitro by food extracts, fractions and purified phytochemicals.

    PubMed

    Martin, Harry; Burgess, Elaine J; Smith, Wendy A; McGhie, Tony K; Cooney, Janine M; Lunken, Rona C M; de Guzman, Erika; Trower, Tania; Perry, Nigel B

    2015-01-01

    We have identified a range of food phytochemicals that inhibit Janus Kinase 2 (JAK2) and Adenosine Monophosphate Kinase (AMPK). A mutated and dysregulated form of JAK2, a tyrosine kinase, is associated with several diseases including Crohn's disease. Using an in vitro, time-resolved fluorescence (TR-FRET) assay, we tested 49 different types of food extracts, plus 10 concentrated fractions of increasing hydrophobicity from each extract, to find foods containing JAK2 inhibitors. The food extracts tested included grains, meat, fish, shellfish, dairy products, herbs, mushrooms, hops, fruits and vegetables. Several fruits were potent inhibitors of JAK2: blackberry, boysenberry, feijoa, pomegranate, rosehip and strawberry, which all contain ellagitannins, known inhibitors of kinases. These fruits are in the Rosales and Myrtales plant orders. No other foods gave >1% of the maximal JAK2 inhibitory activities of these fruits. AMPK, a sensor and regulator of energy metabolism in cells, is a serine-threonine kinase which is reported to be activated by various flavonoid phytochemicals. Using a TR-FRET assay, we tested various fruit extracts for AMPK activation and inhibition. Ellagitannin containing foods scored highly as AMPK inhibitors. Despite several reports of AMPK activation in whole cells by phytochemicals, no extracts or pure compounds activated AMPK in our assay.

  16. Helix bundle loops determine whether histidine kinases autophosphorylate in cis or in trans.

    PubMed

    Ashenberg, Orr; Keating, Amy E; Laub, Michael T

    2013-04-12

    Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.

  17. 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

  18. 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.

  19. 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

  20. Signal transduction by epidermal growth factor and heregulin via the kinase-deficient ErbB3 protein.

    PubMed Central

    Kim, H H; Vijapurkar, U; Hellyer, N J; Bravo, D; Koland, J G

    1998-01-01

    The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys-->Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins. PMID:9693119

  1. Signal transduction by epidermal growth factor and heregulin via the kinase-deficient ErbB3 protein.

    PubMed

    Kim, H H; Vijapurkar, U; Hellyer, N J; Bravo, D; Koland, J G

    1998-08-15

    The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys-->Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.

  2. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-03-15

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.

  3. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed Central

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-01-01

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase. PMID:9148746

  4. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein.

    PubMed Central

    Celenza, J L; Carlson, M

    1989-01-01

    The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability. Images PMID:2557546

  5. 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.

  6. Diacylglycerol kinases in membrane trafficking

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years. PMID:27057419

  7. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  8. Learning sensor models for wireless sensor networks

    NASA Astrophysics Data System (ADS)

    Ertin, Emre

    2007-04-01

    Sensor data generation is a key component of high fidelity design and testing of applications at scale. In addition to its utility in validation of applications and network services, it provides a theoretical basis for the design of algorithms for efficient sampling, compression and exfiltration of the sensor readings. Modeling of the environmental processes that gives rise to sensor readings is the core problem in physical sciences. Sensor modeling for wireless sensor networks combine the physics of signal generation and propagation with models of transducer saturation and fault models for hardware. In this paper we introduce a novel modeling technique for constructing probabilistic models for censored sensor readings. The model is an extension of the Gaussian process regression and applies to continuous valued readings subject to censoring. We illustrate the performance of the proposed technique in modeling wireless propagation between nodes of a wireless sensor network. The model can capture the non-isotropic nature of the propagation characteristics and utilizes the information from the packet reception failures. We use measured data set from the Kansei sensor network testbed using 802.15.4 radios.

  9. Dominant Mutations of Drosophila Map Kinase Kinase and Their Activities in Drosophila and Yeast Map Kinase Cascades

    PubMed Central

    Lim, Y. M.; Tsuda, L.; Inoue, Y. H.; Irie, K.; Adachi-Yamada, T.; Hata, M.; Nishi, Y.; Matsumoto, K.; Nishida, Y.

    1997-01-01

    Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors. PMID:9136016

  10. 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.

  11. Air Sensor Toolbox

    EPA Pesticide Factsheets

    Air Sensor Toolbox provides information to citizen scientists, researchers and developers interested in learning more about new lower-cost compact air sensor technologies and tools for measuring air quality.

  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. 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

  14. Wake Vortex Sensors Requirements Overview

    NASA Technical Reports Server (NTRS)

    Hinton, David A.

    1997-01-01

    The presentation includes discussions of primary wake vortex system requirements, evolution models, sensor evolution, site specific sensor tradeoffs, wake sensor functions, deployment considerations, the operational test bed system and additional sensor requirements.

  15. Calorie restriction: is AMPK as a key sensor and effector?

    PubMed Central

    Cantó, Carles; Auwerx, Johan

    2013-01-01

    Summary Dietary restriction can extend lifespan in most organisms tested to date, suggesting that mechanisms sensing nutrient and energy availability might regulate longevity. The AMP-activated protein kinase (AMPK) has emerged as a key energy sensor with the ability to transcriptionally reprogram the cell and metabolically adapt to external cues. In this review we will discuss the possible role of AMPK in the beneficial effects of calorie restriction on health and lifespan. PMID:21841070

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Directional Acoustic Density Sensor

    DTIC Science & Technology

    2010-09-13

    fluctuations of fluid density at a point . (2) DESCRIPTION OF THE PRIOR ART [0004] Conventional vector sensors measure particle velocity, v (vx,Vytvz...dipole-type or first order sensor that is realized by measuring particle velocity at a point , (which is the vector sensor sensing approach for...underwater sensors), or by measuring the gradient of the acoustic pressure at two closely spaced (less than the wavelength of an acoustic wave) points as it

  1. Targeting chk2 kinase: molecular interaction maps and therapeutic rationale.

    PubMed

    Pommier, Yves; Sordet, Olivier; Rao, V Ashutosh; Zhang, Hongliang; Kohn, Kurt W

    2005-01-01

    Most anticancer drugs presently used clinically target genomic DNA. The selectivity of these anticancer drugs for tumor tissues is probably due to tumor-specific defects suppressing cell cycle checkpoints and DNA repair, and enhancing apoptotic response in the tumor. We will review the molecular interactions within the ATM-Chk2 pathway implicating the DNA damage sensor kinases (ATM, ATR and DNA-PK), the adaptor BRCT proteins (Nbs1, Brca1, 53BP1, MDC1) and the effector kinases (Chk2, Chk1, Plk3, JNK, p38). The molecular interaction map convention (MIM) will be used for presenting this molecular network (http://discover.nci.nih.gov/mim/). A characteristic of the ATM-Chk2 pathway is its redundancy. First, ATM and Chk2 phosphorylate common substrates including p53, E2F1, BRCA1, and Chk2 itself, which suggests that Chk2 (also known as CHECK2, Cds1 in fission yeast, and Dmchk2 or Dmnk or Loki in the fruit fly) acts as a relay for ATM and/or as a salvage pathway when ATM is inactivated. Secondly, redundancy is apparent for the substrates, which can be phosphorylated/activated at similar residues by Chk2, Chk1, and the polo kinases (Plk's). Functionally, Chk2 can activate both apoptosis (via p53, E2F1 and PML) and cell cycle checkpoint (via Cdc25A and Cdc25C, p53, and BRCA1). We will review the short list of published Chk2 inhibitors. We will also propose a novel paradigm for screening interfacial inhibitors of Chk2. Chk2 inhibitors might be used to enhance the tumor selectivity of DNA targeted agents in p53-deficient tumors, and for the treatment of tumors whose growth depends on enhanced Chk2 activity.

  2. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways.

    PubMed

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Lin, Chun-Cheng; Lin, Chun-Hung; Hsu, Jason; Wong, Chi-Huey

    2004-11-15

    We have demonstrated that an extract of Ganoderma lucidum (Reishi or Ling-Zhi) polysaccharides (EORP) exerts immunomodulating activities by stimulating the expression of inflammatory cytokines from mouse spleen cells. Interestingly, via responding to LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, and using TLR4 Ab blockage in human blood-derived monocytic macrophages, we have found that the TLR4, but not complement receptor type 3, is a putative receptor of EORP, mediating the consequent immunomodulating events associated with IL-1 gene expression. Based on our studies of reactive oxygen species production, polymyxin B inhibition, and protein tyrosine kinase (PTK) activity, we ruled out the possibility of LPS contamination in EORP. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with inflammatory cytokine IL-1. In human macrophages and murine macrophage J774A.1 cells, EORP was found to up-regulate IL-1 secretion and pro-IL-1 (precursor of IL-1) as well as IL-1-converting enzyme expression. Specifically, EORP rapidly stimulates PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1 ELISA, we have extensively examined and dissected the role of individual PK in the regulation of pro-IL-1/IL-1. Our findings establish that EORP-mediated signaling pathways are involved in the pro-IL-1/IL-1 regulation: PTK/protein kinase C/MEK1/ERK and PTK/Rac1/p21-activated kinase/p38.

  3. Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity.

    PubMed

    Zhong, Hanbing; Wang, Danyang; Wang, Nan; Rios, Yesenia; Huang, Haigen; Li, Song; Wu, Xinrong; Lin, Shuo

    2011-07-01

    Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)(zn1) transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.

  4. Effects of AMP-activated protein kinase in cerebral ischemia.

    PubMed

    Li, Jun; McCullough, Louise D

    2010-03-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to 'cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed.

  5. Protein engineering of bacterial histidine kinase receptor systems.

    PubMed

    Xie, Wei; Blain, Katherine Y; Kuo, Mario Meng-Chiang; Choe, Senyon

    2010-07-01

    Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

  6. Effects of AMP-activated protein kinase in cerebral ischemia

    PubMed Central

    Li, Jun; McCullough, Louise D

    2010-01-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to ‘cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed. PMID:20010958

  7. 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.

  8. 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.

  9. 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

  10. Micro sun sensor

    NASA Technical Reports Server (NTRS)

    Liebe, C. C.; Mobasser, S.; Wrigley, C. J.; Bae, Y.; Howard, A.; Schroeder, J.

    2002-01-01

    A new generation of sun sensors is emerging. These sun sensors utilize an imaging detector and the sun sensor determines the sun angles based on an image of fringes or centroids on the detector plane. Typically determines the sun angle in two axes.

  11. 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.

  12. 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.

  13. 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

  14. Fetal anaemia due to pyruvate kinase deficiency.

    PubMed Central

    Gilsanz, F; Vega, M A; Gómez-Castillo, E; Ruiz-Balda, J A; Omeñaca, F

    1993-01-01

    Pyruvate kinase deficiency was diagnosed in an infant by umbilical vessel sampling at 30 weeks' gestation. Although three previous hydropic siblings had been stillborn or died in the neonatal period, this infant survived with transfusion dependent haemolytic anaemia. Prompt fetal diagnosis of pyruvate kinase deficiency is feasible and allows better management of hydrops fetalis due to this disorder. PMID:8285758

  15. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

    Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

  16. 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

  17. Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition.

    PubMed

    Zhang, Shumin; Wang, Xu; Iqbal, Shareen; Wang, Yanru; Osunkoya, Adeboye O; Chen, Zhengjia; Chen, Zhuo; Shin, Dong M; Yuan, Hongwei; Wang, Yongqiang A; Zhau, Haiyen E; Chung, Leland W K; Ritenour, Chad; Kucuk, Omer; Wu, Daqing

    2013-01-18

    Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

  18. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

    PubMed

    Gravesen, A; Sørensen, K; Aarestrup, F M; Knøchel, S

    2001-01-01

    A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

  19. 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

  20. Aurora Kinase Inhibitors: Current Status and Outlook.

    PubMed

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions.

  1. Aurora Kinase Inhibitors: Current Status and Outlook

    PubMed Central

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions. PMID:26734566

  2. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

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

    PubMed

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

    2016-03-27

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

  4. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  5. 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.

  6. 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.

  7. 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

  8. Heat Perception and Aversive Learning in Honey Bees: Putative Involvement of the Thermal/Chemical Sensor AmHsTRPA.

    PubMed

    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.

  9. Sensor mount assemblies and sensor assemblies

    DOEpatents

    Miller, David H [Redondo Beach, CA

    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.

  10. Multimission unattended ground sensor

    NASA Astrophysics Data System (ADS)

    Prado, Gervasio; Succi, George P.; Fitzgerald, James; Clapp, Daniel; Gampert, Robert; Martel, Philip O.

    2002-08-01

    Technological advances in a number of fields have allowed SenTech to develop a highly capable Unattended Ground Sensor (UGS) able to perform a number of critical missions such as ground and air vehicle surveillance, personnel detection and tracking and sniper localization. These sensors have also been combined with electro-optic sensors to provide target images and improved tracking accuracy. Processing is done in a highly integrated processing module developed under DARPA's IUGS program. Acoustic sensors have been engineered to achieve a three-pound unit with a 15 day field life and long range VHF communications. These sensors will be delivered in early 2002 for testing during field exercises. Extensive testing of the algorithms and software has been conducted over the last few years at a variety of government-sponsored tests and demonstrations. A Gateway unit has been developed which can manage the operation of an eight-sensor field and perform two-dimensional sensor fusion.

  11. Silicon force sensor

    DOEpatents

    Galambos, Paul C.; Crenshaw, Thomas B.; Nishida, Erik E.; Burnett, Damon J.; Lantz, Jeffrey W.

    2016-07-05

    The various technologies presented herein relate to a sensor for measurement of high forces and/or high load shock rate(s), whereby the sensor utilizes silicon as the sensing element. A plate of Si can have a thinned region formed therein on which can be formed a number of traces operating as a Wheatstone bridge. The brittle Si can be incorporated into a layered structure comprising ductile and/or compliant materials. The sensor can have a washer-like configuration which can be incorporated into a nut and bolt configuration, whereby tightening of the nut and bolt can facilitate application of a compressive preload upon the sensor. Upon application of an impact load on the bolt, the compressive load on the sensor can be reduced (e.g., moves towards zero-load), however the magnitude of the preload can be such that the load on the sensor does not translate to tensile stress being applied to the sensor.

  12. Digital Sensor Technology

    SciTech Connect

    Thomas, Ken D.; Quinn, Edward L.; Mauck, Jerry L.; Bockhorst, Richard M.

    2015-02-01

    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 and reliability. This paper, which refers to a final report issued in 2013, demonstrates these benefits in direct comparisons of digital and analog sensor applications. Improved accuracy results from the superior operating characteristics of digital sensors. These include improvements in sensor accuracy and drift and other related parameters which reduce total loop uncertainty and thereby increase safety and operating margins. An example instrument loop uncertainty calculation for a pressure sensor application is presented to illustrate these improvements. This is a side-by-side comparison of the instrument loop uncertainty for both an analog and a digital sensor in the same pressure measurement application. Similarly, improved sensor reliability is illustrated with a sample calculation for determining the probability of failure on demand, an industry standard reliability measure. This looks at equivalent analog and digital temperature sensors to draw the comparison. The results confirm substantial reliability improvement with the digital sensor, due in large part to ability to continuously monitor the health of a digital sensor such that problems can be immediately identified and corrected. This greatly reduces the likelihood of a latent failure condition of the sensor at the time of a design basis event. Notwithstanding the benefits of digital sensors, there are certain qualification issues that are inherent with digital technology and these are described in the report. One major qualification impediment for digital sensor implementation is software common cause failure (SCCF).

  13. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  14. 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

  15. 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

  16. Src Tyrosine Kinase Alters Gating of Hyperpolarization-Activated HCN4 Pacemaker Channel through Tyr531

    PubMed Central

    Li, Chen-Hong; Zhang, Qi; Teng, Bunyen; Mustafa, S. Jamal; Huang, Jian-Ying; Yu, Han-Gang

    2009-01-01

    We recently discovered that the constitutively active Src tyrosine kinase can enhance the HCN4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, PP2, on HCN4 and its mutant channels ex pressed in HEK293 cells using whole-cell patch clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole-cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr531 and Tyr554. Substituting HCN4-Tyr531 with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr554 by phenylalanine did not abolish the effects of PP2 on voltage-dependent activation, but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole-cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src actions on HCN4 channels is Tyr531. PMID:17977941

  17. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    PubMed

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  18. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    PubMed

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

  19. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  20. HEAT Sensor: Harsh Environment Adaptable Thermionic Sensor

    SciTech Connect

    Limb, Scott J.

    2016-05-31

    This document is the final report for the “HARSH ENVIRONMENT ADAPTABLE THERMIONIC SENSOR” project under NETL’s Crosscutting contract DE-FE0013062. This report addresses sensors that can be made with thermionic thin films along with the required high temperature hermetic packaging process. These sensors can be placed in harsh high temperature environments and potentially be wireless and self-powered.

  1. NDP kinase reactivity towards 3TC nucleotides.

    PubMed

    Kreimeyer, A; Schneider, B; Sarfati, R; Faraj, A; Sommadossi, J P; Veron, M; Deville-Bonne, D

    2001-05-01

    Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.

  2. The ubiquitin-conjugating enzyme, Ubc1, indirectly regulates SNF1 kinase activity via Forkhead-dependent transcription

    PubMed Central

    Jiao, Rubin; Lobanova, Liubov; Waldner, Amanda; Fu, Anthony; Xiao, Linda; Harkness, Troy A.; Arnason, Terra G.

    2016-01-01

    The SNF1 kinase in Saccharomyces cerevisiae is an excellent model to study the regulation and function of the AMP-dependent protein kinase (AMPK) family of serine-threonine protein kinases. Yeast discoveries regarding the regulation of this non-hormonal sensor of metabolic/environmental stress are conserved in higher eukaryotes, including poly-ubiquitination of the α-subunit of yeast (Snf1) and human (AMPKα) that ultimately effects subunit stability and enzyme activity. The ubiquitin-cascade enzymes responsible for targeting Snf1 remain unknown, leading us to screen for those that impact SNF1 kinase function. We identified the E2, Ubc1, as a regulator of SNF1 kinase function. The decreased Snf1 abundance found upon deletion of Ubc1 is not due to increased degradation, but instead is partly due to impaired SNF1 gene expression, arising from diminished abundance of the Forkhead 1/2 proteins, previously shown to contribute to SNF1 transcription. Ultimately, we report that the Fkh1/2 cognate transcription factor, Hcm1, fails to enter the nucleus in the absence of Ubc1. This implies that Ubc1 acts indirectly through transcriptional effects to modulate SNF1 kinase activity. PMID:28357323

  3. Human Gastric Cancer Kinase Profile and Prognostic Significance of MKK4 Kinase

    PubMed Central

    Wu, Chew-Wun; Li, Anna F.-Y.; Chi, Chin-Wen; Huang, Chen Lung; Shen, King-Han; Liu, Wing-Yiu; Lin, Wen-chang

    2000-01-01

    Alterations of protein tyrosine kinase are often associated with uncontrolled cell growth and tumor progression. Knowledge of the overall expression pattern of tyrosine kinases should prove beneficial in understanding the signaling pathways involved in gastric cancer oncogenesis and in providing possible biomarkers for gastric cancer progression. To establish a general tyrosine-kinase expression profile, degenerated polymerase chain reaction primers designed from the consensus catalytic kinase motifs were used to amplify protein tyrosine kinase molecules from gastric cancer tissues. We observed more than 50 tyrosine and serine/threonine kinases from matching pairs of gastric cancer tissue and normal mucosa. Based on this new kinase profile information, we selected the MKK4 gene for further immunohistochemical studies. Statistical analysis of MKK4 protein expression and clinicopathological features indicated that MKK4 kinase expression could serve as a significant prognostic factor for relapse-free survival and for overall survival. We demonstrated a simple and sensitive method for establishing protein tyrosine-kinase expression profiles of human gastric cancer tissues as well as for discovering novel and useful clinical biomarkers from such kinase expression profiles. PMID:10854223

  4. Polo-like kinase-activating kinases: Aurora A, Aurora B and what else?

    PubMed

    Archambault, Vincent; Carmena, Mar

    2012-04-15

    The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G(2), and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.

  5. Solid State Humidity Sensors

    NASA Astrophysics Data System (ADS)

    Chang, Song-Lin

    There are only a few solid state humidity sensors available today. Most of those sensors use a porous oxide material as a principal part of the device. The devices work on the basis of a change in resistance as the moisture in the air varies. In this experiment, two solid state humidity sensors have been developed for use under practical conditions. One is a Polymer Oxide Semiconductor device with a POLYOX film that absorbs the moisture from the air. The amount of water dipoles absorbed by the polymer is a function of relative humidity. This sensor can measure relative humidity from 20% to 90%. The other is a Dew Point sensor. The sensor is in contact with the upper surface of a miniature Peltier cooler. Water molecules deposited on the sensor surface cause the electrical current through the sensor to increase. The operator adjusts the temperature of the Peltier cooler until a saturated current through the sensor is reached. About one min. is required to measure low relative humidities. The Dew Point sensor can measure a range of relative humidities of 30% to 80%.

  6. 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.

  7. Functional analysis of anomeric sugar kinases.

    PubMed

    Conway, Louis P; Voglmeir, Josef

    2016-09-02

    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.

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

    PubMed

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

    2016-06-24

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

  9. The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

    PubMed

    Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

    2009-02-01

    The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

  10. MITRE sensor layer prototype

    NASA Astrophysics Data System (ADS)

    Duff, Francis; McGarry, Donald; Zasada, David; Foote, Scott

    2009-05-01

    The MITRE Sensor Layer Prototype is an initial design effort to enable every sensor to help create new capabilities through collaborative data sharing. By making both upstream (raw) and downstream (processed) sensor data visible, users can access the specific level, type, and quantities of data needed to create new data products that were never anticipated by the original designers of the individual sensors. The major characteristic that sets sensor data services apart from typical enterprise services is the volume (on the order of multiple terabytes) of raw data that can be generated by most sensors. Traditional tightly coupled processing approaches extract pre-determined information from the incoming raw sensor data, format it, and send it to predetermined users. The community is rapidly reaching the conclusion that tightly coupled sensor processing loses too much potentially critical information.1 Hence upstream (raw and partially processed) data must be extracted, rapidly archived, and advertised to the enterprise for unanticipated uses. The authors believe layered sensing net-centric integration can be achieved through a standardize-encapsulate-syndicateaggregate- manipulate-process paradigm. The Sensor Layer Prototype's technical approach focuses on implementing this proof of concept framework to make sensor data visible, accessible and useful to the enterprise. To achieve this, a "raw" data tap between physical transducers associated with sensor arrays and the embedded sensor signal processing hardware and software has been exploited. Second, we encapsulate and expose both raw and partially processed data to the enterprise within the context of a service-oriented architecture. Third, we advertise the presence of multiple types, and multiple layers of data through geographic-enabled Really Simple Syndication (GeoRSS) services. These GeoRSS feeds are aggregated, manipulated, and filtered by a feed aggregator. After filtering these feeds to bring just the type

  11. Liquid level sensor

    SciTech Connect

    Kulkarni, Atul; Karekar, R.N.; Aiyer, R.C.

    2005-10-15

    The article reports an idea of using a simple, cantilever-type load cell with a rod as a level sensor for continuous liquid level measurements. The sensor is based on the principle of the Archimedes buoyancy principle. The density and geometry of the rod govern the choice of the load cell. The length of the rod is governed by the height of the tank. A series of cyclic tests have demonstrated a highly repeatable response of the sensor. The accuracy of this low-cost sensor is field tested and found to be {+-}0.5% of the full range, for a 10 m level of water in a tank, and is working reliably for the period of 18 months. The sensor range can be easily extended to lower and higher tank heights. The sensor is crowned by its easy installation and calibration.

  12. Magnetic current sensor

    NASA Technical Reports Server (NTRS)

    Black, Jr., William C. (Inventor); Hermann, Theodore M. (Inventor)

    1998-01-01

    A current determiner having an output at which representations of input currents are provided having an input conductor for the input current and a current sensor supported on a substrate electrically isolated from one another but with the sensor positioned in the magnetic fields arising about the input conductor due to any input currents. The sensor extends along the substrate in a direction primarily perpendicular to the extent of the input conductor and is formed of at least a pair of thin-film ferromagnetic layers separated by a non-magnetic conductive layer. The sensor can be electrically connected to a electronic circuitry formed in the substrate including a nonlinearity adaptation circuit to provide representations of the input currents of increased accuracy despite nonlinearities in the current sensor, and can include further current sensors in bridge circuits.

  13. Intelligent Sensors Security

    PubMed Central

    Bialas, Andrzej

    2010-01-01

    The paper is focused on the security issues of sensors provided with processors and software and used for high-risk applications. Common IT related threats may cause serious consequences for sensor system users. To improve their robustness, sensor systems should be developed in a restricted way that would provide them with assurance. One assurance creation methodology is Common Criteria (ISO/IEC 15408) used for IT products and systems. The paper begins with a primer on the Common Criteria, and then a general security model of the intelligent sensor as an IT product is discussed. The paper presents how the security problem of the intelligent sensor is defined and solved. The contribution of the paper is to provide Common Criteria (CC) related security design patterns and to improve the effectiveness of the sensor develop