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Sample records for acid kinase family

  1. A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families.

    Science.gov (United States)

    Zhu, Guozhi; Fujii, Koichi; Liu, Yin; Codrea, Vlad; Herrero, Juan; Shaw, Stephen

    2005-10-28

    Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-theta and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  3. Src family kinases in chronic kidney disease.

    Science.gov (United States)

    Wang, Jun; Zhuang, Shougang

    2017-09-01

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

  4. The venus kinase receptor (VKR) family: structure and evolution

    Science.gov (United States)

    2013-01-01

    Background Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. Results Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. Conclusions This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology. PMID:23721482

  5. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

  6. Erythropoietin and interleukin-2 activate distinct JAK kinase family members.

    Science.gov (United States)

    Barber, D L; D'Andrea, A D

    1994-01-01

    The erythropoietin (EPO) receptor and the interleukin-2 (IL-2) receptor beta-chain subunit are members of the cytokine receptor superfamily. They have conserved primary amino acid sequences in their cytoplasmic domains and activate phosphorylation of common substrates, suggesting common biochemical signaling mechanisms. We have generated a cell line, CTLL-EPO-R, that contains functional cell surface receptors for both EPO and IL-2. CTLL-EPO-R cells demonstrated similar growth kinetics in EPO and IL-2. Stimulation with EPO resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK2. In contrast, stimulation with IL-2 or the related cytokine IL-4 resulted in the rapid, dose-dependent tyrosine phosphorylation of JAK1 and an additional 116-kDa protein. This 116-kDa protein was itself immunoreactive with a polyclonal antiserum raised against JAK2 and appears to be a novel member of the JAK kinase family. Immune complex kinase assays confirmed that IL-2 and IL-4 activated JAK1 and EPO activated JAK2. These results demonstrate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R cells and that the EPO and IL-2 receptors interact with distinct JAK kinase family members within the same cellular background. Images PMID:7935373

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

    DEFF Research Database (Denmark)

    Sandrini, Michael; Piskur, Jure

    2005-01-01

    Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kin...

  8. The arabinose kinase, ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family.

    Science.gov (United States)

    Sherson, S; Gy, I; Medd, J; Schmidt, R; Dean, C; Kreis, M; Lecharny, A; Cobbett, C

    1999-03-01

    The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene. ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.

  9. The venus kinase receptor (VKR) family: structure and evolution.

    OpenAIRE

    Vanderstraete, Mathieu; Gouignard, Nadège; Ahier, Arnaud; Morel, Marion; Vicogne, Jérôme; Dissous, Colette

    2013-01-01

    International audience; BACKGROUND: Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be hi...

  10. Demystifying MST family kinases in cell death.

    Science.gov (United States)

    Lehtinen, Maria K; Bonni, Azad

    2008-06-01

    The MST family of protein kinases plays a critical role in the regulation of cell death in diverse organisms including mammals. The intracellular signaling pathways that regulate MST-driven cell death in mammalian cells are the subject of intense investigation. Stress stimuli including oxidative stress and DNA damaging agents trigger the activity of MST in cells. Although the mechanisms by which oxidative stress and DNA damage trigger MST activation remain to be identified, MST activity can be regulated by caspase-induced cleavage as well as interactions with other proteins in cells. Once activated upon oxidative stress, MST induces cell death via phosphorylation and activation of the transcription factor FOXO3 or the histone protein H2B. This review focuses on the currently known upstream activating mechanisms for MST, and explores the downstream signaling pathways that mediate MST's principal function in cell death. Elucidation of MST functions and their regulatory mechanisms in cell death have important implications for our understanding of cellular homeostasis as well as the pathogenesis of diverse diseases.

  11. Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera).

    Science.gov (United States)

    Wang, Gang; Lovato, Arianna; Polverari, Annalisa; Wang, Min; Liang, Ying-Hai; Ma, Yuan-Chun; Cheng, Zong-Ming

    2014-08-27

    Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize. In this study, we performed a genome-wide survey and identified 45 MAPKKK genes in the grapevine genome. Chromosome location, phylogeny, gene structure and conserved protein motifs of MAPKKK family in grapevine have been analyzed to support the prediction of these genes. In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization. This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine.

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

    Science.gov (United States)

    Sandrini, Michael P B; Piskur, Jure

    2005-05-01

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

  13. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes

    NARCIS (Netherlands)

    Beuers, U.; Probst, I.; Soroka, C.; Boyer, J. L.; Kullak-Ublick, G. A.; Paumgartner, G.

    1999-01-01

    The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated.

  14. Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors.

    Science.gov (United States)

    Bowers, Alex J; Boylan, John F

    2004-03-17

    The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression.

  15. Expression of the MAST family of serine/threonine kinases.

    Science.gov (United States)

    Garland, Patrick; Quraishe, Shmma; French, Pim; O'Connor, Vincent

    2008-02-21

    The Microtubule-Associated Serine/Threonine Kinase family (MAST1-4, and MAST-like) is characterised by the presence of a serine/threonine kinase domain and a postsynaptic density protein-95/discs large/zona occludens-1 domain (PDZ). This latter domain gives the MAST family the capacity to scaffold its own kinase activity. In the present study we have profiled the mRNA for each member of the MAST family transcripts across various tissues, with particular focus on rodent brain. Reverse-transcriptase polymerase chain reaction (RT-PCR) has shown equivalent patterns of expression for MAST1 and 2 in multiple tissues. Both MAST3 and 4 show more distinct expression in several tissues, and MAST-like appears to be predominantly expressed in heart and testis. In situ hybridisation reveals overlapping expression of MAST1 and 2 in specific brain regions. In contrast, MAST3 shows selective expression in the striatum and cerebral cortex. MAST4 also exhibits distinct expression in oligodendrocytes of white matter containing brain regions. In keeping with previous results, this family member also shows increased expression in the hippocampus following seizure-like activity. Our analysis of MAST family expression provides support for the role of these kinases in a broad range of neural functions.

  16. X-Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Muckelbauer, Jodi; Sack, John S.; Ahmed, Nazia; Burke, James; Chang, ChiehYing Y.; Gao, Mian; Tino, Joseph; Xie, Dianlin; Tebben, Andrew J. (BMS)

    2012-06-27

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

  17. Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition

    Science.gov (United States)

    Rexer, Brent N.; Ham, Amy-Joan L.; Rinehart, Cammie; Hill, Salisha; Granja-Ingram, Nara de Matos; González, Ana María; Mills, Gordon B.; Dave, Bhuvanesh; Chang, Jenny C.; Liebler, Daniel C.; Arteaga, Carlos L.

    2011-01-01

    Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable whereas active PI3K-Akt and MAPK were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFK) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors. PMID:21499296

  18. Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase

    NARCIS (Netherlands)

    Sprengers, E.D.; Staal, Gerard E.J.

    1979-01-01

    1. 1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Trismaleate shows a loss of positive homotropic interactions, as compared

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region....... These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity....

  20. Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

    Science.gov (United States)

    Negami, A I; Sasaki, H; Yamamura, H

    1985-09-16

    Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

  1. Regulation of Ack-Family Nonreceptor Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Victoria Prieto-Echagüe

    2011-01-01

    Full Text Available Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.

  2. Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.

    Science.gov (United States)

    Jerga, Agoston; Lu, Ying-Jie; Schujman, Gustavo E; de Mendoza, Diego; Rock, Charles O

    2007-07-27

    Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

  3. [Identification of a novel human MAST4 gene, a new member of the microtubule associated serine-threonine kinase family].

    Science.gov (United States)

    Sun, L; Gu, S; Li, X; Sun, Y; Zheng, D; Yu, K; Ji, C; Tang, R; Xie, Y; Mao, Y

    2006-01-01

    Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64%, 63%, 59% and 39% identical aminoacid residues with MAST1, MAST2, MAST3 and MASTL respectively. RT-PCR analysis revealed relatively high expression level of MAST4 in most normal human tissues, with an exception of in testis, small intestine, colon and peripheral blood leukocyte.

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

    Science.gov (United States)

    Dalton, George D; Dewey, William L

    2006-02-01

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

  5. DMPD: Regulation of phagocyte migration and recruitment by Src-family kinases. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18385944 Regulation of phagocyte migration and recruitment by Src-family kinases. B...how Regulation of phagocyte migration and recruitment by Src-family kinases. PubmedID 18385944 Title Regulat...ion of phagocyte migration and recruitment by Src-family kinases. Authors Baruzzi

  6. A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases.

    Science.gov (United States)

    Belham, Christopher; Roig, Joan; Caldwell, Jennifer A; Aoyama, Yumi; Kemp, Bruce E; Comb, Michael; Avruch, Joseph

    2003-09-12

    The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little noncatalytic sequence but bind to the carboxyl-terminal noncatalytic tail of Nercc1/Nek9, a NIMA family protein kinase that is activated in mitosis. Microinjection of anti-Nercc1 antibodies leads to spindle abnormalities and prometaphase arrest or chromosome missegregation. Herein we show that Nek6 is increased in abundance and activity during mitosis; activation requires the phosphorylation of Ser206 on the Nek6 activation loop. This phosphorylation and the activity of recombinant Nek6 is stimulated by coexpression with an activated mutant of Nercc1. Moreover, Nercc1 catalyzes the direct phosphorylation of prokaryotic recombinant Nek6 at Ser206 in vitro concomitant with 20-25-fold activation of Nek6 activity; Nercc1 activates Nek7 in vitro in a similar manner. Nercc1/Nek9 is likely to be responsible for the activation of Nek6 during mitosis and probably participates in the regulation of Nek7 as well. These findings support the conclusion that Nercc1/Nek9 and Nek6 represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression.

  7. Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes.

    Science.gov (United States)

    Kawamura, M; McVicar, D W; Johnston, J A; Blake, T B; Chen, Y Q; Lal, B K; Lloyd, A R; Kelvin, D J; Staples, J E; Ortaldo, J R

    1994-01-01

    Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase. Images PMID:8022790

  8. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Xiong Zhang

    2014-11-01

    Full Text Available Embryonic stem (ES cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent

  9. Versican G1 domain enhances adenoviral-mediated transgene expression and can be modulated by inhibitors of the Janus kinase (JAK)/STAT and Src family kinase pathways.

    Science.gov (United States)

    Akinfenwa, Patricia Y; Bond, Wesley S; Ildefonso, Cristhian J; Hurwitz, Mary Y; Hurwitz, Richard L

    2017-09-01

    To examine the biochemical influences that may contribute to the success of gene therapy for ocular disorders, the role of versican, a vitreous component, in adenoviral-mediated transgene expression was examined. Versican is a large chondroitin sulfate-containing, hyaluronic acid-binding proteoglycan present in the extracellular matrix and in ocular vitreous body. Y79 retinoblastoma cells and CD44-negative SK-N-DZ neuroblastoma cells transduced with adenoviral vectors in the presence of versican respond with an activation of transgene expression. Proteolysis of versican generates a hyaluronan-binding G1 domain. The addition of recombinant versican G1 to SK-N-DZ cells results in a similar activation of transgene expression, and treatment with dasatinib, an inhibitor of Src family kinases, also mimics the effects of versican. Enhancement is accompanied by an increase in signal transducer and activator of transcription 5 (STAT5) phosphorylation and is abrogated by treatment with C188-9, a STAT3/5 inhibitor, or with ruxolitinib, a Janus kinase 1/2 (JAK1/2) inhibitor. These data implicate versican G1 in enhancing adenoviral vector transgene expression in a hyaluronic acid-CD44 independent manner that is down-regulated by inhibitors of the JAK/STAT pathway and enhanced by inhibitors of the Src kinase pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. An amino acid substitution in the Drosophila hopTum-l Jak kinase causes leukemia-like hematopoietic defects.

    Science.gov (United States)

    Luo, H; Hanratty, W P; Dearolf, C R

    1995-01-01

    Proteins of the Jak family of non-receptor kinases play important roles in mammalian hematopoietic signal transduction. They mediate the cellular response to a wide range of cytokines and growth factors. A dominant mutation in a Drosophila Jak kinase, hopscotchTumorous-lethal (hopTum-l), causes hematopoietic defects. Here we conduct a molecular analysis of hopTum-l. We demonstrate that the hopTum-l hematopoietic phenotype is caused by a single amino acid substitution of glycine to glutamic acid at residue 341. We generate a true revertant of the hopTum-l mutation, in which both the molecular lesion and the mutant hematopoietic phenotype revert back to wild type. We also examine the effects of the G341E substitution in transgenic flies. The results indicate that a mutant Jak kinase can cause leukemia-like abnormalities. Images PMID:7729418

  11. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    Science.gov (United States)

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  12. Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin.

    Science.gov (United States)

    Liu, Da Zhi; Waldau, Ben; Ander, Bradley P; Zhan, Xinhua; Stamova, Boryana; Jickling, Glen C; Lyeth, Bruce G; Sharp, Frank R

    2017-07-01

    Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

  13. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-28

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

  15. Identification and sequence analysis of six new members of the NIMA-related kinase family in Chlamydomonas.

    Science.gov (United States)

    Bradley, Brian A; Wagner, James J D; Quarmby, Lynne M

    2004-01-01

    The NIMA kinases are an evolutionarily conserved protein family with enigmatic roles in the regulation of mitosis. We report six new members of this family in Chlamydomonas, in addition to the previously identified NIMA-related kinase, Fa2p. Chlamydomonas NIMA-related kinases (CNKs) 1-6 were sequenced from subclones generated by RT-PCR using information from EST libraries and the recently sequenced Chlamydomonas genome. Phylogenetic and bioinformatic approaches were used to determine the relationships of the six new members with known members of the NIMA-related kinase family. Although humans express at least eleven NIMA-related kinases, the eukaryotic microbes that have been studied to date express only one or two members of the family. Thus, the discovery that Chlamydomonas expresses a total of at least seven NIMA-related kinases is intriguing. Our analyses suggest that members of this family may play roles in the assembly and function of cilia.

  16. Signalling by the phosphoinositide 3-kinase family in immune cells

    Science.gov (United States)

    Okkenhaug, Klaus

    2015-01-01

    Phosphoinositide 3-kinases control many important aspects of immune cell development, differentiation and function. Mammals have eight PI3K catalytic subunits which are divided into 3 classes based on similarities in structure and function. Specific roles for the class I PI3Ks have been broadly investigated and are relatively well understood as is the function of their corresponding phosphatases. More recently, specific roles for the class II and class III PI3Ks have emerged. Through vertebrate evolution and in parallel with the evolution of adaptive immunity, there has been a dramatic increase not only in the genes for PI3K subunits but also in genes for phosphatases that act on 3-phopshoinositides and on 3-phosphoinositide binding proteins. Our understanding of the PI3Ks in immunity is guided by fundamental discoveries made in simpler model organisms as well as by appreciating new adaptions of this signalling module in mammals in general and immune cells in particular. PMID:23330955

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    Directory of Open Access Journals (Sweden)

    Nikita Chopra

    2016-03-01

    Full Text Available Bruton's tyrosine kinase (Btk is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA. Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

  19. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  20. The FGGY carbohydrate kinase family: insights into the evolution of functional specificities.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2011-12-01

    Full Text Available Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose. Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families.

  1. Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

    Science.gov (United States)

    Moroco, Jamie A.; Craigo, Jodi K.; Iacob, Roxana E.; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.

    2014-01-01

    Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. PMID:25144189

  2. Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.

    Directory of Open Access Journals (Sweden)

    Jamie A Moroco

    Full Text Available Src-family kinases (SFKs are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12 to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

  3. A biosensor of SRC family kinase conformation by exposable tetracysteine useful for cell-based screening.

    Science.gov (United States)

    Irtegun, Sevgi; Wood, Rebecca; Lackovic, Kurt; Schweiggert, Jörg; Ramdzan, Yasmin M; Huang, David C S; Mulhern, Terrence D; Hatters, Danny M

    2014-07-18

    We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.

  4. A novel predicted calcium-regulated kinase family implicated in neurological disorders.

    Directory of Open Access Journals (Sweden)

    Małgorzata Dudkiewicz

    Full Text Available The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca(2+-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca(2+-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1 and CXORF36 (DIA1R, are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins.

  5. Erythropoietin and interleukin-2 activate distinct JAK kinase family members.

    OpenAIRE

    Barber, D L; D'Andrea, A D

    1994-01-01

    The erythropoietin (EPO) receptor and the interleukin-2 (IL-2) receptor beta-chain subunit are members of the cytokine receptor superfamily. They have conserved primary amino acid sequences in their cytoplasmic domains and activate phosphorylation of common substrates, suggesting common biochemical signaling mechanisms. We have generated a cell line, CTLL-EPO-R, that contains functional cell surface receptors for both EPO and IL-2. CTLL-EPO-R cells demonstrated similar growth kinetics in EPO ...

  6. Abl family kinases regulate endothelial barrier function in vitro and in mice.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Chislock

    Full Text Available The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg, as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR, Kit, colony stimulating factor 1 receptor (CSF1R, and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far......, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of d......CK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters...

  8. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl...

  9. A sensor kinase recognizing the cell-cell signal BDSF (cis-2-dodecenoic acid) regulates virulence in Burkholderia cenocepacia

    DEFF Research Database (Denmark)

    McCarthy, Y.; Yang, Liang; Twomey, K.B.

    2010-01-01

    P>Burkholderia cenocepacia is an opportunistic human pathogen that uses cis-2-dodecenoic acid (BDSF) as a quorum-sensing signal to control expression of virulence factors. BDSF is a signal molecule of the diffusible signal factor (DSF) family that was first described in the plant pathogen...... with an input domain unrelated to that of RpfC, the DSF sensor found in xanthomonads. Transcriptome profiling established the scope of the BDSF regulon and demonstrated that the sensor controls expression of a subset of these genes. A chimeric sensor kinase in which the input domain of BCAM0227 replaced...

  10. The NIMA-family kinase Nek3 regulates microtubule acetylation in neurons.

    Science.gov (United States)

    Chang, Jufang; Baloh, Robert H; Milbrandt, Jeffrey

    2009-07-01

    NIMA-related kinases (Neks) belong to a large family of Ser/Thr kinases that have critical roles in coordinating microtubule dynamics during ciliogenesis and mitotic progression. The Nek kinases are also expressed in neurons, whose axonal projections are, similarly to cilia, microtubule-abundant structures that extend from the cell body. We therefore investigated whether Nek kinases have additional, non-mitotic roles in neurons. We found that Nek3 influences neuronal morphogenesis and polarity through effects on microtubules. Nek3 is expressed in the cytoplasm and axons of neurons and is phosphorylated at Thr475 located in the C-terminal PEST domain, which regulates its catalytic activity. Although exogenous expression of wild-type or phosphomimic (T475D) Nek3 in cultured neurons has no discernible impact, expression of a phospho-defective mutant (T475A) or PEST-truncated Nek3 leads to distorted neuronal morphology with disturbed polarity and deacetylation of microtubules via HDAC6 in its kinase-dependent manner. Thus, the phosphorylation at Thr475 serves as a regulatory switch that alters Nek3 function. The deacetylation of microtubules in neurons by unphosphorylated Nek3 raises the possibility that it could have a role in disorders where axonal degeneration is an important component.

  11. Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family.

    Science.gov (United States)

    Bataille, Carole J R; Brennan, Méabh B; Byrne, Simon; Davies, Stephen G; Durbin, Matthew; Fedorov, Oleg; Huber, Kilian V M; Jones, Alan M; Knapp, Stefan; Liu, Gu; Nadali, Anna; Quevedo, Camilo E; Russell, Angela J; Walker, Roderick G; Westwood, Robert; Wynne, Graham M

    2017-05-01

    The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75μM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

    Science.gov (United States)

    Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

    2017-01-01

    CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

  13. Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper.

    Science.gov (United States)

    Venkatesh, Jelli; Jahn, Molly; Kang, Byoung-Cheorl

    2016-01-01

    The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance.

  14. The NIMA-family kinase, Nek1 affects the stability of centrosomes and ciliogenesis.

    Science.gov (United States)

    White, Mark C; Quarmby, Lynne M

    2008-06-04

    Mutations in Nek1 (NIMA-Related Kinase 1) are causal in the murine models of polycystic kidney disease kat and kat2J. The Neks are known as cell cycle kinases, but recent work in protists has revealed that in addition to roles in the regulation of cell cycle progression, some Neks also regulate cilia. In most cells, cilia are disassembled prior to mitosis and are regenerated after cytokinesis. We propose that Neks participate in the coordination of ciliogenesis with cell cycle progression. Mammalian Nek1 is a candidate for this activity because renal cysts form in response to dysfunctional ciliary signalling. Here we report that over-expression of full-length mNek1 inhibited ciliogenesis without disrupting centrosomes in the murine renal epithelial cell line IMCD3. In contrast, over-expression of the kinase domain with its associated basic region, but without the acidic domain, caused loss of centrosomes. As expected, these cells also failed to grow cilia. Both defective ciliogenesis in response to too much mNek1 and disassembly of centrosomes in response to expression of the kinase lacking the presumptive regulatory domain was abrogated by kinase-inactivating mutations or by removal of the coiled-coil domain. We observed that kinase-inactive, C-terminal truncations of mNek1 retaining the coiled-coil domain localized to the cilium, and we define a ciliary targeting region within the coiled-coil domain. Based on our data, we propose that Nek1 plays a role in centrosome integrity, affecting both ciliogenesis and centrosome stability.

  15. A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines.

    Science.gov (United States)

    Knecht, Wolfgang; Sandrini, Michael P B; Johansson, Kenth; Eklund, Hans; Munch-Petersen, Birgitte; Piskur, Jure

    2002-04-02

    In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.

  16. SRC family kinases in hamster spermatozoa: evidence for the presence of LCK.

    Science.gov (United States)

    Singh, Durgesh Kumar; Deshmukh, Rohit Kumar; Narayanan, Praveen Kumar; Shivaji, Sisinthy; Siva, Archana Bharadwaj

    2017-05-01

    Sperm capacitation is a prerequisite for successful fertilization. Increase in tyrosine phosphorylation is considered the hallmark of capacitation and attempts to understand its regulation are ongoing. In this regard, we attempted to study the role of SRC family kinases (SFKs) in the hamster sperm functions. Interestingly, we found the presence of the lymphocyte-specific protein tyrosine kinase, LCK, in mammalian spermatozoa and further characterized it in terms of its localization and function. LCK was found in spermatozoa of several species, and its transcript was identified in the hamster testis. Autophosphorylation of LCK at the Y394 residue increased as capacitation progressed, indicating an upregulation of LCK activity during capacitation. Inhibition of LCK (and perhaps the other SFKs) with the use of a specific inhibitor showed a significant decrease in protein tyrosine phosphorylation of several proteins, implying LCK/SFKs as key tyrosine kinase(s) regulating tyrosine phosphorylation during hamster sperm capacitation. Dihydrolipoamide dehydrogenase was identified as a substrate for LCK/SFK. LCK/SFKs inhibition significantly reduced the percentage fertilization ( in vitro ) but had no effect on sperm motility, hyperactivation and acrosome reaction. In summary, this is the first report on the presence of LCK, an SFK of hematopoietic lineage in spermatozoa besides being the first study on the role of SFKs in the spermatozoa of Syrian hamsters. © 2017 Society for Reproduction and Fertility.

  17. Phosphorylation of c-Cbl and p85 PI3K Driven by All-trans Retinoic Acid and CD38 Depends on Lyn Kinase Activity

    OpenAIRE

    Congleton, Johanna; Shen, Miaoqing; Macdonald, Robert; Malavasi, Fabio; Yen, Andrew

    2014-01-01

    The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driv...

  18. AGC family kinase 1 participates in trogocytosis but not in phagocytosis in Entamoeba histolytica.

    Science.gov (United States)

    Somlata; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2017-07-17

    The protozoan parasite Entamoeba histolytica is the aetiologic agent of amoebiasis, an endemic infection in developing countries with considerable morbidity and mortality. Recently, trogocytosis has been recognized as the key step in amoebic cytolysis and invasion, a paradigm shift in understanding pathogenicity of this organism. Here we report that AGC family kinase 1 is specifically involved in trogocytosis of live human cells and does not participate in phagocytosis of dead cells. Live imaging reveals localization of this kinase in the long and thin tunnels formed during trogocytosis but not in the trogosomes (endosomes formed after trogocytosis). Silencing of the specific gene leads to a defect in CHO cell destruction and trogocytosis while other endocytic processes remain unaffected. The results suggest that the trogocytic pathway is likely to be different from phagocytosis though many of the steps and molecules involved may be common. Entamoeba histolytica can kill host cells by trogocytosis, while it ingests dead cells by phagocytosis. Here, Somlata et al. show that EhAGCK1, an AGC family kinase, is specifically involved in trogocytosis, shedding light on the molecular differences between trogocytosis and phagocytosis.

  19. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    Directory of Open Access Journals (Sweden)

    Pene-Dumitrescu Teodora

    2012-03-01

    Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

  20. A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

    Science.gov (United States)

    Ahier, Arnaud; Rondard, Philippe; Gouignard, Nadège; Khayath, Naji; Huang, Siluo; Trolet, Jacques; Donoghue, Daniel J.; Gauthier, Monique; Pin, Jean-Philippe; Dissous, Colette

    2009-01-01

    Background Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases. PMID:19461966

  1. A new family of receptor tyrosine kinases with a venus flytrap binding domain in insects and other invertebrates activated by aminoacids.

    Directory of Open Access Journals (Sweden)

    Arnaud Ahier

    Full Text Available BACKGROUND: Tyrosine kinase receptors (RTKs comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. METHODS AND FINDINGS: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR, were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA(B receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. CONCLUSION: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.

  2. Jak2 tyrosine kinase residues glutamic acid 1024 and arginine 1113 form a hydrogen bond interaction that is essential for Jak-STAT signal transduction.

    Science.gov (United States)

    Sandberg, Eric M; VonDerLinden, Dannielle; Ostrov, David A; Sayeski, Peter P

    2004-10-01

    Angiotensin II is a well-known vasoactive peptide, but it can also act as a potent growth factor, partially through activation of the tyrosine kinase Jak2. Activated Jak2 tyrosine phosphorylates and activates members of the Signal Transducers and Activators of Transcription (STAT) family of cytoplasmic transcription factors. Recently, we demonstrated that tryptophan 1020 and glutamic acid 1024 within the Jak2 activation loop are required for Jak2 tyrosine kinase activity. Here, we sought to elucidate the requirement of glutamic acid 1024 for Jak2 function. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative interaction between glutamic acid 1024 and an arginine at position 1113. We generated a series of charge-based substitution mutations at position 1113 and found that conversion of arginine 1113 to glutamic acid, alanine, or lysine prevented Jak2 autophosphorylation. Furthermore, mutation of arginine 1113 prevented the following angiotensin II-dependent processes from occurring: (1) Jak2 tyrosine phosphorylation, (2) Jak2/AT1receptor co-association, (3) STAT1 recruitment to the Jak2/AT1receptor complex, (4) STAT1 tyrosine phosphorylation, and (5) STAT-mediated gene expression. We determined that the interaction between glutamic acid 1024 and arginine 1113 consists of two distinct hydrogen bonds. We conclude that these hydrogen bond interactions are critical for Jak2 kinase function and subsequent angiotensin II-dependent activation of the Jak/STAT signaling pathway.

  3. Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5.

    Science.gov (United States)

    Bickel, Kathleen G; Mann, Barbara J; Waitzman, Joshua S; Poor, Taylor A; Rice, Sarah E; Wadsworth, Patricia

    2017-09-01

    Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology. © 2017 Wiley Periodicals, Inc.

  4. The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry.

    Directory of Open Access Journals (Sweden)

    Emily Mazanka

    2008-08-01

    Full Text Available Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1's phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine -5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2's partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2's interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation

  5. Lithocholic acid is an Eph-ephrin ligand interfering with Eph-kinase activation.

    Directory of Open Access Journals (Sweden)

    Carmine Giorgio

    Full Text Available Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-transformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki =  49 µM. Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC(50  = 48 and 66 µM, respectively without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.

  6. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  7. Regulation of floral patterning and organ identity by Arabidopsis ERECTA-family receptor kinase genes.

    Science.gov (United States)

    Bemis, Shannon M; Lee, Jin Suk; Shpak, Elena D; Torii, Keiko U

    2013-12-01

    Due to the lack of cell migration, plant organogenesis relies on coordinated cell proliferation, cell growth, and differentiation. A flower possesses a complex structure, with sepals and petals constituting the perianth, and stamens and pistils where male and female gametophytes differentiate. While advances have been made in our understanding of gene regulatory networks controlling flower development, relatively little is known of how cell-cell coordination influences floral organ specification. The Arabidopsis ERECTA (ER)-family receptor kinases, ER, ER-LIKE1 (ERL1), and ERL2, regulate inflorescence architecture, organ shape, and epidermal stomatal patterning. Here it is reported that ER-family genes together regulate floral meristem organization and floral organ identity. The stem cell marker CLAVATA3 exhibits misplaced expression in the floral meristems of the er erl1 erl2 mutant. Strikingly, homeotic conversion of sepals to carpels was observed in er erl1 erl2 flowers. Consistently, ectopic expression of AGAMOUS, which determines carpel identity, was detected in er erl1 erl2 flower primordia. Among the known downstream components of ER-family receptor kinases in stomatal patterning, YODA (YDA) is also required for proper floral patterning. YDA and the ER-family show complex, synergistic genetic interactions: er erl1 erl2 yda quadruple mutant plants become extremely small, callus-like masses. While a constitutively active YDA fully rescues stomatal clustering in er erl1 erl2, it only partially rescues er erl1 erl2 flower defects. The study suggests that ER-family signalling is crucial for ensuring proper expression domains of floral meristem and floral organ identity determinants, and further implies the existence of a non-canonical downstream pathway.

  8. Polo-like kinase 4: the odd one out of the family

    Directory of Open Access Journals (Sweden)

    Bornens Michel

    2010-09-01

    Full Text Available Abstract Polo-like kinase 4 (PLK4 is a unique member of the Polo-like family of kinases that shares little homology with its siblings and has an essential role in centriole duplication. The turn-over of this kinase must be strictly controlled to prevent centriole amplification. This is achieved, in part, by an autoregulatory mechanism, whereby PLK4 autophosphorylates residues in a PEST sequence located carboxy-terminal to its catalytic domain. Phosphorylated PLK4 is subsequently recognized by the SCF complex, ubiquitinylated and targeted to the proteasome for degradation. Recent data have also shown that active PLK4 is restricted to the centrosome, a mechanism that could serve to prevent aberrant centriole assembly elsewhere in the cell. While significant advances have been made in understanding how PLK4 is regulated it is certain that additional regulatory mechanisms exist to safeguard the fidelity of centriole duplication. Here, we overview past and present data discussing the regulation and functions of PLK4.

  9. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyros......BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family...... of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

  10. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, Henrik; Galili, G; Knudsen, S

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the...... as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.......In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance...... the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8...

  11. Indispensable residue for uridine binding in the uridine-cytidine kinase family

    Directory of Open Access Journals (Sweden)

    Fumiaki Tomoike

    2017-09-01

    Full Text Available Uridine-cytidine kinase (UCK, including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.

  12. Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

    Energy Technology Data Exchange (ETDEWEB)

    Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

    2017-05-01

    The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

  13. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    Science.gov (United States)

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  14. Lauric acid and myristic acid from Allium sativum inhibit the growth of Mycobacterium tuberculosis H37Ra: in silico analysis reveals possible binding to protein kinase B.

    Science.gov (United States)

    Muniyan, Rajiniraja; Gurunathan, Jayaraman

    2016-12-01

    The bulb of Allium sativum Linn (Alliaceae) has numerous medicinal values. Though the petroleum ether extract of the bulb has shown to exhibit antimycobacterial activity, the phytochemical(s) responsible for this inhibitory activity is not known. To characterize the bioactive compounds in the petroleum ether extract of Allium sativum (garlic) that inhibit the growth of Mycobacterium tuberculosis H37Ra. Bioactivity-guided fractionation was employed to isolate the bioactive compounds. Antimycobacterial activity was evaluated by well-diffusion method and microplate alamar blue assay (MABA). Infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy were used to characterize the bioactive compounds. Autodock was used to obtain information on molecular recognition, and molecular dynamics simulation was performed using GROMACS. The bioactive compounds that inhibited the growth of M. tuberculosis H37Ra were found to be lauric acid (LA) and myristic acid (MA). The minimal inhibitory concentration of LA and MA was found to be 22.2 and 66.7 μg/mL, respectively. In silico analysis revealed that these fatty acids could bind at the cleft between the N-terminal and C-terminal lobes of the cytosolic domain of serine/threonine protein kinase B (PknB). The inhibition activity was dependent on the alkyl chain length of the fatty acid, and the amino acid residues involved in binding to fatty acid was found to be conserved across the Pkn family of proteins. The study indicates the possibility of using fatty acid derivatives, involving Pkn family of proteins, to inhibit the signal transduction processes in M. tuberculosis.

  15. Members of a Novel Kinase Family (DUF1537) Can Recycle Toxic Intermediates into an Essential Metabolite.

    Science.gov (United States)

    Thiaville, Jennifer J; Flood, Jake; Yurgel, Svetlana; Prunetti, Laurence; Elbadawi-Sidhu, Mona; Hutinet, Geoffrey; Forouhar, Farhad; Zhang, Xinshuai; Ganesan, Venkateswaran; Reddy, Patrick; Fiehn, Oliver; Gerlt, J A; Hunt, John F; Copley, Shelley D; de Crécy-Lagard, Valérie

    2016-08-19

    DUF1537 is a novel family of kinases identified by comparative genomic approaches. The family is widespread and found in all sequenced plant genomes and 16% of sequenced bacterial genomes. DUF1537 is not a monofunctional family and contains subgroups that can be separated by phylogenetic and genome neighborhood context analyses. A subset of the DUF1537 proteins is strongly associated by physical clustering and gene fusion with the PdxA2 family, demonstrated here to be a functional paralog of the 4-phosphohydroxy-l-threonine dehydrogenase enzyme (PdxA), a central enzyme in the synthesis of pyridoxal-5'-phosphate (PLP) in proteobacteria. Some members of this DUF1537 subgroup phosphorylate l-4-hydroxythreonine (4HT) into 4-phosphohydroxy-l-threonine (4PHT), the substrate of PdxA, in vitro and in vivo. This provides an alternative route to PLP from the toxic antimetabolite 4HT that can be directly generated from the toxic intermediate glycolaldehyde. Although the kinetic and physical clustering data indicate that these functions in PLP synthesis are not the main roles of the DUF1537-PdxA2 enzymes, genetic and physiological data suggest these side activities function has been maintained in diverse sets of organisms.

  16. Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion.

    Directory of Open Access Journals (Sweden)

    Deborah Huveldt

    Full Text Available Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM. Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1 activation of Src family kinases (SFKs is common in GBM, 2 the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3 SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.

  17. Phosphorylation and externalization of galectin-4 is controlled by Src family kinases.

    Science.gov (United States)

    Ideo, Hiroko; Hoshi, Ikue; Yamashita, Katsuko; Sakamoto, Masaru

    2013-12-01

    Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconjugates extracellularly. The mechanism of subcellular localization and externalization of galectin-4 has not yet been determined. A preliminary experiment using pervanadate (PV) showed that galectin-4 is tyrosine-phosphorylated in cells and suggested that Src kinases are involved. Cell transfection with galectin-4 and active Src plasmids showed that galectin-4 can be tyrosine phosphorylated by members of the Src kinase family. The C-terminal peptide YVQI of galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2 domains of Src and SHP2 were found to bind to this peptide. Immunofluorescence analysis showed that galectin-4 and phosphorylated proteins were intensely stained in the area of membrane protrusions of PV-treated or Src-activated cells. Furthermore, MUC1 derived from NUGC-4 cells was observed to bind to galectin-4, and externalization of the bound molecules from the cell to the medium increased in the hyperphosphorylated condition. Study of the transfection of the mutant galectin-4 which lacks the C-terminal peptide revealed that the phosphorylation status is important for externalization of galectin-4. These results suggest that externalization of galectin-4 can be regulated by signaling molecules and that it may function intracellularly as an adaptor protein serving to modulate the trafficking of glycoproteins.

  18. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets

    NARCIS (Netherlands)

    Beuers, Ulrich; Denk, Gerald U.; Soroka, Carol J.; Wimmer, Ralf; Rust, Christian; Paumgartner, Gustav; Boyer, James L.

    2003-01-01

    Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of

  19. The role of the atypical kinases ABC1K7 and ABC1K8 in abscisic acid responses

    Directory of Open Access Journals (Sweden)

    Anna eManara

    2016-03-01

    Full Text Available The ABC1K family of atypical kinases (activity of bc1 complex kinase is represented in bacteria, archaea and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling.

  20. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

  1. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway

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    Zeljkica eJandric

    2013-11-01

    Full Text Available Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in Candida glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologues CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species, and required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  2. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway.

    Science.gov (United States)

    Jandric, Zeljkica; Gregori, Christa; Klopf, Eva; Radolf, Martin; Schüller, Christoph

    2013-01-01

    Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  3. The mechanism of MAP kinase activation under acidic condition in feline esophageal smooth muscle cells.

    Science.gov (United States)

    Park, Sun Young; Lee, Young Ju; Min, Youngsil; Kim, Hak Rim; Jeong, Ji Hoon; Sohn, Uy Dong

    2011-10-01

    Reflux esophagitis results from repeated exposure of the esophagus to acidic gastric juice or bile-containing duodenal contents. In Barrett's adenocarcinoma, acid increases proliferation via ERK and p38 MAPK activation. This study was focused on determination of the mechanism(s) underlying MAPKs (ERK 1/2, p38 MAPK, and JNK) activation induced by acidic medium at pH 4 in normal feline primary cultured esophageal smooth muscle cells (FESMCs). We detected ERK 1/2 and p38 MAPK phosphorylation after exposure to pH 4 or neutral media in the presence or absence of several inhibitors and quantified the MAPK levels using western blotting analysis and densitometry. Acidic medium markedly increased the phosphorylation of ERK 1/2 and p38 MAPK within 10 min. Acid-induced ERK 1/2 and p38 MAPK activation was inhibited by pertussis toxin (PTX-sensitive G(i/o) protein inhibitor), DEDA (phospholipase (PL) A(2) inhibitor), ρCMB (PLD inhibitor), GF109203X (protein kinase C (PKC) inhibitor) and D609 (phosphatidylcholine-specific PLC inhibitor). But, genistein (tyrosine kinase inhibitor), forskolin (adenylate cyclase activator) and U73122 (phosphatidylinositol-specific PLC inhibitor) had no effect on acid-induced ERK1/2 and p38 MAPK activation. These findings indicate that the activation of ERK 1/2 and p38 MAPK pathways by acidic conditions, at least in part, may be mediated by activation of the G(i/o) protein coupled receptors, PC-PLC, PLD, PLA(2), and PKC in FESMCs.

  4. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

    Directory of Open Access Journals (Sweden)

    Douglas R Boettner

    2008-01-01

    Full Text Available Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK, was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i incubation of ameba with anti-PATMK antibodies; (ii PATMK mRNA knock-down using a novel shRNA expression system; and (iii expression of a carboxy-truncation of PATMK (PATMK(delta932. Expression of the carboxy-truncation of PATMK(delta932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

  5. Expression of the HER-1-4 family of receptor tyrosine kinases in neuroendocrine tumours.

    Science.gov (United States)

    Srirajaskanthan, Rajaventhan; Shah, Tahir; Watkins, Jennifer; Marelli, Laura; Khan, Korsa; Caplin, Martyn E

    2010-04-01

    The type I receptor tyrosine kinase family comprises four homologous members: Epidermal growth factor receptor (EGFR), HER-2, HER-3 and HER-4. Studies have shown that EGFR and HER-2 play a critical role in oncogenesis. In this study we sought to determine the pattern of expression and the prognostic significance of EGFR, HER-2, HER-3 and HER-4 in a variety of neuroendocrine tumours using immunohistochemistry. HER family receptor expression in 82 paraffin-embedded specimens of neuroendocrine tumours using immunohistochemistry was examined. The pattern and protein expression levels for each receptor were correlated with clinical and pathological parameters. EGFR expression was identified in 86.6% samples, HER-2 was not expressed in any samples, HER-3 was expressed in 8.5% samples and HER-4 was expressed 91.5%. EGFR and HER-4 were co-expressed in 79.3% of cases. HER-3 was correlated with better survival. EGFR was not associated with poor prognosis. This study has demonstrated EGFR, HER-2 and HER-4 expression is not associated with poorer survival. HER-3 expression is correlated with better prognosis. Overexpression of EGFR and HER-4 may offer potential new therapeutic targets.

  6. Nek11, a new member of the NIMA family of kinases, involved in DNA replication and genotoxic stress responses.

    Science.gov (United States)

    Noguchi, Kohji; Fukazawa, Hidesuke; Murakami, Yuko; Uehara, Yoshimasa

    2002-10-18

    DNA replication and genotoxic stresses activate various checkpoint-associated protein kinases, and checkpoint dysfunction often leads to cell lethality. Here, we have identified new members of the mammalian NIMA family of kinases, termed Nek11L and Nek11S (NIMA-related kinase 11 Long and Short isoform) as novel DNA replication/damage stresses-responsive kinases. Molecular cloning and biochemical studies showed that the catalytic domain of Nek11 is most similar to Nek4 and Nek3, and substrate specificity of Nek11L is distinguishable from those of NIMA and Nek2. The expression of nek11L mRNA increased through S to G(2)/M phase, and subcellular localization of Nek11 protein altered between interphase and prometaphase, suggesting multiple roles of Nek11. We found an activation of Nek11 kinase activity when cells were treated with various DNA-damaging agents and replication inhibitors, and this activation of Nek11 was suppressed by caffeine in HeLaS3 cells. The transient expression of wild-type Nek11L enhanced the aphidicolin-induced S-phase arrest, whereas the aphidicolin-induced S-phase arrest was reduced in the U2OS cell lines expressing kinase-negative Nek11L (K61R), and these cells were more sensitive to aphidicolin-induced cell lethality. Collectively, these results suggest that Nek11 has a role in the S-phase checkpoint downstream of the caffeine-sensitive pathway.

  7. Mathematical model of early Reelin-induced Src family kinase-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Helge Hass

    Full Text Available Reelin is a large glycoprotein with a dual role in the mammalian brain. It regulates the positioning and differentiation of postmitotic neurons during brain development and modulates neurotransmission and memory formation in the adult brain. Alterations in the Reelin signaling pathway have been described in different psychiatric disorders. Reelin mainly signals by binding to the lipoprotein receptors Vldlr and ApoER2, which induces tyrosine phosphorylation of the adaptor protein Dab1 mediated by Src family kinases (SFKs. In turn, phosphorylated Dab1 activates downstream signaling cascades, including PI3-kinase-dependent signaling. In this work, a mechanistic model based on ordinary differential equations was built to model early dynamics of the Reelin-mediated signaling cascade. Mechanistic models are frequently used to disentangle the highly complex mechanisms underlying cellular processes and obtain new biological insights. The model was calibrated on time-resolved data and a dose-response measurement of protein concentrations measured in cortical neurons treated with Reelin. It focusses on the interplay between Dab1 and SFKs with a special emphasis on the tyrosine phosphorylation of Dab1, and their role for the regulation of Reelin-induced signaling. Model selection was performed on different model structures and a comprehensive mechanistic model of the early Reelin signaling cascade is provided in this work. It emphasizes the importance of Reelin-induced lipoprotein receptor clustering for SFK-mediated Dab1 trans-phosphorylation and does not require co-receptors to describe the measured data. The model is freely available within the open-source framework Data2Dynamics (www.data2dynamics.org. It can be used to generate predictions that can be validated experimentally, and provides a platform for model extensions both to downstream targets such as transcription factors and interactions with other transmembrane proteins and neuronal signaling

  8. HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer

    KAUST Repository

    Boulbes, Delphine R.

    2014-11-11

    Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2-overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2-overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers.

  9. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Jie Hong

    Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  10. Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties

    DEFF Research Database (Denmark)

    Dan, Ippeita; Ong, Shao-En; Watanabe, Norinobu M

    2002-01-01

    We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK do...

  11. Resequencing analysis of the candidate tyrosine kinase and RAS pathway gene families in multiple myeloma.

    Science.gov (United States)

    Hucthagowder, Vishwanathan; Meyer, Rekha; Mullins, Chelsea; Nagarajan, Rakesh; DiPersio, John F; Vij, Ravi; Tomasson, Michael H; Kulkarni, Shashikant

    2012-09-01

    Multiple myeloma (MM) is an incurable, B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in the understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in the pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138-sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in the KRAS, PIK3CA, INSR, LTK, and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools PolyPhen and Sorting Intolerant from Tolerant (SIFT) were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in the functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might also be useful to establish the recurrent nature of these mutations. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  13. The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes.

    Science.gov (United States)

    Arrighi, Jean-François; Barre, Annick; Ben Amor, Besma; Bersoult, Anne; Soriano, Lidia Campos; Mirabella, Rossana; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal; Ghérardi, Michèle; Huguet, Thierry; Geurts, René; Dénarié, Jean; Rougé, Pierre; Gough, Clare

    2006-09-01

    Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

  14. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable...... to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  15. Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase.

    Science.gov (United States)

    Ofiteru, Augustin; Bucurenci, Nadia; Alexov, Emil; Bertrand, Thomas; Briozzo, Pierre; Munier-Lehmann, Hélène; Gilles, Anne-Marie

    2007-07-01

    Bacterial CMP kinases are specific for CMP and dCMP, whereas the related eukaryotic NMP kinase phosphorylates CMP and UMP with similar efficiency. To explain these differences in structural terms, we investigated the contribution of four key amino acids interacting with the pyrimidine ring of CMP (Ser36, Asp132, Arg110 and Arg188) to the stability, catalysis and substrate specificity of Escherichia coli CMP kinase. In contrast to eukaryotic UMP/CMP kinases, which interact with the nucleobase via one or two water molecules, bacterial CMP kinase has a narrower NMP-binding pocket and a hydrogen-bonding network involving the pyrimidine moiety specific for the cytosine nucleobase. The side chains of Arg110 and Ser36 cannot establish hydrogen bonds with UMP, and their substitution by hydrophobic amino acids simultaneously affects the K(m) of CMP/dCMP and the k(cat) value. Substitution of Ser for Asp132 results in a moderate decrease in stability without significant changes in K(m) value for CMP and dCMP. Replacement of Arg188 with Met does not affect enzyme stability but dramatically decreases the k(cat)/K(m) ratio compared with wild-type enzyme. This effect might be explained by opening of the enzyme/nucleotide complex, so that the sugar no longer interacts with Asp185. The reaction rate for different modified CMP kinases with ATP as a variable substrate indicated that none of changes induced by these amino acid substitutions was 'propagated' to the ATP subsite. This 'modular' behavior of E. coli CMP kinase is unique in comparison with other NMP kinases.

  16. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets.

    Science.gov (United States)

    Beuers, Ulrich; Denk, Gerald U; Soroka, Carol J; Wimmer, Ralf; Rust, Christian; Paumgartner, Gustav; Boyer, James L

    2003-05-16

    Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.

  17. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    Energy Technology Data Exchange (ETDEWEB)

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  18. Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

    Directory of Open Access Journals (Sweden)

    Kyu-Hee eLee

    2013-02-01

    Full Text Available We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2 in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2 with the canonical tyrosine motif (YxxΦ of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1. Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365. Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol in PC-12 cells. The effect of carbachol was inhibited by PP2, a Src family kinase (SFK inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells. These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

  19. ASK Family Kinases Are Required for Optimal NLRP3 Inflammasome Priming.

    Science.gov (United States)

    Place, David E; Samir, Parimal; Karki, Rajendra; Briard, Benoit; Vogel, Peter; Kanneganti, Thirumala-Devi

    2018-01-31

    Activation of the multimeric inflammasome complex leads to inflammatory responses to biotic and abiotic triggers. The inflammasome sensor, Nod-like receptor family pyrin domain containing 3 (NLRP3), is activated by a range of stimuli and is tightly regulated to restrict excessive inflammation. Because NLRP3 responds broadly to cellular insults and regulates cell death similar to the stress-activated apoptosis signal-regulating kinases 1 and 2 (ASK1/2), we hypothesized that ASK1/2 may regulate NLRP3 activity. Although essential for mediating NLRP3 inflammasome activation, ASK1/2 were not required for NLRC4 or absent in melanoma 2 inflammasome activation. ASK1/2 was required for NLRP3 up-regulation after lipopolysaccharide treatment in primary bone marrow-derived macrophages and lung fibroblasts as well as during infection with Burkholderia thailandensis and influenza virus. Consistent with reduced NLRP3 expression in response to B. thailandensis, caspase-1 cleavage and cell death were reduced in infected bone marrow-derived macrophages, and mice lacking ASK1/2 were resistant to Burkholderia intranasal infection. Single knockouts of either ASK1 or ASK2 showed a partial role for both ASK1 and ASK2 in NLRP3 up-regulation in response to lipopolysaccharide or B. thailandensis, but ASK2 was required primarily to mediate lethal pathology during intranasal infection in vivo. Our findings identify the ASK1/2 complex as a regulator of NLRP3 activation and highlight a larger role for ASK2 in lung infection during B. thailandensis infection. Copyright © 2018. Published by Elsevier Inc.

  20. Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

    Science.gov (United States)

    Craddock, Christian P; Adams, Nicolette; Kroon, Johan T M; Bryant, Fiona M; Hussey, Patrick J; Kurup, Smita; Eastmond, Peter J

    2017-01-01

    Coordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN-DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin-dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK-insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  1. Adaptation of Opossum Kidney Cells to Luminal Phosphate: Effects of Phosphonoformic Acid and Kinase Inhibitors

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    Linto Thomas

    2016-05-01

    Full Text Available Background/Aims: Renal reabsorption of inorganic phosphate (Pi is mediated by SLC34 and SLC20 Na+/Pi-cotransporters the abundance of which is under hormonal control. Extracellular Pi itself also regulates the expression of cotransporters and the concentration of Pi-regulating hormones, though the signaling pathways are largely unknown. Here, we explored the mechanisms that allow renal proximal cells to adapt to changes in the concentration of Pi. Methods: opossum kidney (OK cells, a model of proximal epithelia, were incubated with different concentrations of Pi in the absence/presence of phosphonoformic acid (PFA, a Pi-analogue and SLC34-inhibitor, and of inhibitors of kinases involved in hormonal control of Pi-homeostasis; cells cultured in normal media were treated with uncouplers of oxidative phosphorylation. Then, the intracellular concentration of ATP and/or the Pi-transport capacity of the cultures were analyzed. Results: luminal Pi regulates the Pi-transport and the intracellular ATP levels. Changes in ATP seem secondary to alterations in Pi-transport, rather than ATP acting as a signal. Adaptation of Pi-transport to high Pi was not mimicked by PFA. Transport adaptation was blocked by PFA but not by kinase inhibitors. Conclusions: in OK cells, adaptation of Pi-transport to luminal Pi does not depend on the same signaling pathways involved in hormonal regulation.

  2. Promotion of Tumor Invasion by Tumor-Associated Macrophages: The Role of CSF-1-Activated Phosphatidylinositol 3 Kinase and Src Family Kinase Motility Signaling

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    Amy R. Dwyer

    2017-06-01

    Full Text Available Macrophages interact with cells in every organ to facilitate tissue development, function and repair. However, the close interaction between macrophages and parenchymal cells can be subverted in disease, particularly cancer. Motility is an essential capacity for macrophages to be able to carry out their various roles. In cancers, the macrophage’s interstitial migratory ability is frequently co-opted by tumor cells to enable escape from the primary tumor and metastatic spread. Macrophage accumulation within and movement through a tumor is often stimulated by tumor cell production of the mononuclear phagocytic growth factor, colony-stimulating factor-1 (CSF-1. CSF-1 also regulates macrophage survival, proliferation and differentiation, and its many effects are transduced by its receptor, the CSF-1R, via phosphotyrosine motif-activated signals. Mutational analysis of CSF-1R signaling indicates that the major mediators of CSF-1-induced motility are phosphatidyl-inositol-3 kinase (PI3K and one or more Src family kinase (SFK, which activate signals to adhesion, actin polymerization, polarization and, ultimately, migration and invasion in macrophages. The macrophage transcriptome, including that of the motility machinery, is very complex and highly responsive to the environment, with selective expression of proteins and splice variants rarely found in other cell types. Thus, their unique motility machinery can be specifically targeted to block macrophage migration, and thereby, inhibit tumor invasion and metastasis.

  3. Promotion of Tumor Invasion by Tumor-Associated Macrophages: The Role of CSF-1-Activated Phosphatidylinositol 3 Kinase and Src Family Kinase Motility Signaling

    Science.gov (United States)

    Dwyer, Amy R.; Greenland, Eloise L.; Pixley, Fiona J.

    2017-01-01

    Macrophages interact with cells in every organ to facilitate tissue development, function and repair. However, the close interaction between macrophages and parenchymal cells can be subverted in disease, particularly cancer. Motility is an essential capacity for macrophages to be able to carry out their various roles. In cancers, the macrophage’s interstitial migratory ability is frequently co-opted by tumor cells to enable escape from the primary tumor and metastatic spread. Macrophage accumulation within and movement through a tumor is often stimulated by tumor cell production of the mononuclear phagocytic growth factor, colony-stimulating factor-1 (CSF-1). CSF-1 also regulates macrophage survival, proliferation and differentiation, and its many effects are transduced by its receptor, the CSF-1R, via phosphotyrosine motif-activated signals. Mutational analysis of CSF-1R signaling indicates that the major mediators of CSF-1-induced motility are phosphatidyl-inositol-3 kinase (PI3K) and one or more Src family kinase (SFK), which activate signals to adhesion, actin polymerization, polarization and, ultimately, migration and invasion in macrophages. The macrophage transcriptome, including that of the motility machinery, is very complex and highly responsive to the environment, with selective expression of proteins and splice variants rarely found in other cell types. Thus, their unique motility machinery can be specifically targeted to block macrophage migration, and thereby, inhibit tumor invasion and metastasis. PMID:28629162

  4. Pantothenate kinase-associated neurodegeneration initially presenting as postural tremor alone in a Japanese family with homozygous N245S substitutions in the pantothenate kinase gene.

    Science.gov (United States)

    Yamashita, Satoshi; Maeda, Yasushi; Ohmori, Hiroyuki; Uchida, Yuji; Hirano, Teruyuki; Yonemura, Kiminobu; Uyama, Eiichiro; Uchino, Makoto

    2004-10-15

    We describe a 24-year-old Japanese woman with pantothenate kinase-associated neurodegeneration (PKAN) whose only early symptom was postural tremor in the right hand at around 18 years of age, leading to a diagnosis of essential tremor at age 21. Although she was treated with arotinolol hydrochloride and clonazepam, she gradually progressed to extrapyramidal and pyramidal signs several years later. T2-weighted magnetic resonance images (MRI) showed bilaterally marked hypointensity with a central region of hyperintensity in the globus pallidus, or the so-called "eye-of-the-tiger" sign. Six years have passed since the initial appearance of postural tremor, whereas she has not shown choreoathetosis, retinitis pigmentosa, optic atrophy, or seizure. Direct sequencing of the patient's genomic DNA revealed homozygous base substitutions in the pantothenate kinase gene (PANK2): the A764-->G substitution (N245S) due to consanguinity of her parents. Although the heterozygous form of this mutation has already been reported among several families, this is the first report of the homozygous mutation in a patient with atypical-type PKAN. This detailed description of the clinical features of a Japanese patient with PKAN arising from homozygous N245S mutations in PANK2 would be useful for elucidating the pathogenesis of PKAN.

  5. A new Apicomplexa-specific protein kinase family : multiple members in Plasmodium falciparum, all with an export signature

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    Mercereau-Puijalon Odile

    2005-03-01

    Full Text Available Abstract Background Malaria caused by protozoan parasites of the genus Plasmodium spp. is a major health burden in tropical countries. The development of new control tools, including vaccines and drugs, is urgently needed. The availability of genome sequences from several malaria parasite species provides a basis on which to identify new potential intervention targets. Database mining for orthologs to the Plasmodium falciparum trophozoite protein R45, a vaccine candidate, led us identify a new gene family. Results Orthologs to the P. falciparum trophozoite protein R45 were detected exclusively in protozoan parasites of the phylum Apicomplexa, including several Plasmodium spp., Toxoplasma gondii and Cryptosporidium parvum. All family members are hybrid genes with a conserved C-terminal protein kinase domain of a novel type, recently called FIKK kinase, associated with a non conserved N-terminal region without any known functional signature. While a single copy gene was detected in most species, considerable gene expansion was observed in P. falciparum and its closest phylogenic relative P. reichenowi, with 20 and six copies, respectively, each with a distinct N-terminal domain. Based on full length protein sequence, pairs of orthologs were observed in closely related species, such as P. berghei and P.y. yoelii, P. vivax and P. knowlesi, or P. reichenowi and P. falciparum. All 20 P. falciparum paralogs possess a canonical Plasmodium export element downstream of a signal / anchor sequence required for exportation outside the parasitophorous vacuole. This is consistent with the reported association of the trophozoite protein R45, the only paralog characterised to date, with the infected red blood cell membrane. Interestingly, most genes are located in the subtelomeric region of chromosomes, in association with other multigene families contributing to the remodelling of the infected red blood cell membrane, in particular the ring erythrocyte surface

  6. Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

    Science.gov (United States)

    Min, Xiaoshan; Ungureanu, Daniela; Maxwell, Sarah; Hammarén, Henrik; Thibault, Steve; Hillert, Ellin-Kristina; Ayres, Merrill; Greenfield, Brad; Eksterowicz, John; Gabel, Chris; Walker, Nigel; Silvennoinen, Olli; Wang, Zhulun

    2015-11-06

    JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Cinnamic acid, from the bark of Cinnamomum cassia, regulates glucose transport via activation of GLUT4 on L6 myotubes in a phosphatidylinositol 3-kinase-independent manner.

    Science.gov (United States)

    Lakshmi, Baddireddi Subhadra; Sujatha, Sundaresan; Anand, Singaravelu; Sangeetha, Kadapakkam Nandhabalan; Narayanan, Rangarajan Badri; Katiyar, Chandrakanth; Kanaujia, Anil; Duggar, Rajeev; Singh, Yogendra; Srinivas, Konasubrahmanya; Bansal, Vinay; Sarin, Simi; Tandon, Ruchi; Sharma, Suchitra; Singh, Suchita

    2009-06-01

    Cinnamomum cassia (Family: Lauraceae) is an Ayurvedic medicinal plant used traditionally for the treatment of a number of diseases, including diabetes. The hypoglycemic effect of this plant has been established in vivo. However, the effects of cinnamic acid, isolated from C. cassia, on the insulin signaling cascade in an in vitro model have not been elucidated. Hence, the aim of the present study was to evaluate the anti-diabetic effect of cinnamic acid on glucose transport by L6 myotubes. The mechanism of action of cinnamic acid was determined using specific targets in the insulin signaling pathway, including protein tyrosine phosphatase (PTP) 1B, phosphatidylinositol 3-kinase (PI3-K) and the glucose transporter GLUT4. After differentiation of myoblast to myotubes, the cells were serum deprived for 5 h and then treated with 1 ng/mL cinnamic acid and 50 μmol/L rosiglitazone for 18 h and 100 nmol/L insulin for 20 min for gene expression studies. Expression of GLUT4 mRNA was increased following treatment of L6 myotubes with 1 ng/mL cinnamic acid. Furthermore, cinnamic acid inhibited PTP1B activity (by 96.5%), but had no significant effect on PI3-K activity. On the basis of the results of the present study, we postulate that cinnamic acid isolated from the hydro-alcoholic extract of Cinnamomum cassia activates glucose transport by a PI3-K-independent pathway. However, the detailed mechanism of action requires further analysis.

  8. Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

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    Nadine Veith

    Full Text Available Pyruvate kinase (PYK is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric

  9. The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

  10. Acetylsalicylic acid and salicylic acid decrease tumor cell viability and glucose metabolism modulating 6-phosphofructo-1-kinase structure and activity.

    Science.gov (United States)

    Spitz, Guilherme A; Furtado, Cristiane M; Sola-Penna, Mauro; Zancan, Patricia

    2009-01-01

    The common observation that cancer cells present higher glycolytic rates when compared to control cells leads to the proposal of glycolysis as a potential target for the development of anti-tumoral agents. Anti-inflammatory drugs, such as acetylsalicylic acid (ASA) and salicylic acid (SA), present anti-tumoral properties, inducing apoptosis and altering tumor glucose utilization. The present work aims at evaluating whether ASA could directly decrease cell glycolysis through inhibition of the major regulatory enzyme within this pathway, 6-phosphofructo-1-kinase (PFK). We show that ASA and SA inhibit purified PFK in a dose-dependent manner, and that this inhibition occurs due to the modulation of the enzyme quaternary structure. ASA and SA promote the dissociation of the enzyme active tetramers into quite inactive dimers, a common regulatory mechanism of this enzyme. The inhibitory effects of ASA and SA on PFK are fully reversible and can be prevented or reverted by the binding of the enzyme to the actin filaments. Both drugs are also able to decrease glucose consumption by human breast cancer cell line MCF-7, as well as its viability, which decrease parallelly to the inhibition of PFK on these cells. In the end, we demonstrate the ability of ASA and SA to directly modulate an important regulatory intracellular enzyme, and propose that this is one of their mechanisms promoting anti-tumoral effects.

  11. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  12. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Tamm, Christoffer, E-mail: christoffer.tamm@imbim.uu.se; Galito, Sara Pijuan, E-mail: sara.pijuan@imbim.uu.se; Anneren, Cecilia, E-mail: cecilia.anneren@imbim.uu.se

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  13. Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

    Directory of Open Access Journals (Sweden)

    Kap Lim

    Full Text Available The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP, was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170, which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

  14. Unconjugated secondary bile acids activate the unfolded protein response and induce golgi fragmentation via a src-kinase-dependant mechanism

    Science.gov (United States)

    Sharma, Ruchika; Quilty, Francis; Gilmer, John F.; Long, Aideen; Byrne, Anne-Marie

    2017-01-01

    Bile acids are components of gastro-duodenal refluxate and regarded as causative agents in oesophageal disease but the precise mechanisms are unknown. Here we demonstrate that a specific subset of physiological bile acids affect the protein secretory pathway by inducing ER stress, activating the Unfolded Protein Response (UPR) and causing disassembly of the Golgi apparatus in oesophageal cells. Deoxycholic acid (DCA), Chemodeoxycholic acid (CDCA) and Lithocholic acid (LCA) activated the PERK arm of the UPR, via phosphorylation of eIF2α and up-regulation of ATF3, CHOP and BiP/GRP78. UPR activation by these bile acids is mechanistically linked with Golgi fragmentation, as modulating the UPR using a PERK inhibitor (GSK2606414) or salubrinal attenuated bile acid-induced effects on Golgi structure. Furthermore we demonstrate that DCA, CDCA and LA activate Src kinase and that inhibition of this kinase attenuated both bile acid-induced BiP/GRP78 expression and Golgi fragmentation. This study highlights a novel mechanism whereby environmental factors (bile acids) impact important cellular processes regulating cell homeostasis, including the UPR and Golgi structure, which may contribute to cancer progression in the oesophagus. PMID:27888615

  15. Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor.

    Science.gov (United States)

    Denda, Kimitoshi; Nakao-Wakabayashi, Kanako; Okamoto, Naoki; Kitamura, Naomi; Ryu, Je-Young; Tagawa, Yoh-ichi; Ichisaka, Tomoko; Yamanaka, Shinya; Komada, Masayuki

    2011-08-19

    The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

  16. Butyric acid tolerance of rice mutant M4 families

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    Mauricio Marini Kopp

    2007-01-01

    Full Text Available Hydromorphic soils have a low drainage capacity and are used mainly for the cultivation of irrigated rice.This condition favors the development of anaerobic microorganisms that produce phytotoxic substances. The objective of thisstudy was to evaluate the response of rice mutants to the phytotoxicity caused by butyric acid under anaerobic conditions. Theexperiment consisted of four treatments arranged in a randomized block design. Plants of 40 families were grown in ahydroponic system and the measured variables were root length and length of aerial part (LAP, number of roots (NR androot dry matter (RDM and aerial part dry matter (DMAP. The analysis of variance was performed, the relative performancecalculated and linear regressions were fitted. Only the treatment effect for NR and effect of interaction for LAP were notsignificant. Root length was most affected by the acid and the regressions expressed positive as well as negative effects for acidtolerance in the mutant families.

  17. Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C.

    Science.gov (United States)

    Haag, M; Leonard, F; Magada, O N; Kruger, M C

    2000-03-01

    Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.

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

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    Kenyon Colin P

    2012-03-01

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

  19. Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators.

    Science.gov (United States)

    Jarillo, J A; Capel, J; Leyva, A; Martínez-Zapater, J M; Salinas, J

    1994-07-01

    We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.

  20. Hydroxysafflor yellow A suppress oleic acid-induced acute lung injury via protein kinase A

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    Wang, Chaoyun [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China); Huang, Qingxian [Department of Hepatobiliary Surgery, Yantai Yuhuangding Hospital, Yantai, Shandong 264000 (China); Wang, Chunhua; Zhu, Xiaoxi; Duan, Yunfeng; Yuan, Shuai [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China); Bai, Xianyong, E-mail: xybai2012@163.com [School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, Shandong 264003 (China)

    2013-11-01

    Inflammation response and oxidative stress play important roles in acute lung injury (ALI). Activation of the cAMP/protein kinase A (PKA) signaling pathway may attenuate ALI by suppressing immune responses and inhibiting the generation of reactive oxygen species (ROS). Hydroxysafflor yellow A (HSYA) is a natural flavonoid compound that reduces oxidative stress and inflammatory cytokine-mediated damage. In this study, we examined whether HSYA could protect the lungs from oleic acid (OA)-induced injury, which was used to mimic ALI, and determined the role of the cAMP/PKA signaling pathway in this process. Arterial oxygen tension (PaO{sub 2}), carbon dioxide tension, pH, and the PaO{sub 2}/fraction of inspired oxygen ratio in the blood were detected using a blood gas analyzer. We measured wet/dry lung weight ratio and evaluated tissue morphology. The protein and inflammatory cytokine levels in the bronchoalveolar lavage fluid and serum were determined using enzyme-linked immunoassay. The activities of superoxide dismutase, glutathione peroxidase, PKA, and nicotinamide adenine dinucleotide phosphate oxidase, and the concentrations of cAMP and malondialdehyde in the lung tissue were detected using assay kits. Bcl-2, Bax, caspase 3, and p22{sup phox} levels in the lung tissue were analyzed using Western blotting. OA increased the inflammatory cytokine and ROS levels and caused lung dysfunction by decreasing cAMP synthesis, inhibiting PKA activity, stimulating caspase 3, and reducing the Bcl-2/Bax ratio. H-89 increased these effects. HSYA significantly increased the activities of antioxidant enzymes, inhibited the inflammatory response via cAMP/PKA pathway activation, and attenuated OA-induced lung injury. Our results show that the cAMP/PKA signaling pathway is required for the protective effect of HSYA against ALI. - Highlights: • Oleic acid (OA) cause acute lung injury (ALI) via inhibiting cAMP/PKA signal pathway. • Blocking protein kinase A (PKA) activation may

  1. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression.

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    Roig, Joan; Mikhailov, Alexei; Belham, Christopher; Avruch, Joseph

    2002-07-01

    The protein kinase NIMA is an indispensable pleiotropic regulator of mitotic progression in Aspergillus. Although several mammalian NIMA-like kinases (Neks) are known, none appears to have the broad importance for mitotic regulation attributed to NIMA. Nercc1 is a new NIMA-like kinase that regulates chromosome alignment and segregation in mitosis. Its NIMA-like catalytic domain is followed by a noncatalytic tail containing seven repeats homologous to those of the Ran GEF, RCC1, a Ser/Thr/Pro-rich segment, and a coiled-coil domain. Nercc1 binds to another NIMA-like kinase, Nek6, and also binds specifically to the Ran GTPase through both its catalytic and its RCC1-like domains, preferring RanGDP in vivo. Nercc1 exists as a homooligomer and can autoactivate in vitro by autophosphorylation. Nercc1 is a cytoplasmic protein that is activated during mitosis and is avidly phosphorylated by active p34(Cdc2). Microinjection of anti-Nercc1 antibodies in prophase results in spindle abnormalities and/or chromosomal misalignment. In Ptk2 cells the outcome is prometaphase arrest or aberrant chromosome segregation and aneuploidy, whereas in CFPAC-1 cells prolonged arrest in prometaphase is the usual response. Nercc1 and its partner Nek6 represent a new signaling pathway that regulates mitotic progression.

  2. Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase.

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    Buttery, Shawnna M; Kono, Keiko; Stokasimov, Ema; Pellman, David

    2012-10-01

    Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.

  3. The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

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    Jennifer Pöhlmann

    2014-09-01

    Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

  4. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L..

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    Zongyong Tong

    Full Text Available Alfalfa (Medicago sativa L. is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs, such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  5. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tong, Zongyong; Xie, Can; Ma, Lei; Liu, Liping; Jin, Yongsheng; Dong, Jiangli; Wang, Tao

    2014-01-01

    Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  6. Diverse Transcriptional Programs Associated with Environmental Stress and Hormones in the Arabidopsis Receptor-Like Kinase Gene Family

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    Chae, Lee; Sudat, Sylvia; Dudoit, Sandrine; Zhu, Tong; Luan, Sheng

    2009-01-01

    The genome of Arabidopsis thaliana encodes more than 600 receptor-like kinase (RLK) genes, by far the dominant class of receptors found in land plants. Although similar to the mammalian receptor tyrosine kinases, plant RLKs are serine/threonine kinases that represent a novel signaling innovation unique to plants and, consequently, an excellent opportunity to understand how extracellular signaling evolved and functions in plants as opposed to animals. RLKs are predicted to be major components of the signaling pathways that allow plants to respond to environmental and developmental conditions. However, breakthroughs in identifying these processes have been limited to only a handful of individual RLKs. Here, we used a Syngenta custom Arabidopsis GeneChip array to compile a detailed profile of the transcriptional activity of 604 receptor-like kinase genes after exposure to a cross-section of known signaling factors in plants, including abiotic stresses, biotic stresses, and hormones. In the 68 experiments comprising the study, we found that 582 of the 604 RLK genes displayed a two-fold or greater change in expression to at least one of 12 types of treatments, thereby providing a large body of experimental evidence for targeted functional screens of individual RLK genes. We investigated whether particular subfamilies of RLK genes are responsive to specific types of signals and found that each subfamily displayed broad ranges of expression, as opposed to being targeted towards particular signal classes. Finally, by analyzing the divergence of sequence and gene expression among the RLK subfamilies, we present evidence as to the functional basis for the expansion of the RLKs and how this expansion may have affected conservation and divergences in their function. Taken as a whole, our study represents a preliminary, working model of processes and interactions in which the members of the RLK gene family may be involved, where such information has remained elusive for so many

  7. Phosphorylation of c-Cbl and p85 PI3K Driven by All-trans Retinoic Acid and CD38 Depends on Lyn Kinase Activity

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    Congleton, Johanna; Shen, Miaoqing; MacDonald, Robert; Malavasi, Fabio; Yen, Andrew

    2014-01-01

    The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48 hours followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48 hours following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48 hours of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation. PMID:24686085

  8. Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity.

    Science.gov (United States)

    Congleton, Johanna; Shen, Miaoqing; MacDonald, Robert; Malavasi, Fabio; Yen, Andrew

    2014-07-01

    The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists.

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    Kathryn S Carpentier

    2016-10-01

    Full Text Available During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm PKR (F489S is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.

  10. Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent

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    Liu W

    2015-07-01

    Full Text Available Wei Liu,1,* Jin-Feng Ning,2,* Qing-Wei Meng,1 Jing Hu,1 Yan-Bin Zhao,1 Chao Liu,3 Li Cai11The Fourth Department of Medical Oncology, Harbin Medical University Cancer Hospital, 2The Thoracic Surgery Department, Harbin Medical University Cancer Hospital, Harbin, People’s Republic of China; 3General Surgery Department, Mudanjiang Guanliju Central Hospital, Mishan, Heilongjiang Province, People’s Republic of China*These authors contributed equally to this workAbstract: The epidermal growth factor receptor (EGFR family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC, particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB. Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10 against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase–ligand interaction space in the PDB.Keywords: EGFR, kinase

  11. Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms.

    Science.gov (United States)

    Wloga, Dorota; Camba, Amy; Rogowski, Krzysztof; Manning, Gerard; Jerka-Dziadosz, Maria; Gaertig, Jacek

    2006-06-01

    The genome of Tetrahymena thermophila contains 39 loci encoding NIMA-related kinases (NRKs), an extraordinarily large number for a unicellular organism. Evolutionary analyses grouped these sequences into several subfamilies, some of which have orthologues in animals, whereas others are protist specific. When overproduced, NRKs of three subfamilies caused rapid shortening of cilia. Ultrastructural studies revealed that each NRK triggered ciliary resorption by a distinct mechanism that involved preferential depolymerization of a subset of axonemal microtubules, at either the distal or proximal end. Overexpression of a kinase-inactive variant caused lengthening of cilia, indicating that constitutive NRK-mediated resorption regulates the length of cilia. Each NRK preferentially resorbed a distinct subset of cilia, depending on the location along the anteroposterior axis. We also show that normal Tetrahymena cells maintain unequal length cilia. We propose that ciliates used a large number of NRK paralogues to differentially regulate the length of specific subsets of cilia in the same cell.

  12. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-activated Protein Kinase Alpha in vivo and in vitro

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    Hara Lim

    2016-12-01

    Full Text Available Chrysophanic acid (CA is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ and CCAAT/enhancer-binding protein alpha (C/EBPα in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α, the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK inhibitor, CA was able to restore the activation of AMPKα in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases we suggest the new potential of CA as an anti-obese pharmacotherapy.

  13. Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

    Science.gov (United States)

    Sen, Hrishiraj; Aggarwal, Nikhil; Ishionwu, Chibueze; Hussain, Nosheen; Parmar, Chandni; Jamshad, Mohammed; Bavro, Vassiliy N; Lund, Peter A

    2017-09-15

    The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this. IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps

  14. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

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    Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (Purdue); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-{angstrom} resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases.

  15. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

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    Hitomi Maruta

    Full Text Available Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4 genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A, which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  16. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    Science.gov (United States)

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  17. Rosmarinic acid plays a protective role in the embryogenesis of zebrafish exposed to food colours through its influence on aurora kinase A level.

    Science.gov (United States)

    Swarnalatha, Y; Jerrine Joseph, I S; Jayakrishna, Tippabathani

    2017-05-01

    To evaluate the protective nature of the rosmarinic acid from Sphaeranthus amaranthoides during zebra fish embryogenesis. Rosmarinic acid was isolated from the S. amaranthoides. An accurate, sensitive and simple LC-MS analysis was performed to determine the rosmarinic acid from S. amaranthoides. In the present study, zebrafish embryos were exposed to crimson red and sunset yellow at a concentration of 0.1 and 0.5mg/l and the effect of these food colours on the levels of aurora kinase A was studied individually. Aurora kinase A levels are crucial for embryogenesis in zebrafish which is used as model in this study. The decrease of aurora kinase A levels in food colour treated embryos influences the embryogenesis, resulting in short and bent trunk leading to cell death and growth retardation. Elevated levels of aurora kinase A in rosmarinic acid treated groups can be attributed to the restoration of normal growth in zebra fish embryos with well developed brain and eyes. Further insilico docking studies were carried out and target was identified as rosmarinic acid. From the docking studies the docking poses and binding energy confirms that aurora kinase A is the target for rosmarinic acid. Rosmarinic acid was found to play a protective role in the embryogenesis of zebra fish exposed to food colours (crimson red and sunset yellow) through its influence on aurora kinase A levels. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Diacylglycerol Kinases: Shaping Diacylglycerol and Phosphatidic Acid Gradients to Control Cell Polarity

    Directory of Open Access Journals (Sweden)

    Gianluca Baldanzi

    2016-11-01

    Full Text Available Diacylglycerol kinases (DGKs terminate diacylglycerol (DAG signaling and promote phosphatidic acid (PA production. Isoform specific regulation of DGKs activity and localization allows DGKs to shape the DAG and PA gradients. The capacity of DGKs to constrain the areas of DAG signaling is exemplified by their role in defining the contact interface between T cells and antigen presenting cells: the immune synapse. Upon T cell receptor engagement, both DGK α and ζ metabolize DAG at the immune synapse thus constraining DAG signaling. Interestingly, their activity and localization are not fully redundant because DGKζ activity metabolizes the bulk of DAG in the cell, whereas DGKα limits the DAG signaling area localizing specifically at the periphery of the immune synapse.When DGKs terminate DAG signaling, the local PA production defines a new signaling domain, where PA recruits and activates a second wave of effector proteins. The best-characterized example is the role of DGKs in protrusion elongation and cell migration. Indeed, upon growth factor stimulation, several DGK isoforms, such as α, ζ, and γ, are recruited and activated at the plasma membrane. Here, local PA production controls cell migration by finely modulating cytoskeletal remodeling and integrin recycling. Interestingly, DGK-produced PA also controls the localization and activity of key players in cell polarity such as aPKC, Par3, and integrin β1. Thus, T cell polarization and directional migration may be just two instances of the general contribution of DGKs to the definition of cell polarity by local specification of membrane identity signaling.

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

    Science.gov (United States)

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

    2016-01-01

    The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

  20. Effect of oncogene activating mutations and kinase inhibitors on amino acid metabolism of human isogenic breast cancer cells.

    Science.gov (United States)

    Kim, Eung-Sam; Samanta, Animesh; Cheng, Hui Shan; Ding, Zhaobing; Han, Weiping; Toschi, Luisella; Chang, Young Tae

    2015-12-01

    We investigated the changes in amino acid (AA) metabolism induced in MCF10A, a human mammary epithelial cell line, by the sequential knock-in of K-Ras and PI3K mutant oncogenes. Differentially regulated genes associated to AA pathways were identified on comparing gene expression patterns in the isogenic cell lines. Additionally, we monitored the changes in the levels of AAs and transcripts in the cell lines treated with kinase inhibitors (REGO: a multi-kinase inhibitor, PI3K-i: a PI3K inhibitor, and MEK-i: a MEK inhibitor). In total, 19 AAs and 58 AA-associated transcripts were found to be differentially regulated by oncogene knock-in and by drug treatment. In particular, the multi-kinase and MEK inhibitor affected pathways in K-Ras mutant cells, whereas the PI3K inhibitor showed a major impact in the K-Ras/PI3K double mutant cells. These findings may indicate the dependency of AA metabolism on the oncogene mutation pattern in human cancer. Thus, future therapy might include combinations of kinase inhibitors and drug targeting enzymes of AA pathways.

  1. Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways during Apoptosis Induction in Human Pancreatic β-Cells Is Inhibited by Unsaturated Oleic Acid

    Directory of Open Access Journals (Sweden)

    Jan Šrámek

    2017-11-01

    Full Text Available It has been shown that saturated fatty acids (FAs have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP kinase and endoplasmic reticulum (ER stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA affects the effect of saturated stearic acid (SA on the p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinase (ERK pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

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

    Science.gov (United States)

    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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Tomato ABSCISIC ACID STRESS RIPENING (ASR gene family revisited.

    Directory of Open Access Journals (Sweden)

    Ido Golan

    Full Text Available Tomato ABSCISIC ACID RIPENING 1 (ASR1 was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each, whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons. ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA. Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.

  4. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Science.gov (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  5. Transcriptional regulation of human mucin MUC4 by bile acids in oesophageal cancer cells is promoter-dependent and involves activation of the phosphatidylinositol 3-kinase signalling pathway.

    Science.gov (United States)

    Mariette, Christophe; Perrais, Michaël; Leteurtre, Emmanuelle; Jonckheere, Nicolas; Hémon, Brigitte; Pigny, Pascal; Batra, Surinder; Aubert, Jean-Pierre; Triboulet, Jean-Pierre; Van Seuningen, Isabelle

    2004-02-01

    Abnormal gastro-oesophageal reflux and bile acids have been linked to the presence of Barrett's oesophageal premalignant lesion associated with an increase in mucin-producing goblet cells and MUC4 mucin gene overexpression. However, the molecular mechanisms underlying the regulation of MUC4 by bile acids are unknown. Since total bile is a complex mixture, we undertook to identify which bile acids are responsible for MUC4 up-regulation by using a wide panel of bile acids and their conjugates. MUC4 apomucin expression was studied by immunohistochemistry both in patient biopsies and OE33 oesophageal cancer cell line. MUC4 mRNA levels and promoter regulation were studied by reverse transcriptase-PCR and transient transfection assays respectively. We show that among the bile acids tested, taurocholic, taurodeoxycholic, taurochenodeoxycholic and glycocholic acids and sodium glycocholate are strong activators of MUC4 expression and that this regulation occurs at the transcriptional level. By using specific pharmacological inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase A and protein kinase C, we demonstrate that bile acid-mediated up-regulation of MUC4 is promoter-specific and mainly involves activation of phosphatidylinositol 3-kinase. This new mechanism of regulation of MUC4 mucin gene points out an important role for bile acids as key molecules in targeting MUC4 overexpression in early stages of oesophageal carcinogenesis.

  6. The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

    2017-01-01

    Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer......-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.......o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro...

  7. Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

    Science.gov (United States)

    Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

    2014-04-01

    Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

  8. Intracellular calcium-release and protein kinase C-activation stimulate sonic hedgehog gene expression during gastric acid secretion

    Science.gov (United States)

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L.

    2010-01-01

    Introduction Hypochlorhydria during Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2+i-dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)-activation. Method We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H+/K+-β-cholera-toxin overexpressing mice (Ctox). Results Mice that expressed sHip-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression was also repressed in the hyperchlorhydric Ctox model with elevated cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca2+i-release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin- and carbachol-mediated release of Ca2+i induced Shh expression. Ca2+-chelation with BAPTA+EGTA reduced Shh expression. Overexpression of PKC-α, -β and -δ (but not PKC-ε) induced Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Conclusion Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca2+i-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin. PMID:20816837

  9. Lipoic acid attenuates inflammation via cAMP and protein kinase A signaling.

    Directory of Open Access Journals (Sweden)

    Sonemany Salinthone

    2010-09-01

    Full Text Available Abnormal regulation of the inflammatory response is an important component of diseases such as diabetes, Alzheimer's disease and multiple sclerosis (MS. Lipoic acid (LA has been shown to have antioxidant and anti-inflammatory properties and is being pursued as a therapy for these diseases. We first reported that LA stimulates cAMP production via activation of G-protein coupled receptors and adenylyl cyclases. LA also suppressed NK cell activation and cytotoxicity. In this study we present evidence supporting the hypothesis that the anti-inflammatory properties of LA are mediated by the cAMP/PKA signaling cascade. Additionally, we show that LA oral administration elevates cAMP levels in MS subjects.We determined the effects of LA on IL-6, IL-17 and IL-10 secretion using ELISAs. Treatment with 50 µg/ml and 100 µg/ml LA significantly reduced IL-6 levels by 19 and 34%, respectively, in T cell enriched PBMCs. IL-17 levels were also reduced by 35 and 50%, respectively. Though not significant, LA appeared to have a biphasic effect on IL-10 production. Thymidine incorporation studies showed LA inhibited T cell proliferation by 90%. T-cell activation was reduced by 50% as measured by IL-2 secretion. Western blot analysis showed that LA treatment increased phosphorylation of Lck, a downstream effector of protein kinase A. Pretreatment with a peptide inhibitor of PKA, PKI, blocked LA inhibition of IL-2 and IFN gamma production, indicating that PKA mediates these responses. Oral administration of 1200 mg LA to MS subjects resulted in increased cAMP levels in PBMCs four hours after ingestion. Average cAMP levels in 20 subjects were 43% higher than baseline.Oral administration of LA in vivo resulted in significant increases in cAMP concentration. The anti-inflammatory effects of LA are mediated in part by the cAMP/PKA signaling cascade. These novel findings enhance our understanding of the mechanisms of action of LA.

  10. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

    NARCIS (Netherlands)

    Beilina, Alexandria; Rudenko, Iakov N.; Kaganovich, Alice; Civiero, Laura; Chau, Hien; Kalia, Suneil K.; Kalia, Lorraine V.; Lobbestael, Evy; Chia, Ruth; Ndukwe, Kelechi; Ding, Jinhui; Nalls, Mike A.; Olszewski, Maciej; Hauser, David N.; Kumaran, Ravindran; Lozano, Andres M.; Baekelandt, Veerle; Greene, Lois E.; Taymans, Jean-Marc; Greggio, Elisa; Cookson, Mark R.; Plagnol, Vincent; Martinez, Maria; Hernandez, Dena G.; Sharma, Manu; Sheerin, Una-Marie; Saad, Mohamad; Simón-Sánchez, Javier; Schulte, Claudia; Lesage, Suzanne; Sveinbjörnsdóttir, Sigurlaug; Arepalli, Sampath; Barker, Roger; Ben-Shlomo, Yoav; Berendse, Henk W.; Berg, Daniela; Bhatia, Kailash; de Bie, Rob M. A.; Biffi, Alessandro; Bloem, Bas; Bochdanovits, Zoltan; Bonin, Michael; Bras, Jose M.; Brockmann, Kathrin; Brooks, Janet; Burn, David J.; Charlesworth, Gavin; Chen, Honglei; Chong, Sean; Clarke, Carl E.; Cooper, J. Mark; Corvol, Jean Christophe; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T.; van Dijk, Karin D.; Dillman, Allissa; Durif, Frank; Dürr, Alexandra; Edkins, Sarah; Evans, Jonathan R.; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Gibbs, J. Raphael; Goate, Alison; Gray, Emma; Guerreiro, Rita; Gústafsson, Omar; Harris, Clare; van Hilten, Jacobus J.; Hofman, Albert; Hollenbeck, Albert; Holton, Janice; Hu, Michele; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E.; Huttenlocher, Johanna; Illig, Thomas; München, Helmholtz Zentrum; Jónsson, Pálmi V.; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morris, Huw R.; Morrison, Karen E.; Mudanohwo, Ese; O'Sullivan, Sean S.; Pearson, Justin; Perlmutter, Joel S.; Pétursson, Hjörvar; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Shaw, Karen; Shoulson, Ira; Sidransky, Ellen; Smith, Colin; Spencer, Chris C. A.; Stefánsson, Hreinn; Steinberg, Stacy; Stockton, Joanna D.; Strange, Amy; Talbot, Kevin; Tanner, Carlie M.; Tashakkori-Ghanbaria, Avazeh; Tison, François; Trabzuni, Daniah; Traynor, Bryan J.; Uitterlinden, André G.; Velseboer, Daan; Vidailhet, Marie; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams, Nigel; Williams-Gray, Caroline H.; Winder-Rhodes, Sophie; Stefánsson, Kári; Hardy, John; Heutink, Peter; Brice, Alexis; Gasser, Thomas; Singleton, Andrew B.; Wood, Nicholas W.; Chinnery, Patrick F.; Ferrucci, Luigi; Johnson, Robert; Longo, Dan L.; Majounie, Elisa; Nalls, Michael A.; O'Brien, Richard; Troncoso, Juan; van der Brug, Marcel; Zielke, H. Ronald; Zonderman, Alan B.

    2014-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and

  11. Diastereomeric mixture of calophyllic acid and isocalophyllic acid stimulates glucose uptake in skeletal muscle cells: involvement of PI-3-kinase- and ERK1/2-dependent pathways.

    Science.gov (United States)

    Prasad, Janki; Maurya, Chandan Kumar; Pandey, Jyotsana; Jaiswal, Natasha; Madhur, Gaurav; Srivastava, Arvind Kumar; Narender, Tadigoppula; Tamrakar, Akhilesh Kumar

    2013-05-06

    The diastereomeric mixture of calophyllic acid and isocalophyllic acid (F015) isolated from the leaves of Calophyllum inophyllum was investigated for the metabolic effect on glucose transport in skeletal muscle cells. In L6 myotubes, F015 dose-dependently stimulated glucose uptake by increasing translocation of glucose transporter4 (GLUT4) to plasma membrane without affecting their gene expression. The effects on glucose uptake were additive to insulin. Inhibitors analyses revealed that F015-induced glucose uptake was dependent on the activation of phosphatidylinositol-3-kinase (PI-3-K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), while independent to the activation of 5'AMP-activated kinase (AMPK). F015 significantly increased the phosphorylation of AKT, AS160 and ERK1/2, account for the augmented glucose transport capacity in L6 myotubes. Furthermore, F015 improved glucose tolerance and enhanced insulin sensitivity in skeletal muscle of dexamethasone-induced insulin resistant mice. Our findings demonstrate that F015 activates glucose uptake in skeletal muscle cells through PI-3-K- and EKR1/2-dependent mechanisms and can be a potential lead for the management of diabetes and obesity. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer cell growth by activating Src family kinases.

    Science.gov (United States)

    Leroy, C; Shen, Q; Strande, V; Meyer, R; McLaughlin, M E; Lezan, E; Bentires-Alj, M; Voshol, H; Bonenfant, D; Alex Gaither, L

    2015-10-29

    The transmembrane glycoprotein, CUB (complement C1r/C1s, Uegf, Bmp1) domain-containing protein 1 (CDCP1) is overexpressed in several cancer types and is a predictor of poor prognosis for patients on standard of care therapies. Phosphorylation of CDCP1 tyrosine sites is induced upon loss of cell adhesion and is thought to be linked to metastatic potential of tumor cells. Using a tyrosine-phosphoproteomics screening approach, we characterized the phosphorylation state of CDCP1 across a panel of breast cancer cell lines. We focused on two phospho-tyrosine pTyr peptides of CDCP1, containing Tyr707 and Tyr806, which were identified in all six lines, with the human epidermal growth factor 2-positive HCC1954 cells showing a particularly high phosphorylation level. Pharmacological modulation of tyrosine phosphorylation indicated that, the Src family kinases (SFKs) were found to phosphorylate CDCP1 at Tyr707 and Tyr806 and play a critical role in CDCP1 activity. We demonstrated that CDCP1 overexpression in HEK293 cells increases global phosphotyrosine content, promotes anchorage-independent cell growth and activates several SFK members. Conversely, CDCP1 downregulation in multiple solid cancer cell lines decreased both cell growth and SFK activation. Analysis of primary human tumor samples demonstrated a correlation between CDCP1 expression, SFK and protein kinase C (PKC) activity. Taken together, our results suggest that CDCP1 overexpression could be an interesting therapeutic target in multiple solid cancers and a good biomarker to stratify patients who could benefit from an anti-SFK-targeted therapy. Our data also show that multiple tyrosine phosphorylation sites of CDCP1 are important for the functional regulation of SFKs in several tumor types.

  13. Casein kinase 1-Like 3 is required for abscisic acid regulation of ...

    African Journals Online (AJOL)

    The homozygous T-DNA mutant of a casein kinase 1-Like 3 (ckl3) was identified. The quantitative realtime polymerase chain reaction (QRT-PCR) results showed that CKL3 was highly expressed in flowers and roots, but less in stems, leaves and leafstalks. It was found that CKL3 gene was induced by abscisic (ABA).

  14. A Stepwise Approach for the Synthesis of Folic Acid Conjugates with Protein Kinase Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Krajčovičová, S.; Gucký, Tomáš; Hendrychová, Denisa; Kryštof, Vladimír; Soural, M.

    2017-01-01

    Roč. 82, č. 24 (2017), s. 13530-13541 ISSN 0022-3263 R&D Projects: GA ČR(CZ) GA15-15264S; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : CYCLIN-DEPENDENT KINASES * FOLATE RECEPTOR * REGIOSELECTIVE SYNTHESIS Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.849, year: 2016

  15. Casein kinase 1-Like 3 is required for abscisic acid regulation of ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... different cell types and in several subcellular compart- ments, including the membranes, nucleus and cytoplasm ... priming by another kinase. CKI also phosphorylates a related unprimed site, which ... CK1 isoforms function as regulators of multiple signal transduction pathways in most eukaryotic cell types.

  16. Cajaninstilbene acid relaxes rat renal arteries: roles of Ca2+ antagonism and protein kinase C-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Dong-Mei Zhang

    Full Text Available Cajaninstilbene acid (CSA is a major active component present in the leaves of Cajanus cajan (L. Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1-10 µM produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F(2α (U46619, and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with N(G-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl(2, glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl(2 (0.01-5 mM in Ca(2+-free 60 mM KCl solution and by 30 nM (--Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca(2+ influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate (PMA, protein kinase C agonist in Ca(2+-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC at Ser19 and myosin phosphatase target subunit 1 (MYPT1 at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.

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

    CSIR Research Space (South Africa)

    Kenyon, CP

    2012-03-01

    Full Text Available , with the concomitant re-protonation of C8. Groups 8 There is only one member in each of Group 8, Group 9, Group 10 and Group 11 kinases. Group 8 consists of ribo- flavin kinase from Schizosaccharomyces pombe and Homo sapiens (flavokinase family) (Additional file 1... 3) in that the latter two are pyrophosphotrans- ferases transferring a pyrophosphate group from a nucleo- side triphosphate, such as ATP to the hydroxyl of thiamine. All enzymes in Group 7 are homo-dimeric, with the active site comprising amino...

  18. Intracellular calcium release and protein kinase C activation stimulate sonic hedgehog gene expression during gastric acid secretion.

    Science.gov (United States)

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L

    2010-12-01

    Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation. We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-β-cholera-toxin-overexpressing mice. Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-β-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-α, -β, and -δ (but not PKC-ϵ) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Secretagogues that stimulate

  19. Expression of multiple Src family kinases in sea urchin eggs and their function in Ca2+ release at fertilization.

    Science.gov (United States)

    Townley, Ian K; Schuyler, Erin; Parker-Gür, Michelle; Foltz, Kathy R

    2009-03-15

    Egg activation at fertilization in deuterostomes requires a rise in intracellular Ca(2+), which is released from the egg's endoplasmic reticulum. In sea urchins, a Src Family Kinase (SpSFK1) is necessary for the PLCgamma-mediated signaling event that initiates this Ca(2+) release (Giusti, A.F., O'Neill, F.J., Yamasu, K., Foltz, K.R. and Jaffe, L.A., 2003. Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization. Dev. Biol. 256, 367-378.). Annotation of the Strongylocentrotus purpuratus genome sequence led to the identification of additional, predicted SFKs (Bradham, C.A., Foltz, D.R., Beane, W.S., Amone, M.I., Rizzo, F., Coffman, J.A., Mushegian, A., Goel, M., Morales, J., Geneviere, A.M., Lapraz, F., Robertson, A.J., Kelkar, H., Loza-Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lepage, T., Gache, C., McClay, D.R., Manning, G., 2006. The sea urchin kinome: a first look. Dev. Biol. 300, 180-193.; Roux, M.M., Townley, I.K., Raisch, M., Reade, A., Bradham, C., Humphreys, G., Gunaratne, H.J., Killian, C.E., Moy, G., Su, Y.H., Ettensohn, C.A., Wilt, F., Vacquier, V.D., Burke, R.D., Wessel, G. and Foltz, K.R., 2006. A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation. Dev. Biol. 300, 416-433.). Here, we describe the cloning and characterization of these 4 additional SFKs and test their function during the initial Ca(2+) release at fertilization using the dominant-interfering microinjection method coupled with Ca(2+) recording. While two of the new SFKs (SpFrk and SpSFK3) are necessary for Ca(2+) release, SpSFK5 appears dispensable for early egg to embryo transition events. Interestingly, SpSFK7 may be involved in preventing precocious release of Ca(2+). Binding studies indicate that only SpSFK1 is capable of direct interaction with PLCgamma. Immunolocalization studies suggest that one or more SpSFK and PLCgamma are localized to the egg cortex and at the site of sperm-egg interaction

  20. Amino Acids Involved in Polyphosphate Synthesis and Its Mobilization Are Distinct in Polyphosphate Kinase-1 from Mycobacterium tuberculosis

    Science.gov (United States)

    Mittal, Payal; Karthikeyan, Subramanian; Chakraborti, Pradip K.

    2011-01-01

    Background In bacteria polyphosphates (poly-P) are involved in cellular metabolism and development especially during stress. The enzyme, principally involved in polyphosphate biosynthesis and its mobilization leading to generation of NTPs, is known as polyphosphate kinase (PPK). Principal Findings Among two genes of polyphosphate kinases present in Mycobacterium tuberculosis, we cloned and expressed PPK1 in Escherichia coli as histidine-tagged protein. This ∼86 kDa protein is capable of autophosphorylation and synthesis of poly-P as well as NTP. Among 22 conserved histidine residues, we found only His-491 is autophosphorylated and crucial for any enzymatic activity. Substitution of His-510 caused mPPK1 protein deficient but not defective in autophosphorylation, thereby contrary to earlier reports negating any role of this residue in the process. However, mutation of His-510 with either Ala or Gln affected ATP or poly-P synthesis depending on the substitution; while such effects were severe with H510A but mild with H510Q. Furthermore, mPPK1 also renders auxiliary nucleotide diphosphate kinase function by synthesizing virtually all NTPs/dNTPs from their cognate NDPs/dNDPs by utilizing poly-P as the phosphate donor albeit with varied efficiency. To assess the influence of other catalytic domain residues of mPPK1 towards its functionality, we designed mutations based on E. coli PPK1 crystal structure since it owes 68% amino acid sequence similarity with mPPK1. Interestingly, our results revealed that mutations in mPPK1 affecting poly-P synthesis always affected its ATP synthesizing ability; however, the reverse may not be true. Conclusions/Significance We conclude that amino acid residues involved in poly-P and ATP synthesizing activities of mPPK1 are distinct. Considering conserved nature of PPK1, it seems our observations have broader implications and not solely restricted to M. tuberculosis only. PMID:22110640

  1. The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth and signal via protein kinase C.

    Science.gov (United States)

    Loers, Gabriele; Astafiev, Steven; Hapiak, Yuliya; Saini, Vedangana; Mishra, Bibhudatta; Gul, Sheraz; Kaur, Gurcharan; Schachner, Melitta; Theis, Thomas

    2017-08-01

    Polysialic acid (PSA) is a large, negatively charged, linear homopolymer of alpha2-8-linked sialic acid residues. It is generated by two polysialyltransferases and attached to N- and/or O-linked glycans, and its main carrier is the neural cell adhesion molecule (NCAM). PSA controls the development and regeneration of the nervous system by enhancing cell migration, axon pathfinding, synaptic targeting, synaptic plasticity, by regulating the differentiation of progenitor cells and by modulating cell-cell and cell-matrix adhesions. In the adult, PSA plays a role in the immune system, and PSA mimetics promote functional recovery after nervous system injury. In search for novel small molecule mimetics of PSA that are applicable for therapy, we identified idarubicin, an antineoplastic anthracycline, and irinotecan, an antineoplastic agent of the topoisomerase I inhibitor class, as PSA mimetics using a competition enzyme-linked immunosorbent assay. Idarubicin and irinotecan compete with the PSA-mimicking peptide and colominic acid, the bacterial analog of PSA, for binding to the PSA-specific monoclonal antibody 735. Idarubicin and irinotecan stimulate neurite outgrowth and survival of cultured cerebellar neurons after oxidative stress via protein kinase C and Erk1/2 in a similar manner as colominic acid, whereas Fyn, casein kinase II and the phosphatase and tensin homolog are only involved in idarubicin and irinotecan-stimulated neurite outgrowth. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds irinotecan and idarubicin which trigger the same signaling cascades as PSA, thus introducing the possibility of retargeting these drugs to treat nervous system injuries. © 2017 International Society for Neurochemistry.

  2. Dynamics of PLCγ and Src family kinase 1 interactions during nuclear envelope formation revealed by FRET-FLIM.

    Directory of Open Access Journals (Sweden)

    Richard D Byrne

    Full Text Available The nuclear envelope (NE breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1 during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.

  3. Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress†

    OpenAIRE

    Lawrence, Clare L.; Botting, Catherine H.; Antrobus, Robin; Coote, Peter J.

    2004-01-01

    Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite mino...

  4. The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes1[W

    Science.gov (United States)

    Arrighi, Jean-François; Barre, Annick; Ben Amor, Besma; Bersoult, Anne; Soriano, Lidia Campos; Mirabella, Rossana; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal; Ghérardi, Michèle; Huguet, Thierry; Geurts, René; Dénarié, Jean; Rougé, Pierre; Gough, Clare

    2006-01-01

    Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known. PMID:16844829

  5. Crystal structure of Clostridium acetobutylicum aspartate kinase (CaAk: An important allosteric enzyme for amino acids production

    Directory of Open Access Journals (Sweden)

    Babu A. Manjasetty

    2014-09-01

    Full Text Available Aspartate kinase (AK is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-l-aspartate from l-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase (CaAK; E.C. 2.7.2.4; Mw = 48,030 Da; 437aa; SwissProt: Q97MC0 has been determined to 3 Å resolution. CaAK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (<30%. It is composed of two domains: an N-terminal catalytic domain (kinase domain and a C-terminal regulatory domain further comprised of two small domains belonging to the ACT domain family. Pairwise comparison of 12 molecules in the asymmetric unit helped to identify the bending regions which are in the vicinity of ATP binding site involved in domain movements between the catalytic and regulatory domains. All 12 CaAK molecules adopt fully open T-state conformation leading to the formation of three tetramers unique among other similar AK structures. On the basis of comparative structural analysis, we discuss tetramer formation based on the large conformational changes in the catalytic domain associated with the lysine binding at the regulatory domains. The structure described herein is homologous to a target in wide-spread pathogenic (toxin producing bacteria such as Clostridium tetani (64% sequence identity suggesting the potential of the structure solved here to be applied for modeling drug interactions. CaAK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry.

  6. Development of Poly Lactic/Glycolic Acid (PLGA) Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor.

    Science.gov (United States)

    Koda, Sho; Okumura, Naoki; Kitano, Junji; Koizumi, Noriko; Tabata, Yasuhiko

    2017-01-01

    The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7-10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.

  7. Development of Poly Lactic/Glycolic Acid (PLGA Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor

    Directory of Open Access Journals (Sweden)

    Sho Koda

    2017-01-01

    Full Text Available Purpose. The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA as a drug delivery carrier of Rho kinase (ROCK inhibitor for the treatment of corneal endothelial disease. Method. ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1, and a double emulsion [(W1/O/W2] was formed with dichloromethane (O and polyvinyl alcohol (W2. Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Results. Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. Conclusions. ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7–10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.

  8. Solute carrier family 35 member F2 is indispensable for papillary thyroid carcinoma progression through activation of transforming growth factor-β type I receptor/apoptosis signal-regulating kinase 1/mitogen-activated protein kinase signaling axis.

    Science.gov (United States)

    He, Jing; Jin, Yiting; Zhou, Mingxia; Li, Xiaoyan; Chen, Wanna; Wang, Yiwei; Gu, Siwen; Cao, Yun; Chu, Chengyu; Liu, Xiuping; Zou, Qiang

    2017-12-22

    Solute carrier family members control essential physiological functions and are tightly linked to human diseases. Solute carrier family 35 member F2 (SLC35F2) is aberrantly activated in several malignancies. However, the biological function and molecular mechanism of SLC35F2 in papillary thyroid carcinoma (PTC) are yet to be fully explored. Here, we showed that SLC35F2 was prominently upregulated in PTC tissues at both protein and mRNA expression level compared with matched adjacent normal tissues. Besides, the high expression of SLC35F2 was significantly associated with lymph node metastasis in patients with PTC. CRISPR/Cas9-mediated knockout of SLC35F2 attenuated the tumorigenic properties of PTC, including cell proliferation, migration and invasion and induced G1 phase arrest. In contrast, ectopic expression of SLC35F2 brought about aggressive malignant phenotypes of PTC cells. Moreover, SLC35F2 expedited the proliferation and migration of PTC cells by targeting transforming growth factor-β type I receptor (TGFBR1) and phosphorylation of apoptosis signal-regulating kinase 1 (p-ASK-1), thereby activating the mitogen-activated protein kinase signaling pathway. The malignant behaviors induced by overexpression of SLC35F2 could be abrogated by silencing of TGFBR1 using a specific inhibitor. We conducted the first study on SLC35F2 in thyroid cancer with the aim of elucidating the functional significance and molecular mechanism of SLC35F2. Our findings suggest that SLC35F2 exerts its oncogenic effect on PTC progression through the mitogen-activated protein kinase pathway, with dependence on activation of TGFBR-1 and apoptosis signal-regulating kinase 1. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  9. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4

    DEFF Research Database (Denmark)

    Brodersen, Klaus Peter; Petersen, Morten; Nielsen, Henrik Bjørn

    2006-01-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective...

  10. Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion

    Directory of Open Access Journals (Sweden)

    Ivan Quétier

    2016-01-01

    Full Text Available In animals, the protein kinase C (PKC family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10, with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.

  11. Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

    Science.gov (United States)

    Watanabe, Daisuke; Takagi, Hiroshi

    2017-06-01

    Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G2/M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

  12. Whole Exome Sequencing, Familial Genomic Triangulation, and Systems Biology Converge to Identify a Novel Nonsense Mutation in TAB2-encoded TGF-beta Activated Kinase 1 in a Child with Polyvalvular Syndrome.

    Science.gov (United States)

    Ackerman, Jaeger P; Smestad, John A; Tester, David J; Qureshi, Muhammad Y; Crabb, Beau A; Mendelsohn, Nancy J; Ackerman, Michael J

    2016-09-01

    To use whole exome sequencing (WES) of a family trio to identify a genetic cause for polyvalvular syndrome. A male child was born with mild pulmonary valve stenosis and mild aortic root dilatation, and an atrial septal defect, ventricular septal defect, and patent ductus arteriosus that were closed surgically. Subsequently, the phenotype of polyvalvular syndrome with involvement of both semilunar and both atrioventricular valves emerged. His family history was negative for congenital heart disease. Because of hypotonia, myopia, soft pale skin, joint hypermobility, and mild facial dysmorphism, either Noonan syndrome- or William syndrome-spectrum disorders were suspected clinically. However, chromosomal analysis was normal and commercially available Noonan syndrome and William syndrome genetic tests were negative. Whole exome sequencing of the patient and both parents was performed. Variants were analyzed by sporadic and autosomal recessive inheritance models. A sporadic mutation, annotated as c.1491 T > A, in TAB2, resulting in a nonsense mutation, p.Y497X, in the TAB2-encoded TGF-beta activated kinase 1 (TAK1) was identified as the most likely disease-susceptibility gene. This mutation results in elimination of the terminal 197 amino acids, including the C-terminal binding motif critical for interactions with TRAF6 and TAK1. The combination of WES, genomic triangulation, and systems biology has uncovered perturbations in TGF-beta activated kinase 1 signaling as a novel pathogenic substrate for polyvalvular syndrome. © 2016 Wiley Periodicals, Inc.

  13. A new regulatory switch in a JAK protein kinase.

    Science.gov (United States)

    Tsui, Vickie; Gibbons, Paul; Ultsch, Mark; Mortara, Kyle; Chang, Christine; Blair, Wade; Pulk, Rebecca; Stanley, Mark; Starovasnik, Melissa; Williams, David; Lamers, Maria; Leonard, Phillip; Magnuson, Steven; Liang, Jun; Eigenbrot, Charles

    2011-02-01

    Members of the JAK family of protein kinases mediate signal transduction from cytokine receptors to transcription factor activation. Over-stimulation of these pathways is causative in immune disorders like rheumatoid arthritis, psoriasis, lupus, and Crohn's disease. A search for selective inhibitors of a JAK kinase has led to our characterization of a previously unknown kinase conformation arising from presentation of Tyr962 of TYK2 to an inhibitory small molecule via an H-bonding interaction. A small minority of protein kinase domains has a Tyrosine residue in this position within the αC-β4 loop, and it is the only amino acid commonly seen here with H-bonding potential. These discoveries will aid design of inhibitors that discriminate among the JAK family and more widely among protein kinases. © 2010 Wiley-Liss, Inc.

  14. The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice.

    Directory of Open Access Journals (Sweden)

    Laurent L Reber

    Full Text Available Gouty arthritis is caused by the deposition of monosodium urate (MSU crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid (PLGA nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

  15. Serum-dependent selective expression of EhTMKB1-9, a member of Entamoeba histolytica B1 family of transmembrane kinases.

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    Shiteshu Shrimal

    Full Text Available Entamoeba histolytica transmembrane kinases (EhTMKs can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (>or=95%. Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell

  16. Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

    Directory of Open Access Journals (Sweden)

    Bin Shan

    Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

  17. The P21-activated kinase PAK4 is implicated in fatty-acid potentiation of insulin secretion downstream of free fatty acid receptor 1.

    Science.gov (United States)

    Bergeron, Valérie; Ghislain, Julien; Poitout, Vincent

    2016-11-01

    Free fatty acid receptor 1 (FFA1/GPR40) plays a key role in the potentiation of glucose-stimulated insulin secretion by fatty acids in pancreatic β cells. We previously demonstrated that GPR40 signaling leads to cortical actin remodeling and potentiates the second phase of insulin secretion. In this study, we examined the role of p21 activated kinase 4 (PAK4), a known regulator of cytoskeletal dynamics, in GPR40-dependent potentiation of insulin secretion. The fatty acid oleate induced PAK4 phosphorylation in human islets, in isolated mouse islets and in the insulin secreting cell line INS832/13. However, oleate-induced PAK4 phosphorylation was not observed in GPR40-null mouse islets. siRNA-mediated knockdown of PAK4 in INS832/13 cells abrogated the potentiation of insulin secretion by oleate, whereas PAK7 knockdown had no effect. Our results indicate that PAK4 plays an important role in the potentiation of insulin secretion by fatty acids downstream of GPR40.

  18. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport.

    Science.gov (United States)

    Cleland, P J; Appleby, G J; Rattigan, S; Clark, M G

    1989-10-25

    Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.

  19. Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression.

    Science.gov (United States)

    Kwon, Hye-Sook; Huang, Boli; Ho Jeoung, Nam; Wu, Pengfei; Steussy, Calvin N; Harris, Robert A

    2006-01-01

    Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor alpha (RXRalpha) and retinoic acid receptor alpha (RARalpha) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRalpha/RARalpha/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRalpha/RARalpha/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

  20. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  1. Pyrazole carboxamides and carboxylic acids as protein kinase inhibitors in aberrant eukaryotic signal transduction

    DEFF Research Database (Denmark)

    Persson, Tobias; Yde, Christina W.; Rasmussen, Jakob Ewald

    2007-01-01

    Densely functionalised pyrazole carboxamides and carboxylic acids were synthesised in an expedient manner through saponification and transamidation, respectively, of ester-functionalised pyrazoles. This synthetic protocol allowed for three diversifying steps in which appendages on the pyrazole sc...

  2. New insights into the metabolism of aspartate-family amino acids in plant seeds.

    Science.gov (United States)

    Wang, Wenyi; Xu, Mengyun; Wang, Guoping; Galili, Gad

    2018-02-05

    Aspartate-family amino acids. Aspartate (Asp)-family pathway, via several metabolic branches, leads to four key essential amino acids: Lys, Met, Thr, and Ile. Among these, Lys and Met have received the most attention, as they are the most limiting amino acid in cereals and legumes crops, respectively. The metabolic pathways of these four essential amino acids and their interactions with regulatory networks have been well characterized. Using this knowledge, extensive efforts have been devoted to augmenting the levels of these amino acids in various plant organs, especially seeds, which serve as the main source of human food and livestock feed. Seeds store a number of storage proteins, which are utilized as nutrient and energy resources. Storage proteins are composed of amino acids, to guarantee the continuation of plant progeny. Thus, understanding the seed metabolism, especially with respect to the accumulation of aspartate-derived amino acids Lys and Met, is a crucial factor for sustainable agriculture. In this review, we summarized the Asp-family pathway, with some new examples of accumulated Asp-family amino acids, particularly Lys and Met, in plant seeds. We also discuss the recent advances in understanding the roles of Asp-family amino acids during seed development.

  3. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  4. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  5. Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain

    NARCIS (Netherlands)

    Julkowska, M.M.; McLoughlin, F.; Galvan-Ampudia, C.S.; Rankenberg, J.M.; Kawa, D.; Klimecka, M.; Haring, M.A.; Munnik, T.; Kooijman, E.E.; Testerink, C.

    2015-01-01

    Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate

  6. Bacteriocins of lactic acid bacteria : extending the family

    NARCIS (Netherlands)

    Alvarez-Sieiro, Patricia; Montalbán-López, Manuel; Mu, Dongdong; Kuipers, Oscar P

    2016-01-01

    Lactic acid bacteria (LAB) constitute a heterogeneous group of microorganisms that produce lactic acid as the major product during the fermentation process. LAB are Gram-positive bacteria with great biotechnological potential in the food industry. They can produce bacteriocins, which are

  7. Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase.

    Science.gov (United States)

    Yoshioka, Aya; Murata, Kousaku; Kawai, Shigeyuki

    2014-11-01

    Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong Hoon; Jeon, Byeong Tak; Jeong, Eun Ae [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of); Kim, Joon Soo; Cho, Yong Woon [Department of Neurosurgery, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, Gyeongnam 630-723 (Korea, Republic of); Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of); Roh, Gu Seob, E-mail: anaroh@gnu.ac.kr [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of)

    2010-03-12

    Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P{sub 1}) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P{sub 1} in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P{sub 1} proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P{sub 1} are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P{sub 1} signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.

  9. Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids*

    Science.gov (United States)

    Novellasdemunt, Laura; Tato, Irantzu; Navarro-Sabate, Aurea; Ruiz-Meana, Marisol; Méndez-Lucas, Andrés; Perales, Jose Carlos; Garcia-Dorado, David; Ventura, Francesc; Bartrons, Ramon; Rosa, Jose Luis

    2013-01-01

    Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked. PMID:23457334

  10. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    Science.gov (United States)

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  11. Isolation and characterization of a Paramecium cDNA clone encoding a putative serine/threonine protein kinase.

    Science.gov (United States)

    Wada, Satoru; Watanabe, Tsuyoshi

    2007-11-01

    Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan--Paramecium caudatum--using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.

  12. The Gibberellic Acid Stimulated-Like Gene Family in Maize and Its Role in Lateral Root Development

    National Research Council Canada - National Science Library

    Roman Zimmermann; Hajime Sakai; Frank Hochholdinger

    2010-01-01

    .... mays Gibberellic Acid Stimulated-Like), were identified. The ZmGSL family encodes small proteins of 75 to 128 amino acids, which are characterized by a conserved 59 to 64 amino acid C-terminal domain...

  13. A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance.

    Science.gov (United States)

    Makrantoni, Vasso; Dennison, Paul; Stark, Michael J R; Coote, Peter J

    2007-12-01

    In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

  14. Activation of phosphatidylinositol-3 kinase, AMP-activated kinase and Akt substrate-160 kDa by trans-10, cis-12 conjugated linoleic acid mediates skeletal muscle glucose uptake.

    Science.gov (United States)

    Mohankumar, Suresh K; Taylor, Carla G; Siemens, Linda; Zahradka, Peter

    2013-02-01

    Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

    DEFF Research Database (Denmark)

    Karlsson, Richard; Engström, Maria; Jönsson, Maria

    2003-01-01

    Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood....... Using two myeloid progenitor cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL-mediated survival is dependent on the activation of phosphatidylinositol-3 (PI-3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL...

  16. Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade.

    Science.gov (United States)

    Hill, C S; Oh, S Y; Schmidt, S A; Clark, K J; Murray, A W

    1994-01-01

    Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 microM LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases. Images Figure 1 Figure 3 Figure 6 PMID:7980407

  17. Regulation of serum-responsive transmembrane kinase EhTMKB1-9 by an unsaturated lipid, oleic acid in protistan parasite Entamoeba histolytica.

    Science.gov (United States)

    Saha, Arpita; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-11-01

    Transmembrane kinases of Entamoeba histolytica are known to play a wide range of roles from virulence, phagocytosis, and proliferation to stress response. Transmembrane kinase EhTMKB1-9 is thought to be involved in early proliferative response and it was originally identified as a serum inducible gene. Ability to stimulate EhTMKB1 expression of serum starved cells resides in unsaturated fatty acids associated with albumin fraction of serum and the mechanism of stimulation follows activation of EhTMKB1-9 promoter. Gel shift assay showed the presence of proteins that bind to the specific site of EhTMKB1-9 upstream region and the concentration of these protein(s) go down on serum starvation, but level of binding protein(s) go up on serum or fatty acid replenishment. This increase in concentration of binding molecule(s) is due to new synthesis rather than activation of existing molecule(s) as a protein synthesis inhibitor blocked enhanced level of gel shifted material on replenishment. The stimulating activity resides in the fatty acyl chain, but not in the head group. Moreover, the fatty acid initiates signaling through class I PI3 kinases that result in activation of EhTMKB1-9 expression. These results suggest a novel mechanism of gene regulation in E. histolytica, and unsaturated fatty acids as potential new signaling molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Constitutively active Arabidopsis MAP Kinase 3 triggers defense responses involving salicylic acid and SUMM2 resistance protein

    KAUST Repository

    Genot, Baptiste

    2017-04-12

    Mitogen-activated protein kinases (MAPKs) are important regulators of plant immunity. Most of the knowledge about the function of these pathways is derived from loss-of-function approaches. Using a gain-of-function approach, we investigated the responses controlled by a constitutively active (CA) MPK3 in Arabidopsis thaliana. CA-MPK3 plants are dwarfed and display a massive de-repression of defense genes associated with spontaneous cell death as well as accumulation of reactive oxygen species (ROS), phytoalexins and the stress-related hormones ethylene and salicylic acid (SA). Remarkably CA-MPK3/sid2 and CA-MPK3/ein2-50 lines which are impaired in SA synthesis and ethylene signaling, respectively, retain most of the CA-MPK3-associated phenotypes, indicating that constitutive activity of MPK3 can bypass SA and ethylene signaling to activate defense responses. A comparative analysis of the molecular phenotypes of CA-MPK3 and mpk4 autoimmunity suggested convergence between the MPK3 and MPK4-guarding modules. In support of this model, CA-MPK3 crosses with summ1 and summ2, two known suppressors of mpk4, resulted in a partial reversion of the CA-MPK3 phenotypes. Overall, our data unravel a novel mechanism by which the MAPK signaling network contributes to a robust defense response system.

  19. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Ju, Tsai-Kai [Instrumentation Center, National Taiwan University, Taipei, Taiwan, ROC (China); Technology Commons, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Huang, Yuan-Li [Department of Biotechnology, Asia University, Taichung, Taiwan, ROC (China); Lee, Ming-Shyue [Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC (China); Chen, Jiun-Hong [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Lee, Hsinyu, E-mail: hsinyu@ntu.edu.tw [Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan, ROC (China); Center for Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan, ROC (China)

    2013-11-01

    Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

  20. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  1. Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1*

    Science.gov (United States)

    Vina-Vilaseca, Arnau; Bender-Sigel, Julia; Sorkina, Tatiana; Closs, Ellen Ildicho; Sorkin, Alexander

    2011-01-01

    Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC. PMID:21212261

  2. Role of mitogen-activated protein kinases in tauroursodeoxycholic acid-induced bile formation in cholestatic rat liver.

    Science.gov (United States)

    Denk, Gerald Ulrich; Hohenester, Simon; Wimmer, Ralf; Böhland, Claudia; Rust, Christian; Beuers, Ulrich

    2008-07-01

    Ursodeoxycholic acid exerts anticholestatic effects in various cholestatic disorders and experimental models of cholestasis. Its taurine conjugate (TUDCA) stimulates bile salt secretion in isolated perfused rat livers (IPRL) under physiological, non-cholestatic conditions, in part by mitogen-activated protein kinase (MAPK)-dependent mechanisms. The role of MAPK in the anticholestatic effect of TUDCA, however, is unclear. Therefore, we studied the role of MAPK in the anticholestatic effect of TUDCA in IPRL and isolated rat hepatocytes (IRH) in taurolithocholic acid (TLCA)-induced cholestasis. Bile flow, biliary levels of 2,4-dinitrophenyl-S-glutathione (GS-DNP) as a marker of hepatobiliary organic anion secretion and activity of lactate dehydrogenase (LDH) in hepatovenous effluate as a marker of hepatocellular damage in IPRL perfused with TUDCA and/or TLCA were determined in the presence or absence of MAPK inhibitors. In addition, phosphorylation of Erk 1/2 and p38(MAPK) induced by TUDCA and/or TLCA was studied by Western immunoblot in IPRL and IRH. TUDCA-induced bile flow was impaired by the Erk 1/2 inhibitor PD98059 in normal livers (-28%), but not in livers made cholestatic by TLCA. GS-DNP secretion was unaffected by PD98059 under both conditions. TUDCA-induced bile formation and organic anion secretion both in the presence and absence of TLCA were unaffected by the p38(MAPK) inhibitor SB202190. Erk 1/2 phosphorylation in liver tissue was unchanged after bile salt exposure for 70 min, but was transiently enhanced by TUDCA in IRH. MAPK do not mediate the anticholestatic effects of TUDCA in TLCA-induced cholestasis.

  3. Bacteriocins of lactic acid bacteria: extending the family.

    Science.gov (United States)

    Alvarez-Sieiro, Patricia; Montalbán-López, Manuel; Mu, Dongdong; Kuipers, Oscar P

    2016-04-01

    Lactic acid bacteria (LAB) constitute a heterogeneous group of microorganisms that produce lactic acid as the major product during the fermentation process. LAB are Gram-positive bacteria with great biotechnological potential in the food industry. They can produce bacteriocins, which are proteinaceous antimicrobial molecules with a diverse genetic origin, posttranslationally modified or not, that can help the producer organism to outcompete other bacterial species. In this review, we focus on the various types of bacteriocins that can be found in LAB and the organization and regulation of the gene clusters responsible for their production and biosynthesis, and consider the food applications of the prototype bacteriocins from LAB. Furthermore, we propose a revised classification of bacteriocins that can accommodate the increasing number of classes reported over the last years.

  4. Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families.

    Science.gov (United States)

    Melis, M A; Cau, M; Muntoni, F; Mateddu, A; Galanello, R; Boccone, L; Deidda, F; Loi, D; Cao, A

    1998-01-01

    This study reports three children from three unrelated families, aged from 9 to 12 years, who were investigated because of the incidental finding of elevated serum creatine kinase (CK) levels and were found to have a dystrophinopathy. The molecular defect consisted of a deletion of variable extent within the central rod domain of the dystrophin gene, involving either exons 32-44 or 48-51 or 48-53. In each family we found the same deletion in at least one adult male relative aged from 40 to 77 years, who was either completely asymptomatic or had very mild muscle involvement (thin muscles and/or mild scoliosis), with normal or borderline CK levels. This study suggests once again that deletions of the central rod domain of dystrophin may be associated with elevation of serum CK as the only manifestation and that prediction of the clinical severity based solely on the molecular findings should be interpreted with caution.

  5. Using amino acid correlation and community detection algorithms to identify functional determinants in protein families.

    Directory of Open Access Journals (Sweden)

    Lucas Bleicher

    Full Text Available Correlated mutation analysis has a long history of interesting applications, mostly in the detection of contact pairs in protein structures. Based on previous observations that, if properly assessed, amino acid correlation data can also provide insights about functional sub-classes in a protein family, we provide a complete framework devoted to this purpose. An amino acid specific correlation measure is proposed, which can be used to build networks summarizing all correlation and anti-correlation patterns in a protein family. These networks can be submitted to community structure detection algorithms, resulting in subsets of correlated amino acids which can be further assessed by specific parameters and procedures that provide insight into the relationship between different communities, the individual importance of community members and the adherence of a given amino acid sequence to a given community. By applying this framework to three protein families with contrasting characteristics (the Fe/Mn-superoxide dismutases, the peroxidase-catalase family and the C-type lysozyme/α-lactalbumin family, we show how our method and the proposed parameters and procedures are related to biological characteristics observed in these protein families, highlighting their potential use in protein characterization and gene annotation.

  6. Plant Cyclin-Dependent Kinase Inhibitors of the KRP Family: Potent Inhibitors of Root-Knot Nematode Feeding Sites in Plant Roots

    Directory of Open Access Journals (Sweden)

    Paulo Vieira

    2017-09-01

    Full Text Available Root-knot nematodes (RKN, Meloidogyne spp., are distributed worldwide and impose severe economic damage to many agronomically important crops. The plant cell cycle machinery is considered one of the pivotal components for the formation of nematode feeding sites (NFSs or galls. These feeding sites contain five to nine hypertrophied giant cells (GC resulting from developmental reprogramming of host root cells by this pathogen. GC undergo synchronous waves of mitotic activity uncoupled from cytokinesis giving rise to large multinucleate cells. As development of the NFS progresses, multiple rounds of DNA synthesis occur in the nuclei of GC, coupled with nuclear and cellular expansion. These cells are highly metabolically active and provide the nematode with nutrients necessary for its development and completion of its life cycle. In Arabidopsis seven cyclin dependent kinase inhibitors (CKIs belonging to the interactors/inhibitors of the cyclin dependent kinases (ICK family, also referred as Kip-Related Proteins (KRPs have been identified. Interactions of KRPs with CDK/Cyclin complexes decrease CDK activity, affecting both cell cycle progression and DNA content in a concentration-dependent manner. We performed the functional analysis of all Arabidopsis KRP gene members during RKN interaction in Arabidopsis to obtain more insight into their role during gall development. We demonstrated that three members of this family (KRP2, KRP5, and KRP6 were highly expressed in galls and were important for cell cycle regulation during NFS development as shown by their different modes of action. We also pointed out that cell cycle inhibition through overexpression of all members of the KRP family can affect NFS development and consequently compromise the nematode’s life cycle. In this review we summarized our recent understanding of the KRP family of genes, and their role in controlling cell cycle progression at the RKN feeding site.

  7. The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation.

    Science.gov (United States)

    Palazzo, A; Bluteau, O; Messaoudi, K; Marangoni, F; Chang, Y; Souquere, S; Pierron, G; Lapierre, V; Zheng, Y; Vainchenker, W; Raslova, H; Debili, N

    2016-12-01

    Essentials The role of the cytoskeleton during megakaryocyte differentiation was examined. Human megakaryocytes are derived from in vitro cultured CD34(+) cells. Cell division control protein 42 (CDC42) positively regulates proplatelet formation (PPF). Neural Wiskott-Aldrich syndrome protein, the main effector of CDC42 with Src positively regulates PPF. Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34(+) cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP. © 2016 International Society on Thrombosis and

  8. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016 (China); Wang, Jianwei, E-mail: wangjianwei1968@gmail.com [Department of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016 (China); Gu, Tieguang [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences, Sydney, NSW 2000 Australia (Australia); Yamahara, Johji [Pharmafood Institute, Kyoto 602-8136 (Japan); Li, Yuhao, E-mail: yuhao@sitcm.edu.au [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences, Sydney, NSW 2000 Australia (Australia)

    2014-06-01

    Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in

  9. ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

    Science.gov (United States)

    Weckwerth, Philipp; Ehlert, Britta; Romeis, Tina

    2015-03-01

    Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize. © 2014 John Wiley & Sons Ltd.

  10. Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH/3T3 cells.

    Science.gov (United States)

    Denys, A; Hichami, A; Maume, B; Khan, N A

    2001-08-01

    Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFalpha) in NIH/3T3 cells. We observed that both PMA and TGFalpha induced ERK1/ERK2 phosphorylation within 5 min of stimulation. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFalpha did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA- but not TGFalpha-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKC alpha and PKC epsilon, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKC delta isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKC alpha and epsilon isoforms.

  11. Diverse roles for the ror-family receptor tyrosine kinases in neurons and glial cells during development and repair of the nervous system.

    Science.gov (United States)

    Endo, Mitsuharu; Minami, Yasuhiro

    2017-05-03

    The Ror-family of receptor tyrosine kinases (RTKs) are involved critically in tissue genesis and organogenesis during development. In mammals, Ror1 and Ror2, members of the Ror-family RTKs, have been shown to mediate cell polarity, migration, proliferation, and differentiation through the activation of noncanonical Wnt signaling by acting as receptors or co-receptors for Wnt5a. Nematodes bearing mutations within the cam-1 gene, encoding a Ror2 ortholog, exhibit defects in various developmental processes of the nervous system, including neuronal cell migration, polarization, axonal extension, and synaptic transmission. In mice, Ror2 and/or Ror1 are also shown to play roles in regulating neurite extension, synapse formation, and synaptic transmission of hippocampal neurons, indicating that the Ror-family RTKs have evolutionarily conserved functions at least in part in neurons during development. Furthermore, Ror2 and/or Ror1 are expressed in neural stem/progenitor cells of the developing brain and in astrocytes of the adult brain after injury, and they play important roles in regulating cell proliferation under these different contexts. In this article, we overview recent advances in our understanding of the roles of the Ror-family RTKs in the development and repair of the nervous system and discuss their potential for therapeutic targets to neurodegenerative diseases. Developmental Dynamics, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Sphingosine Kinase-1 Involves the Inhibitory Action of HIF-1α by Chlorogenic Acid in Hypoxic DU145 Cells

    Directory of Open Access Journals (Sweden)

    Myoung-Sun Lee

    2017-02-01

    Full Text Available Hypoxia enhances cancer development in a solid tumor. Hypoxia-inducible factor-1 α (HIF-1α is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor that regulates tumor. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, anti-apoptosis and cell proliferation as well as imparts resistance to cancer treatment. In this study, we assessed Crataegus Pinnatifida Bunge var. typical Schneider ethanol extract (CPE for its anti-cancer effects in hypoxia-induced DU145 human prostate cancer cell line. CPE decreased the abundance of HIF-1α and sphingosine kinase-1 (SPHK-1 in hypoxia-induced prostate cancer DU145 cells. CPE decreased HIF-1α and SPHK-1 as well as SPHK-1 activity. Chlorogenic acid (CA is one of four major compounds of CPE. Compared to CPE, CA significantly decreased the expression of HIF-1α and SPHK-1 as well as SPHK-1 activity in hypoxia-induced DU145 cells. Furthermore, CA decreased phosphorylation AKT and GSK-3β, which are associated with HIF-1α stabilization and affected SPHK-1 in a concentration-dependent manner. We confirmed the mechanism of CA-induced inhibition of HIF-1α by SPHK-1 signaling pathway using SPHK-1 siRNA and SPHK inhibitor (SKI. CA decreased the secretion and cellular expression of VEGF, thus inhibiting hypoxia-induced angiogenesis. Treatment of DU145cells with SPHK1 siRNA and CA for 48 h decreased cancer cell growth, and the inhibitory action of SPHK siRNA and CA on cell growth was confirmed by decrease in the abundance of Proliferating cell nuclear antigen (PCNA.

  13. Amino Acid Sensor Kinase Gcn2 Is Required for Conidiation, Secondary Metabolism, and Cell Wall Integrity in the Taxol-Producer Pestalotiopsis microspora

    Directory of Open Access Journals (Sweden)

    Dan Wang

    2017-09-01

    Full Text Available The canonical Gcn2/Cpc1 kinase in fungi coordinates the expression of target genes in response to amino acid starvation. To investigate its possible role in secondary metabolism, we characterized a gcn2 homolog in the taxol-producing fungus Pestalotiopsis microspora. Deletion of the gene led to severe physiological defects under amino acid starvation, suggesting a conserved function of gcn2 in amino acid sensing. The mutant strain Δgcn2 displayed retardation in vegetative growth. It generated dramatically fewer conidia, suggesting a connection between amino acid metabolism and conidiation in this fungus. Importantly, disruption of the gene altered the production of secondary metabolites by HPLC profiling. For instance, under amino acid starvation, the deletion strain Δgcn2 barely produced secondary metabolites including the known natural product pestalotiollide B. Even more, we showed that gcn2 played critical roles in the tolerance to several stress conditions. Δgcn2 exhibited a hypersensitivity to Calcofluor white and Congo red, implying a role of Gcn2 in maintaining the integrity of the cell wall. This study suggests that Gcn2 kinase is an important global regulator in the growth and development of filamentous fungi and will provide knowledge for the manipulation of secondary metabolism in P. microspora.

  14. A new family of extraterrestrial amino acids in the Murchison meteorite.

    Science.gov (United States)

    Koga, Toshiki; Naraoka, Hiroshi

    2017-04-04

    The occurrence of extraterrestrial organic compounds is a key for understanding prebiotic organic synthesis in the universe. In particular, amino acids have been studied in carbonaceous meteorites for almost 50 years. Here we report ten new amino acids identified in the Murchison meteorite, including a new family of nine hydroxy amino acids. The discovery of mostly C 3 and C 4 structural isomers of hydroxy amino acids provides insight into the mechanisms of extraterrestrial synthesis of organic compounds. A complementary experiment suggests that these compounds could be produced from aldehydes and ammonia on the meteorite parent body. This study indicates that the meteoritic amino acids could be synthesized by mechanisms in addition to the Strecker reaction, which has been proposed to be the main synthetic pathway to produce amino acids.

  15. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    Science.gov (United States)

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  16. Analysis of the Ileal Bile Acid Transporter Gene, SLC10A2, in Subjects With Familial Hypertriglyceridemia

    National Research Council Canada - National Science Library

    Love, Martha W; Craddock, Ann L; Angelin, Bo; Brunzell, John D; Duane, William C; Dawson, Paul A

    2001-01-01

    Familial hypertriglyceridemia (FHTG), a disease characterized by elevated plasma very low density lipoprotein triglyceride levels, has been associated with impaired intestinal absorption of bile acids...

  17. Agp2, a member of the yeast amino acid permease family, positively regulates polyamine transport at the transcriptional level.

    Directory of Open Access Journals (Sweden)

    Mustapha Aouida

    Full Text Available Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.

  18. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha

    2013-06-03

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

  19. Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase*

    OpenAIRE

    Chen, Qingfeng; Westfall, Corey S.; Hicks, Leslie M.; Wang, Shiping; Jez, Joseph M.

    2010-01-01

    The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemi...

  20. Dihydrolipoic Acid Inhibits Lysosomal Rupture and NLRP3 Through Lysosome-Associated Membrane Protein-1/Calcium/Calmodulin-Dependent Protein Kinase II/TAK1 Pathways After Subarachnoid Hemorrhage in Rat.

    Science.gov (United States)

    Zhou, Keren; Enkhjargal, Budbazar; Xie, Zhiyi; Sun, Chengmei; Wu, Lingyun; Malaguit, Jay; Chen, Sheng; Tang, Jiping; Zhang, Jianmin; Zhang, John H

    2018-01-01

    The NLRP3 (nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome is a crucial component of the inflammatory response in early brain injury after subarachnoid hemorrhage (SAH). In this study, we investigated a role of dihydrolipoic acid (DHLA) in lysosomal rupture, NLRP3 activation, and determined the underlying pathway. SAH was induced by endovascular perforation in male Sprague-Dawley rats. DHLA was administered intraperitoneally 1 hour after SAH. Small interfering RNA for lysosome-associated membrane protein-1 and CaMKIIα (calcium/calmodulin-dependent protein kinase II α) was administered through intracerebroventricular 48 hours before SAH induction. SAH grade evaluation, short- and long-term neurological function testing, Western blot, and immunofluorescence staining experiments were performed. DHLA treatment increased the expression of lysosome-associated membrane protein-1 and decreased phosphorylated CaMKIIα and NLRP3 inflammasome, thereby alleviating neurological deficits after SAH. Lysosome-associated membrane protein-1 small interfering RNA abolished the neuroprotective effects of DHLA and increased the level of phosphorylated CaMKIIα, p-TAK1 (phosphorylated transforming growth factor-β-activated kinase), p-JNK (phosphorylated c-Jun-N-terminal kinase), and NLRP3 inflammasome. CaMKIIα small interfering RNA downregulated the expression of p-TAK1, p-JNK, and NLRP3 and improved the neurobehavior after SAH. DHLA treatment improved neurofunction and alleviated inflammation through the lysosome-associated membrane protein-1/CaMKII/TAK1 pathway in early brain injury after SAH. DHLA may provide a promising treatment to alleviate early brain injury after SAH. © 2017 American Heart Association, Inc.

  1. Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family.

    Science.gov (United States)

    Zubieta, Chloe; Ross, Jeannine R; Koscheski, Paul; Yang, Yue; Pichersky, Eran; Noel, Joseph P

    2003-08-01

    Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.

  2. Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Collins, Morgan E; Black, Joshua J; Liu, Zhengchang

    2017-07-01

    Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation.IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. Copyright © 2017 American Society

  3. Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells.

    Science.gov (United States)

    Eguchi, Ryoji; Fujita, Yumiko; Tabata, Chiharu; Ogawa, Hiroyasu; Wakabayashi, Ichiro; Nakano, Takashi; Fujimori, Yoshihiro

    2015-11-01

    Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.

  4. The pan-HER family tyrosine kinase inhibitor afatinib overcomes HER3 ligand heregulin-mediated resistance to EGFR inhibitors in non-small cell lung cancer

    Science.gov (United States)

    Yonesaka, Kimio; Kudo, Keita; Nishida, Satomi; Takahama, Takayuki; Iwasa, Tsutomu; Yoshida, Takeshi; Tanaka, Kaoru; Takeda, Masayuki; Kaneda, Hiroyasu; Okamoto, Isamu; Nishio, Kazuto; Nakagawa, Kazuhiko

    2015-01-01

    Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance. PMID:26418897

  5. Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12-oxo-phytodienoic acid, mitogen-activated protein kinase 4 and cytokinin perception.

    Science.gov (United States)

    Meza-Canales, Ivan D; Meldau, Stefan; Zavala, Jorge A; Baldwin, Ian T

    2017-07-01

    Herbivory-induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore-specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (A C ) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1-5 ribulose-1,5-bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion-elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12-oxo-phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen-activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen-activated protein kinase 4 in CO 2 -mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid-mediated defence responses, also elicit 12-oxo-phytodienoic acid-mediated changes in stomatal conductance and A C , an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack. © 2016 John Wiley & Sons Ltd.

  6. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    Science.gov (United States)

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  7. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Bräuner-Osborne, Hans

    2009-01-01

    Family C of human G-protein-coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two gamma-aminobutyric acid type B (GABA(B1-2)) subunits forming the heterodimeric GABA(B) receptor, the calcium-sensing receptor, three taste1 receptors (T1R1-3), a promiscuous L...

  8. Autosomal dominant polycystic liver disease in a family without polycystic kidney disease associated with a novel missense protein kinase C substrate 80K-H mutation.

    Science.gov (United States)

    Peces, Ramón; Drenth, Joost P H; Te Morsche, Rene H M; González, Pedro; Peces, Carlos

    2005-12-28

    Polycystic liver disease (PLD) is characterized by the presence of multiple bile duct-derived epithelial cysts scattered in the liver parenchyma. PLD can manifest itself in patients with severe autosomal dominant polycystic kidney disease (ADPKD). Isolated autosomal dominant polycystic liver disease (ADPLD) is genetically distinct from PLD associated with ADPKD, although it may have similar pathogenesis and clinical manifestations. Recently, mutations in two causative genes for ADPLD, independently from ADPKD, have been identified. We report here a family (a mother and her daughter) with a severe form of ADPLD not associated with ADPKD produced by a novel missense protein kinase C substrate 80K-H (PRKCSH) mutation (R281W). This mutation causes a severe phenotype, since the two affected subjects manifested signs of portal hypertension. Doppler sonography, computed tomography (CT) and magnetic resonance (MR) imaging are effective in documenting the underlying lesions in a non-invasive way.

  9. Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae

    Science.gov (United States)

    Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

    2015-01-01

    The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. PMID:25628332

  10. Docosahexaenoic acid downregulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the c-Jun NH2-terminal kinase mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Lu, Chia-Yang; Li, Chien-Chun; Liu, Kai-Li; Lii, Chong-Kuei; Chen, Haw-Wen

    2009-03-01

    Mitogen-activated protein kinase (MAPK) pathways play central roles in the transduction of extracellular stimuli into cells and the regulation of expression of numerous genes. Docosahexaenoic acid (DHA) was shown to be involved in the regulation of expression of drug metabolizing enzymes (DMEs) in rat primary hepatocytes in response to xenobiotics. Cytochrome P450 2B1 (CYP 2B1) is a DME that is dramatically induced by phenobarbital-type inducers. The constitutive androstane receptor (CAR) plays a critical role in regulating the expression of DMEs, and the phosphorylation/dephosphorylation of CAR is an important event in CYP 2B1 expression. In the present study, we determined the effect of DHA on MAPK transactivation and its role in CYP 2B1 expression induced by phenobarbital. c-Jun NH2-terminal kinase (JNK) JNK1/2 and ERK1/2 were activated by phenobarbital in a dose-dependent manner. DHA (100 muM) inhibited JNK1/2 and ERK2 activation induced by phenobarbital in a time-dependent manner. Both SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited CYP 2B1 protein and mRNA expression induced by phenobarbital. SB203580 significantly increased the intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration compared with a control group (p expression induced by phenobarbital.

  11. Role of exercise-induced calmodulin protein kinase (CAMK)II activation in the regulation of omega-6 fatty acids and lipid metabolism genes in rat skeletal muscle.

    Science.gov (United States)

    Joseph, J S; Ayeleso, A O; Mukwevho, E

    2017-09-22

    Activation of calmodulin dependent protein kinase (CaMK)II by exercise is beneficial in controlling membrane lipids associated with type 2 diabetes and obesity. Regulation of lipid metabolism is crucial in the improvement of type 2 diabetes and obesity associated symptoms. The role of CaMKII in membrane associated lipid metabolism was the focus of this study. Five to six weeks old male Wistar rats were used in this study. GC×GC-TOFMS technique was used to determine the levels of polyunsaturated fatty acids (linoleic acid, arachidonic acid and 11,14-eicosadienoic acid). Carnitine palmitoyltransferase (Cpt-1) and acetyl-CoA carboxylase (Acc-1) genes expression were assessed using quantitative real time PCR (qPCR). From the results, CaMKII activation by exercise increased the levels of arachidonic acid and 11, 14-eicosadienoic acid while a decrease in the level of linolenic acid was observed in the skeletal muscle. The results indicated that exercise-induced CaMKII activation increased CPT-1 expression and decreased ACC-1 expression in rat skeletal muscle. All the observed increases with activation of CaMKII by exercise were aborted when KN93, an inhibitor of CaMKII was injected in exercising rats. This study demonstrated that CaMKII activation by exercise regulated lipid metabolism. This study suggests that CaMKII can be a vital target of therapeutic approach in the management of diseases such as type 2 diabetes and obesity that have increased to epidemic proportions recently.

  12. Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.

    Science.gov (United States)

    Fukuda, Yasuhisa; Abe, Asuka; Tamura, Takashi; Kishimoto, Takahide; Sogabe, Atsushi; Akanuma, Satoshi; Yokobori, Shin-Ichi; Yamagishi, Akihiko; Imada, Katsumi; Inagaki, Kenji

    2016-05-01

    Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

    Directory of Open Access Journals (Sweden)

    Zhen Cai

    2017-04-01

    Full Text Available As well as their importance to nutrition, fatty acids (FA represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK, RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp and catalytic ATP-binding (CA domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

  14. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

    Science.gov (United States)

    Cai, Zhen; Yuan, Zhi-Hui; Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li; Qian, Wei

    2017-04-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

  15. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence

    Science.gov (United States)

    Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li

    2017-01-01

    As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication. PMID:28369120

  16. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxybenzoic Acid

    Directory of Open Access Journals (Sweden)

    Kuan-Han Lee

    2014-01-01

    Full Text Available Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxybenzoic acid (TMPBA and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 µM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4',6-diamidino-2-phenylindole (DAPI nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP kinases, 5' adenosine monophosphate-activated protein kinase (AMPK, and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

  17. Resveratrol prevents impairment in activation of retinoic acid receptors and MAP kinases in the embryos of a rodent model of diabetic embryopathy.

    Science.gov (United States)

    Singh, Chandra K; Kumar, Ambrish; LaVoie, Holly A; DiPette, Donald J; Singh, Ugra S

    2012-09-01

    Diabetes induces impairments in gene expression during embryonic development that leads to premature and improper tissue specialization. Retinoic acid receptors (RARs and retinoid X receptor [RXRs]) and mitogen-activated protein kinases (MAPKs) play crucial roles during embryonic development, and their suppression or activation has been shown as a determinant of the fate of embryonic organogenesis. We studied the activation of RARs and MAPKs in embryonic day 12 (E12) in embryos of rats under normal, diabetic, and diabetic treated with resveratrol ([RSV]; 100 mg/kg body weight) conditions. We found downregulation of RARs and RXRs expressions as well as their DNA-binding activities in the embryos exhibiting developmental delays due to diabetes. Furthermore, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was decreased and phosphorylation of c-Jun N-terminal kinase (JNK) 1/2 and p38 was increased. Interestingly, embryos of diabetic rats treated with RSV showed normalized patterns of RARs, RXRs, neuronal markers, and ERK, JNK and p38 phosphorylation.

  18. Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria.

    Science.gov (United States)

    Xu, Yunjian; Johansson, Magnus; Karlsson, Anna

    2008-01-18

    Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis.

  19. Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs.

    Directory of Open Access Journals (Sweden)

    Tapan Kumar Mohanta

    Full Text Available The mitogen-activated protein kinase (MAPK is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest.

  20. An Improved Mouse Model of Atopic Dermatitis and Suppression of Skin Lesions by an Inhibitor of Tec Family Kinases

    Directory of Open Access Journals (Sweden)

    Yuko Kawakami

    2007-01-01

    Conclusions: We established a highly efficient, highly reproducible protocol to induce skin lesions in NC/Nga mice and successfully applied it to show the efficacy of terreic acid in treating skin lesions. This mouse model of atopic dermatitis will be useful to study the pathogenetic processes of atopic dermatitis and to evaluate the efficacy of drug candidates.

  1. Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

    Science.gov (United States)

    Michaelidis; Pallidou; Vakouftsi

    1999-06-01

    The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

  2. Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression.

    Science.gov (United States)

    Sinnberg, Tobias; Wang, Jun; Sauer, Birgit; Schittek, Birgit

    2016-08-03

    We previously identified CK1α as a novel tumor suppressor in melanoma and reported that the loss of CK1α leads to increased proliferation and invasive growth of melanoma cells by strong activation of the Wnt/β-catenin signaling pathway. In this study we analyzed expression and the functional effects of the dominantly expressed CK1- isoforms α, δ and ε in melanoma cells by quantitative real-time PCR, western blot and immunohistochemistry. We down-regulated CK1 kinase activity with isoform specific siRNAs and small molecule inhibitors. Vice versa we overexpressed the CK1 isoforms α, δ and ε using viral vectors and tested the biological effects on melanoma cell proliferation, migration and invasion. We show that protein expression of all three CK1-isoforms is downregulated in metastatic melanoma cells compared to benign melanocytic cells. Furthermore, the CK1δ and ε isoforms are able to negatively regulate expression of each other, whereas CK1α expression is independently regulated in melanoma cells. Inhibition of the expression and activity of CK1δ or CK1ε by specific inhibitors or siRNAs had no significant effect on the growth and survival of metastatic melanoma cells. Moreover, the over-expression of CK1δ or CK1ε in melanoma cells failed to induce cell death and cell cycle arrest although p53 signaling was activated. This is in contrast to the effects of CK1α where up-regulated expression induces cell death and apoptosis in metastatic melanoma cells. These data indicate that CK1α has a dominant and non-redundant function in melanoma cells and that the CK1δ and ε isoforms are not substantially involved in melanoma progression.

  3. Activation of ERK signalling by Src family kinases (SFKs) in DRG neurons contributes to hydrogen peroxide (H2O2)-induced thermal hyperalgesia.

    Science.gov (United States)

    Singh, Ajeet Kumar; Vinayak, Manjula

    2017-10-01

    Concomitant generation of reactive oxygen species during tissue inflammation has been recognised as a major factor for the development and the maintenance of hyperalgesia, out of which H2O2 is the major player. However, molecular mechanism of H2O2 induced hyperalgesia is still obscure. The aim of present study is to analyse the mechanism of H2O2-induced hyperalgesia in rats. Intraplantar injection of H2O2 (5, 10 and 20 µmoles/paw) induced a significant thermal hyperalgesia in the hind paw, confirmed by increased c-Fos activity in dorsal horn of spinal cord. Onset of hyperalgesia was prior to development of oxidative stress and inflammation. Rapid increase in phosphorylation of extracellular signal regulated kinase (ERK) was observed in neurons of dorsal root ganglia after 20 min of H2O2 (10 µmoles/paw) administration, which gradually returned towards normal level within 24 h, following the pattern of thermal hyperalgesia. The expression of TNFR1 followed the same pattern and colocalised with pERK. ERK phosphorylation was observed in NF-200-positive and -negative neurons, indicating the involvement of ERK in C-fibres as well as in A-fibres. Intrathecal preadministration of Src family kinases (SFKs) inhibitor (PP1) and MEK inhibitor (PD98059) prevented H2O2 induced augmentation of ERK phosphorylation and thermal hyperalgesia. Pretreatment of protein tyrosine phosphatases (PTPs) inhibitor (sodium orthovanadate) also diminished hyperalgesia, although it further increased ERK phosphorylation. Combination of orthovanadate with PP1 or PD98059 did not exhibit synergistic antihyperalgesic effect. The results demonstrate SFKs-mediated ERK activation and increased TNFR1 expression in nociceptive neurons during H2O2 induced hyperalgesia. However, the role of PTPs in hyperalgesic behaviour needs further molecular analysis.

  4. Advanced glycation end products induce brain-derived neurotrophic factor release from human platelets through the Src-family kinase activation.

    Science.gov (United States)

    Furukawa, Kazuo; Fuse, Ichiro; Iwakura, Yuriko; Sotoyama, Hidekazu; Hanyu, Osamu; Nawa, Hiroyuki; Sone, Hirohito; Takei, Nobuyuki

    2017-02-08

    Brain-derived neurotrophic factor (BDNF) exerts beneficial effects not only on diabetic neuropathies but also on cardiovascular injury. There is argument regarding the levels of serum BDNF in patients with diabetes mellitus (DM). Because BDNF in peripheral blood is rich in platelets, this may represent dysregulation of BDNF release from platelets. Here we focused on advanced glycation end products (AGEs), which are elevated in patients with DM and have adverse effects on cardiovascular functions. The aim of this study is to elucidate the role of AGEs in the regulation of BDNF release from human platelets. Platelets collected from peripheral blood of healthy volunteers were incubated with various concentrations of AGE (glycated-BSA) at 37 °C for 5 min with or without BAPTA-AM, a cell permeable Ca(2+) chelator, or PP2, a potent inhibitor of Src family kinases (SFKs). Released and cellular BDNF were measured by ELISA and calculated. Phosphorylation of Src and Syk, a downstream kinase of SFKs, in stimulated platelets was examined by Western blotting and immunoprecipitation. AGE induced BDNF release from human platelets in a dose-dependent manner, which was dependent on intracellular Ca(2+) and SFKs. We found that AGE induced phosphorylation of Src and Syk. AGE induces BDNF release from human platelets through the activation of the Src-Syk-(possibly phospholipase C)-Ca(2+) pathway. Considering the toxic action of AGEs and the protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes. Chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM.

  5. Development of an Antivirulence Drug against Streptococcus mutans: Repression of Biofilm Formation, Acid Tolerance, and Competence by a Histidine Kinase Inhibitor, Walkmycin C ▿

    Science.gov (United States)

    Eguchi, Yoko; Kubo, Norihiro; Matsunaga, Hiroko; Igarashi, Masayuki; Utsumi, Ryutaro

    2011-01-01

    Two-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential of Streptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors of S. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK in Bacillus subtilis, inhibited the in vitro autophosphorylation activity of three purified S. mutans HKs, i.e., VicK, CiaH, and LiaS. Although S. mutans does not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 μg/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes of S. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs. PMID:21282451

  6. HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

    Directory of Open Access Journals (Sweden)

    Kenji Nakashima

    Full Text Available Hepatitis C virus (HCV infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV implied that NS5A was tyrosine phosphorylated by pervanadate (PV treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST-fusion proteins of various Src homology 2 (SH2 domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3 domain. Substitution of Arg(176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

  7. Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A.

    Science.gov (United States)

    Yamagishi, S I; Edelstein, D; Du, X L; Kaneda, Y; Guzmán, M; Brownlee, M

    2001-07-06

    Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. The plasma concentrations are correlated with body mass index, and are reported to be high in patients with insulin resistance, which is one of the major risk factors for cardiovascular disease. However, the direct effect of leptin on vascular wall cells is not fully understood. In this study, we investigated the effects of leptin on reactive oxygen species (ROS) generation and expression of monocyte chemoattractant protein-1 (MCP-1) in bovine aortic endothelial cells (BAEC). We found that leptin increases ROS generation in BAEC in a dose-dependent manner and that its effects are additive with those of glucose. Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), uncoupling protein-1 (UCP1) HVJ-liposomes, or manganese superoxide dismutase (MnSOD) HVJ-liposomes completely prevented the effect of leptin, suggesting that ROS arise from mitochondrial electron transport. Leptin increased fatty acid oxidation by stimulating the activity of carnitine palmitoyltransferase-1 (CPT-1) and inhibiting that of acetyl-CoA carboxylase (ACC), pace-setting enzymes for fatty acid oxidation and synthesis, respectively. Leptin-induced ROS generation, CPT-1 activation, ACC inhibition, and MCP-1 overproduction were found to be completely prevented by either genistein, a tyrosine kinase inhibitor, H-89, a protein kinase A (PKA) inhibitor, or tetradecylglycidate, a CPT-1 inhibitor. Leptin activated PKA, and the effects of leptin were inhibited by the cAMP antagonist Rp-cAMPS. These results suggest that leptin induces ROS generation by increasing fatty acid oxidation via PKA activation, which may play an important role in the progression of atherosclerosis in insulin-resistant obese diabetic patients.

  8. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    Science.gov (United States)

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

    Science.gov (United States)

    Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

    2015-12-25

    Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors

    Directory of Open Access Journals (Sweden)

    Rüegg Markus A

    2007-07-01

    Full Text Available Abstract Background Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ, agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs. Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs and protein tyrosine kinases (PTKs such as those of the Src-family may be essential in stabilizing clusters of AChRs. Results We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein. Conclusion Our data

  11. Protein sequence alignment with family-specific amino acid similarity matrices

    Science.gov (United States)

    2011-01-01

    Background Alignment of amino acid sequences by means of dynamic programming is a cornerstone sequence comparison method. The quality of alignments produced by dynamic programming critically depends on the choice of the alignment scoring function. Therefore, for a specific alignment problem one needs a way of selecting the best performing scoring function. This work is focused on the issue of finding optimized protein family- and fold-specific scoring functions for global similarity matrix-based sequence alignment. Findings I utilize a comprehensive set of reference alignments obtained from structural superposition of homologous and analogous proteins to design a quantitative statistical framework for evaluating the performance of alignment scoring functions in global pairwise sequence alignment. This framework is applied to study how existing general-purpose amino acid similarity matrices perform on individual protein families and structural folds, and to compare them to family-specific and fold-specific matrices derived in this work. I describe an adaptive alignment procedure that automatically selects an appropriate similarity matrix and optimized gap penalties based on the properties of the sequences being aligned. Conclusions The results of this work indicate that using family-specific similarity matrices significantly improves the quality of the alignment of homologous sequences over the traditional sequence alignment based on a single general-purpose similarity matrix. However, using fold-specific similarity matrices can only marginally improve sequence alignment of proteins that share the same structural fold but do not share a common evolutionary origin. The family-specific matrices derived in this work and the optimized gap penalties are available at http://taurus.crc.albany.edu/fsm. PMID:21846354

  12. Protein kinase C regulates mitochondrial targeting of Nur77 and its family member Nor-1 in thymocytes undergoing apoptosis.

    Science.gov (United States)

    Thompson, Jennifer; Burger, Megan L; Whang, Hannah; Winoto, Astar

    2010-07-01

    Nur77 orphan steroid receptor and its family member Nor-1 are required for apoptosis of developing T cells. In thymocytes, signals from the TCR complex induce Nur77 and Nor-1 expression followed by translocation from the nucleus to mitochondria. Nur77 and Nor-1 associate with Bcl-2 in the mitochondria, resulting in a conformation change that exposes the Bcl-2 BH3 domain, a presumed pro-apoptotic molecule of Bcl-2. As Nur77 and Nor-1 are heavily phosphorylated, we examined the requirement of Nur77 and Nor-1 phosphorylation in mitochondria translocation and Bcl-2 BH3 exposure. We found that HK434, a PKC agonist, in combination with calcium ionophore, can induce Nur77 and Nor-1 phosphorylation, translocation, Bcl-2 BH3 exposure and thymocyte apoptosis. Inhibitors of both classical and novel forms of PKC were able to block this process. In contrast, only the general but not classical PKC-specific inhibitors were able to block the same process initiated by PMA, a commonly used PKC agonist. These data demonstrate a differential activation of PKC isoforms by PMA and HK434 in thymocytes, and show the importance of PKC in mitochondria translocation of Nur77/Nor-1 and Bcl-2 conformation change during TCR-induced thymocyte apoptosis.

  13. Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, Mesembryanthemum crystallinum.

    Science.gov (United States)

    Chehab, E Wassim; Patharkar, O Rahul; Cushman, John C

    2007-03-01

    McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydrationstress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin super-family that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTIl1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton.

  14. Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE.

    Science.gov (United States)

    Lin, Yuan; Liu, Tengfei; Liu, Jun; Liu, Xun; Ou, Yongbin; Zhang, Huiling; Li, Meng; Sonnewald, Uwe; Song, Botao; Xie, Conghua

    2015-08-01

    Slowing down cold-induced sweetening (CIS) of potato (Solanum tuberosum) tubers is of economic importance for the potato industry to ensure high-quality products. The conversion of sucrose to reducing sugars by the acid invertase StvacINV1 is thought to be critical for CIS. Identification of the specific StvacINV1 inhibitor StInvInh2B and the α- and β-subunits of the interacting protein SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE from the wild potato species Solanum berthaultii (SbSnRK1) has led to speculation that invertase activity may be regulated via a posttranslational mechanism that remains to be elucidated. Using bimolecular fluorescence complementation assays, this study confirmed the protein complex by pairwise interactions. In vitro kinase assays and protein phosphorylation analysis revealed that phosphorylation of SbSnRK1α is causal for StvacINV1 activity and that its active form blocks the inhibition of StInvInh2B by SbSnRK1β, whereas its inactive form restores the function of SbSnRK1β that prevents StInvInh2B from repressing StvacINV1. Overexpression of SbSnRK1α in CIS-sensitive potato confirmed that SbSnRK1α has significant effects on acid invertase-associated sucrose degradation. A higher level of SbSnRK1α expression was accompanied by elevated SbSnRK1α phosphorylation, reduced acid invertase activity, a higher sucrose-hexose ratio, and improved chip color. Our results lend new insights into a subtle regulatory mode of invertase activity and provide a novel approach for potato CIS improvement. © 2015 American Society of Plant Biologists. All Rights Reserved.

  15. Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1

    Science.gov (United States)

    Liu, Tengfei; Zhang, Huiling; Li, Meng; Song, Botao

    2015-01-01

    Slowing down cold-induced sweetening (CIS) of potato (Solanum tuberosum) tubers is of economic importance for the potato industry to ensure high-quality products. The conversion of sucrose to reducing sugars by the acid invertase StvacINV1 is thought to be critical for CIS. Identification of the specific StvacINV1 inhibitor StInvInh2B and the α- and β-subunits of the interacting protein SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE from the wild potato species Solanum berthaultii (SbSnRK1) has led to speculation that invertase activity may be regulated via a posttranslational mechanism that remains to be elucidated. Using bimolecular fluorescence complementation assays, this study confirmed the protein complex by pairwise interactions. In vitro kinase assays and protein phosphorylation analysis revealed that phosphorylation of SbSnRK1α is causal for StvacINV1 activity and that its active form blocks the inhibition of StInvInh2B by SbSnRK1β, whereas its inactive form restores the function of SbSnRK1β that prevents StInvInh2B from repressing StvacINV1. Overexpression of SbSnRK1α in CIS-sensitive potato confirmed that SbSnRK1α has significant effects on acid invertase-associated sucrose degradation. A higher level of SbSnRK1α expression was accompanied by elevated SbSnRK1α phosphorylation, reduced acid invertase activity, a higher sucrose-hexose ratio, and improved chip color. Our results lend new insights into a subtle regulatory mode of invertase activity and provide a novel approach for potato CIS improvement. PMID:26134163

  16. Src Family Kinases Mediate Betel Quid-Induced Oral Cancer Cell Motility and Could Be a Biomarker for Early Invasion in Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Jeff Yi-Fu Chen

    2008-12-01

    Full Text Available Betel quid (BQ-chewing oral cancer is a prevalent disease in many countries of Southeast Asia. Yet, the precise disease mechanism remains largely unknown. Here, we show that BQ extract-induced cell motility in three oral cancer cells (Ca9-22, SAS, and SCC9 presumably involves the Src family kinases (SFKs. Besides, BQ extract can markedly induce cell migration of wild type mouse embryonic fibroblasts (MEFs but not MEFs lacking three SFK members, namely, Src, Yes, and Fyn, indicating the requirement of SFKs for BQ-induced cell motility. Betel quid extract can also elevate cellular SFK activities because phosphorylation of tyrosine 416 at the catalytic domain is increased, which in turn promotes phosphorylation of an in vitro substrate, enolase. Furthermore, we identified that areca nut, a major component of BQ, is the key factor accounting for BQ-induced cell migration and invasion through SFKs-mediated signaling pathways. Immunohistochemistry revealed that, particularly in BQ-chewing cases, the activity of SFKs was significantly higher in tumor-adjacent mucosa than that in solid tumor areas (P < .01. These results suggest a possible role of SFKs in tumor-host interface and thus in early tumor invasion in vivo. Consistent with this is the observation that activation of SFKs is colocalized with invasive tumor fronts in oral squamous cell carcinoma. Together, we conclude that SFKs may represent a potential biomarker of invasion and therapeutic target in BQ-induced oral cancer.

  17. FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

    Directory of Open Access Journals (Sweden)

    Kim Doré

    Full Text Available Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

  18. Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway

    DEFF Research Database (Denmark)

    Leffers, H; Madsen, Peder; Rasmussen, H H

    1993-01-01

    tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium......We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human...

  19. Stimulation of G protein-coupled bile acid receptor enhances vascular endothelial barrier function via activation of protein kinase A and Rac1.

    Science.gov (United States)

    Kida, Taiki; Omori, Keisuke; Hori, Masatoshi; Ozaki, Hiroshi; Murata, Takahisa

    2014-01-01

    Bile acids are end products of cholesterol metabolism, and they constantly exist at high concentrations in the blood. Since vascular endothelial cells express G protein-coupled bile acid receptor (GPBAR), bile acids potentially modulate endothelial function. Here, we investigated whether and how GPBAR agonism affects endothelial barrier function. In bovine aortic endothelial cells (BAECs), treatment with a GPBAR agonist, taurolithocholic acid (TLCA) increased the transendothelial electrical resistance. In addition, TLCA suppressed the thrombin-induced dextran infiltration through the endothelial monolayer. Knockdown of GPBAR abolished the inhibitory effect of TLCA on hyperpermeability. These results indicate that stimulation of GPBAR enhances endothelial barrier function. TLCA increased intracellular cAMP production in BAECs. Inhibition of protein kinase A (PKA) or Rac1 significantly attenuated the TLCA-induced endothelial barrier protection. TLCA induced cortical actin polymerization, which was attenuated by a Rac1 inhibitor. In vivo, local administration of TLCA into the mouse ear significantly inhibited vascular leakage and edema formation induced by croton oil or vascular endothelial growth factor. These results indicate that stimulation of GPBAR enhances endothelial barrier function by cAMP/PKA/Rac1-dependent cytoskeletal rearrangement.

  20. Global trophic position comparison of two dominant mesopelagic fish families (Myctophidae, Stomiidae) using amino acid nitrogen isotopicanalyses

    Science.gov (United States)

    We examined the biogeochemical and ecological mechanisms responsible for variability in bulk tissue and amino acid (AA) stable nitrogen isotope compositions in two groups of important mesopelagic fish families, Myctophidae (lanternfishes) and Stomiidae (dragonfishes), from five d...

  1. Bile acid metabolism in hereditary forms of hypertriglyceridemia: evidence for an increased synthesis rate in monogenic familial hypertriglyceridemia.

    OpenAIRE

    Angelin, B; Hershon, K S; Brunzell, J D

    1987-01-01

    This study was undertaken to characterize bile acid metabolism in hereditary forms of hypertriglyceridemia. Ten hypertriglyceridemic patients (type IV phenotype) with familial combined hyperlipidemia and 7 patients with monogenic familial hypertriglyceridemia (FHTG) were compared with 18 healthy controls; all subjects were males. Pool size, synthesis rate, and fractional catabolic rate of cholic and chenodeoxycholic acids were determined with an isotope dilution technique. Patients with FHTG ...

  2. Identification of a common amino acid polymorphism in the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

    DEFF Research Database (Denmark)

    Hansen, Torben; Andersen, C B; Echwald, Søren Morgenthaler

    1997-01-01

    Phosphatidylinositol 3-kinase (PI3-K) may regulate the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool in addition to being an insulin signal for translocation of glucose transporters to the plasma membrane. The objectives of the pres......Phosphatidylinositol 3-kinase (PI3-K) may regulate the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool in addition to being an insulin signal for translocation of glucose transporters to the plasma membrane. The objectives...... of the present study were to examine for genetic variability in the human regulatory p85alpha subunit of PI3-K, to look for an association between gene variants and NIDDM in a case-control study, and to relate identified variability to potential changes in whole-body insulin sensitivity and glucose turnover.......13-0.19]). No difference in glucose disappearance constant (KG), insulin sensitivity index (SI), and glucose effectiveness (SG) was observed between wildtype and heterozygous subjects. However, compared with the combined values for wildtype and heterozygous carriers, KG was reduced by 40% (P = 0.004) and SG by 23% (P = 0...

  3. Crystal structure of Clostridium acetobutylicum Aspartate kinase (CaAK): An important allosteric enzyme for amino acids production.

    Science.gov (United States)

    Manjasetty, Babu A; Chance, Mark R; Burley, Stephen K; Panjikar, Santosh; Almo, Steven C

    2014-09-01

    Aspartate kinase (AK) is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase (CaAK; E.C. 2.7.2.4; Mw=48,030Da; 437aa; SwissProt: Q97MC0) has been determined to 3Å resolution. CaAK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (bacteria such as Clostridium tetani (64% sequence identity) suggesting the potential of the structure solved here to be applied for modeling drug interactions. CaAK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry.

  4. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells.

    Science.gov (United States)

    Zhu, Jingying; Qian, Wenyi; Wang, Yixin; Gao, Rong; Wang, Jun; Xiao, Hang

    2015-12-01

    Microglial activation is closely related to the pathogenesis of neurodegenerative diseases by producing proinflammatory cytokines. Perfluorooctane sulfonic acid (PFOS), known as an emerging persistent organic pollutant, is reported to disturb human immune homeostasis; however, whether it affects cytokine production or the immune response in the central nervous system remains unclear. The present study was aimed to explore whether PFOS contributed to inflammatory action and to investigate the corresponding mechanisms in BV2 microglia. PFOS-mediated morphologic changes, cytokine responses and signaling events were examined by light microscopy, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot assays. Our results indicated that PFOS increased BV2 cells activation and simultaneously increased tumor necrosis factor alpha and interleukin-6 expression. In addition, the c-Jun N-terminal protein kinase inhibitor (SP600125), as well as ERK1/2 blocker (PD98059), transcriptionally at least, displayed anti-inflammatory properties on PFOS-elicited cytokine responses. Moreover, the inflammatory transcription factor NF-κB was specifically activated by PFOS as well. These results, taken together, suggested that PFOS exerts its functional effects on the response of microglial cell activation via, in part, the c-Jun N-terminal protein kinase, ERK and NF-κB signaling pathways with its subsequent influence on proinflammatory action. Copyright © 2015 John Wiley & Sons, Ltd.

  5. Ursolic acid-induced AMP-activated protein kinase (AMPK) activation contributes to growth inhibition and apoptosis in human bladder cancer T24 cells.

    Science.gov (United States)

    Zheng, Qing-you; Jin, Feng-suo; Yao, Chen; Zhang, Tong; Zhang, Guo-hui; Ai, Xing

    2012-03-23

    Ursolic acid (UA) has shown the anti-tumor properties against a number of human cancers both in vivo and in vitro, however, its effect in bladder cancer and the corresponding mechanisms of action remain largely unknown. Here we found that UA dose-dependently induced growth inhibition and apoptosis in human bladder cancer T24 cells, and activation of AMP-activated protein kinase (AMPK) may contribute to the process. Our Western-blot results demonstrated a significant AMPK activation after UA treatment in T24 cells. Notably, knockdown of AMPKα by the targeted shRNA largely inhibited UA-induced T24 cell growth inhibition and apoptosis, while an AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or a constitutively active form of AMPK mimic UA's effect. We found the ceramide level was increased after UA treatment in T24 cells, and UA-induced AMPK activation and T24 cell apoptosis were inhibited by ceramide synthase inhibitor fumonisin B1, and was enhanced by exogenously adding cell permeable short-chain ceramide (C6), suggesting that ceramide might serve as an upstream signal for AMPK activation. Further, activation of AMPK by UA promoted c-Jun N-terminal kinase (JNK) activation, but inhibited mTOR complex 1 (mTORC1) signaling to cause survivin down-regulation. Our study suggests that activation of AMPK by UA contributes to growth inhibition and apoptosis in human bladder cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A Src-family-tyrosine kinase, Lyn, is required for efficient IFN-β expression in pattern recognition receptor, RIG-I, signal pathway by interacting with IPS-1.

    Science.gov (United States)

    Lim, Young Ju; Koo, Jung Eun; Hong, Eun-Hee; Park, Zee-Yong; Lim, Kyung-Min; Bae, Ok-Nam; Lee, Joo Young

    2015-03-01

    Retinoic acid-inducible gene I (RIG-I) plays an important role in antiviral immunity as a cytosolic receptor recognizing invading viruses. The activation of downstream signaling pathways led by IFN-β promoter stimulator-1 (IPS-1), an adaptor, is known to culminate in the activation of IRFs and the expression of type I interferons. However, the role of Src-family-tyrosine kinases (STKs) in the RIG-I signaling pathway has not been fully evaluated. Through a combined approach of immunoprecipitation and micro reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis, we established that Lyn, one of the STKs, is associated with RIG-I in macrophages. The association of Lyn and RIG-I was confirmed by co-immunoprecipitation study with 293T cells overexpressing Lyn and RIG-I. Suppression of Lyn by siRNA knockdown or a pharmacological inhibitor (PP2) resulted in the attenuation of IRF3 activation and IFN-β expression induced by short poly I:C, a RIG-I agonist, in macrophages. Lyn activation, as determined by phosphorylation of Tyr396 residue, was observed upon short poly I:C stimulation in the mitochondria of macrophages. Short poly I:C induced the formation of speckle-like aggregates of Lyn, which are prominent in mitochondria. Lyn associated with IPS-1, an adaptor protein of RIG-I, which resides on mitochondria membrane. Helicase domain of RIG-I and CARD of IPS-1 are responsible for the interaction with Lyn while SH3 and SH2 domains in Lyn are required for the association with RIG-I and IPS-1. Collectively, our results indicate that Lyn plays a positive regulatory role in RIG-I-mediated interferon expression as a downstream component of IPS-1. They provide further information as to how tyrosine kinases such as STKs play a role in the regulation of antiviral immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

    Directory of Open Access Journals (Sweden)

    Kunal Shah B

    2012-03-01

    Full Text Available Abstract Background In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG. The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. Methods Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. Results and discussion The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416] and PDE as well as decreased expression of SR-B1

  8. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

    Science.gov (United States)

    Seffernick, Jennifer L; Erickson, Jasmine S; Cameron, Stephan M; Cho, Seunghee; Dodge, Anthony G; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2012-09-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

  9. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine...

  10. KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

    Science.gov (United States)

    Yustein, J T; Li, D; Robinson, D; Kung, H J

    2000-02-03

    The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

  11. Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases, ERK, and c-Jun N-terminal Kinase*

    Science.gov (United States)

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

    2010-01-01

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells. PMID:20829350

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

    Science.gov (United States)

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

    2010-11-12

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

  13. Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß

    Directory of Open Access Journals (Sweden)

    Abeer Abousaab

    2016-12-01

    Full Text Available Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1 or EAAT4 (SLC1A6 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (IEAAT. Results: Appreciable IEAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of GSK3ß but not by coexpression of K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal IEAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM exposure for 24 hours decreased IEAAT in EAAT3 and GSK3ß expressing oocytes to values similar to IEAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify IEAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.

  14. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

    Directory of Open Access Journals (Sweden)

    J Samuel Zigler

    Full Text Available A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs, leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body, and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of

  15. Role of mitogen-activated protein kinases in tauroursodeoxycholic acid-induced bile formation in cholestatic rat liver

    NARCIS (Netherlands)

    Denk, Gerald Ulrich; Hohenester, Simon; Wimmer, Ralf; Boehland, Claudia; Rust, Christian; Beuers, Ulrich

    2008-01-01

    Aim: Ursodeoxycholic acid exerts anticholestatic effects in various cholestatic disorders and experimental models of cholestasis. Its taurine conjugate (TUDCA) stimulates bile salt secretion in isolated perfused rat livers (IPRL) under physiological, non-cholestatic conditions, in part by

  16. α-Lipoic Acid Attenuates LPS-Induced Inflammatory Responses by Activating the Phosphoinositide 3-Kinase/Akt Signaling Pathway

    National Research Council Canada - National Science Library

    Wei-Jian Zhang; Hao Wei; Tory Hagen; Balz Frei

    2007-01-01

    .... We previously observed that the metabolic thiol antioxidant α-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells...

  17. Cyclin-dependent kinase inhibitor, flavopiridol, induces apoptosis and inhibits tumor growth in drug-resistant osteosarcoma and Ewing's family tumor cells.

    Science.gov (United States)

    Li, Yan; Tanaka, Kazuhiro; Li, Xu; Okada, Takamitsu; Nakamura, Tomoyuki; Takasaki, Minoru; Yamamoto, Shunsaku; Oda, Yoshinao; Tsuneyoshi, Masazumi; Iwamoto, Yukihide

    2007-09-15

    Multimodal therapies play important roles in the treatment of osteosarcoma (OS) and Ewing's family of tumors (EFTs), two most frequent malignant bone tumors. Although the clinical outcome of primary OS and EFTs is greatly improved, the relapsed cases often are associated with multidrug resistance of the tumors and the prognosis of these patients is still poor. Flavopiridol, a pan cyclin-dependent kinase (CDK) inhibitor is a novel antitumor agent that can induce cell cycle arrest and apoptosis in many cancer cells. However, there have been no studies about the effects of flavopiridol on drug-resistant OS and EFTs. Here, we demonstrated that flavopiridol induced the cleavage of poly-ADP-ribose polymerase (PARP) in a time and dose dependent manner in adriamycin-resistant OS and EFTs cells expressing P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP(1)) as effectively as in their parental cells. Our data also showed that flavopiridol caused the release of mitochondrial cytochrome c and the activation of caspase-9, caspase-8 and caspase-3, with an increase ratio of the proapoptotic protein level (Bax) to the antiapoptotic protein level (Bcl-2 and Bcl-X(L)), while apoptosis was inhibited by pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK), not by caspase-8 inhibitor (Z-IETD-FMK). The treatment with flavopiridol further inhibited the tumor growth in mouse models of the drug-resistant OS and EFTs. These results suggest that flavopiridol might be promising in clinical therapy for the relapsed OS and EFTs. (c) 2007 Wiley-Liss, Inc.

  18. Polymorphisms in the Janus kinase 2 (JAK)/signal transducer and activator of transcription (STAT) genes: putative association of the STAT gene region with familial breast cancer.

    Science.gov (United States)

    Vaclavicek, Annika; Bermejo, Justo Lorenzo; Schmutzler, Rita K; Sutter, Christian; Wappenschmidt, Barbara; Meindl, Alfons; Kiechle, Marion; Arnold, Norbert; Weber, Bernhard H F; Niederacher, Dieter; Burwinkel, Barbara; Bartram, Claus R; Hemminki, Kari; Försti, Asta

    2007-06-01

    The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway mediates the signals of a wide range of cytokines, growth factors and hormones. Thus, aberrant activation of the JAK/STAT pathway may predispose to malignancy due to deregulation of proliferation, differentiation or apoptosis. In this study, we investigated whether genetic variation in the JAK2 gene and the STAT gene region (STAT3, STAT5A and STAT5B) is associated with breast cancer (BC) risk. We carried out a case-control study using a German sample set with 441 familial, unrelated BC cases and 552 controls matched by age, ethnicity and geographical region. A second similar set (381 cases, 460 controls) was applied to validate the findings. Haplotypes in the JAK2 gene were not associated with the risk of BC. In the STAT gene region, the rare haplotype CAGCC containing the variant alleles of each single nucleotide polymorphism (SNP) was associated with an increased risk odds ratio (OR = 5.83, 95% confidence interval (CI) 1.51-26.28). According to Akaike's information criterion, the best model to describe the relationship between the haplotypes and BC was based on the SNPs rs6503691 (STAT5B) and rs7211777 (STAT3). Carriers of the AC haplotype, which represents the variant alleles of both SNPs, were at an increased risk (OR = 1.41, 95% CI 1.09-1.82). A decreased risk was observed for carriers of the AT haplotype (OR = 0.60, 95% CI 0.38-0.94). Furthermore, individuals with the AC/GC diplotype were at a significantly increased risk (OR = 1.88, 95% CI 1.13-3.14). The observed genetic variation may also influence the inter-individual variation in response to STAT-signalling targeted therapy.

  19. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK Gene Family in Brassica rapa.

    Directory of Open Access Journals (Sweden)

    Kun Lu

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D. Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa.

  20. Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa.

    Science.gov (United States)

    Lu, Kun; Guo, Wenjin; Lu, Junxing; Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa.

  1. A combinatorial approach to detect coevolved amino acid networks in protein families of variable divergence.

    Directory of Open Access Journals (Sweden)

    Julie Baussand

    2009-09-01

    Full Text Available Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.

  2. A combinatorial approach to detect coevolved amino acid networks in protein families of variable divergence.

    Science.gov (United States)

    Baussand, Julie; Carbone, Alessandra

    2009-09-01

    Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.

  3. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  4. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver

    NARCIS (Netherlands)

    Beuers, U.; Bilzer, M.; Chittattu, A.; Kullak-Ublick, G. A.; Keppler, D.; Paumgartner, G.; Dombrowski, F.

    2001-01-01

    Ursodeoxycholic acid (UDCA) exerts anticholestatic effects by undefined mechanisms. Previous work suggested that UDCA stimulates biliary exocytosis via Ca(++)- and protein kinase C (PKC)-dependent mechanisms. Therefore, the effect of taurine-conjugated UDCA (TUDCA) was studied in the experimental

  5. Involvement of atypical protein kinase C in the regulation of cardiac glucose and long-chain fatty acid uptake

    Directory of Open Access Journals (Sweden)

    Daphna D.J. Habets

    2012-09-01

    Full Text Available Aim: The signaling pathways involved in the regulation of cardiac GLUT4 translocation/glucose uptake and CD36 translocation/ long-chain fatty acid uptake are not fully understood. We compared in heart/muscle-specific PKC-λ knockout mice the roles of atypical PKCs (PKC-ζ and PKC-λ in regulating cardiac glucose and fatty acid uptake. Results: Neither insulin-stimulated nor AMPK-mediated glucose and fatty acid uptake were inhibited upon genetic PKC-λ ablation in cardiomyocytes. In contrast, myristoylated PKC-ζ pseudosubstrate inhibited both insulin-stimulated and AMPK-mediated glucose and fatty acid uptake by >80% in both wild-type and PKC-λ-knockout cardiomyocytes. In PKC-λ knockout cardiomyocytes, PKC-ζ is the sole remaining atypical PKC isoform, and its expression level is not different from wild-type cardiomyocytes, in which it contributes to 29% and 17% of total atypical PKC expression and phosphorylation, respectively. Conclusion: Taken together, atypical PKCs are necessary for insulin-stimulated and AMPK-mediated glucose uptake into the heart, as well as for insulin-stimulated and AMPK-mediated fatty acid uptake. However, the residual PKC-ζ activity in PKC-λ-knockout cardiomyocytes is sufficient to allow optimal stimulation of glucose and fatty acid uptake, indicating that atypical PKCs are necessary but not rate-limiting in the regulation of cardiac substrate uptake and that PKC-λ and PKC-ζ have interchangeable functions in these processes.

  6. Eicosapentaenoic acid ameliorates palmitate-induced lipotoxicity via the AMP kinase/dynamin-related protein-1 signaling pathway in differentiated H9c2 myocytes.

    Science.gov (United States)

    Sakamoto, Atsushi; Saotome, Masao; Hasan, Prottoy; Satoh, Terumori; Ohtani, Hayato; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi; Hayashi, Hideharu

    2017-02-01

    Emerging evidence suggested the preferable effects of eicosapentaenoic acid (EPA; n-3 polyunsaturated fatty acid) against cardiac lipotoxicity, which worsens cardiac function by means of excessive serum free fatty acids due to chronic adrenergic stimulation under heart failure. Nonetheless, the precise molecular mechanisms remain elusive. In this study, we focused on dynamin-related protein-1 (Drp1) as a possible modulator of the EPA-mediated cardiac protection against cardiac lipotoxicity, and investigated the causal relation between AMP-activated protein kinase (AMPK) and Drp1. When differentiated H9c2 myocytes were exposed to palmitate (PAL; saturated fatty acid, 400µM) for 24h, these myocytes showed activation of caspases 3 and 7, enhanced caspase 3 cleavage, depolarized mitochondrial membrane potential, depleted intracellular ATP, and enhanced production of intracellular reactive oxygen species. These changes suggested lipotoxicity due to excessive PAL. PAL enhanced mitochondrial fragmentation with increased Drp1 expression, as well. EPA (50µM) restored the PAL-induced apoptosis, mitochondrial dysfunction, and mitochondrial fragmentation with increased Drp1 expression by PAL. EPA activated phosphorylation of AMPK, and pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleotide ameliorated the PAL-induced apoptosis, mitochondrial dysfunction, and downregulated Drp1. An AMPK knockdown via RNA interference enhanced Drp1 expression and attenuated the protective effects of EPA against the PAL-induced lipotoxicity. EPA ameliorates the PAL-induced lipotoxicity via AMPK activation, which subsequently suppresses mitochondrial fragmentation and Drp1 expression. Our findings may provide new insights into the molecular mechanisms of EPA-mediated myocardial protection in heart failure. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Involvement of atypical protein kinase C in the regulation of cardiac glucose and long-chain fatty acid uptake

    DEFF Research Database (Denmark)

    Habets, Daphna D J; Luiken, Joost J F P; Ouwens, Margriet

    2012-01-01

    Aim: The signaling pathways involved in the regulation of cardiac GLUT4 translocation/glucose uptake and CD36 translocation/long-chain fatty acid uptake are not fully understood. We compared in heart/muscle-specific PKC-¿ knockout mice the roles of atypical PKCs (PKC-¿ and PKC-¿) in regulating...... to allow optimal stimulation of glucose and fatty acid uptake, indicating that atypical PKCs are necessary but not rate-limiting in the regulation of cardiac substrate uptake and that PKC-¿ and PKC-¿ have interchangeable functions in these processes....

  8. ω-3 Fatty Acid Ethyl Esters Diminish Postprandial Lipemia in Familial Hypercholesterolemia.

    Science.gov (United States)

    Chan, Dick C; Pang, Jing; Barrett, P Hugh R; Sullivan, David R; Burnett, John R; van Bockxmeer, Frank M; Watts, Gerald F

    2016-10-01

    Impaired postprandial chylomicron metabolism induces hypertriglyceridemia and may increase the risk of atherosclerotic cardiovascular disease. Omega-3 fatty acid ethyl ester (ω-3 FAEE) supplementation decreases plasma triglycerides. However, its effect on postprandial chylomicron metabolism in familial hypercholesterolemia (FH) has not yet been investigated. We aimed to examine the effect of ω-3 FAEE supplementation on postprandial responses in plasma triglycerides, very-low-density lipoprotein (VLDL) apolipoprotein B (apoB)-100, and apoB-48 in FH patients receiving standard cholesterol-lowering treatment. We carried out an 8-week open-label, randomized, crossover intervention trial to test the effect of oral supplementation with 4 g/d ω-3 FAEE (46% eicosapentaenoic acid and 38% docosahexaenoic acid) on postprandial triglyceride, VLDL-apoB-100, and apoB-48 responses in FH patients after ingestion of an oral fat load. Plasma total and incremental triglyceride, VLDL-apoB-100, and apoB-48 0- to 10-hour area under the curve (AUC). ω-3 FAEE supplementation significantly (P lipemia in FH patients receiving standard care; this may have implications for further reducing atherosclerotic cardiovascular disease in this high-risk patient group.

  9. Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan.

    Science.gov (United States)

    Faulds, C B; Mandalari, G; Lo Curto, R B; Bisignano, G; Christakopoulos, P; Waldron, K W

    2006-08-01

    The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1-->4)-beta-D-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5' form of diferulic acid from arabinoxylan (AX) derived from brewers' spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.

  10. Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase.

    NARCIS (Netherlands)

    Arisz, S.A.; van Wijk, R.; Roels, W.; Zhu, J.K.; Haring, M.A.; Munnik, T.

    2013-01-01

    Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using (32)P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid (32)P-PtdOH

  11. [Modulation by some fatty acids of protein kinase C-dependent NADPH oxidase in human adherent monocyte: mechanism of action, possible implication in atherogenesis].

    Science.gov (United States)

    Léger, C L; Kadri-Hassani, N

    1995-01-01

    It is largely admitted nowadays that the early stage of the atherosclerotic lesion involves formation of oxidized (and minimally oxidized) low-density lipoprotein. Their properties are briefly reviewed. It is recalled that a lipolytic process also takes place both at the lumenal surface and in the subendothelial space of the vessels implying lipoprotein lipase (LpL) activity. Recent studies emphasize the role of LpL in accumulating LDL in the vascular tissue (Rutledge & Golberg, J. Lipid Res., 1994, 35, 1152-1160), but the role of LpL-generated unesterified fatty acids (UEFA) in these two locations and their possible implication in atherogenesis are largely neglected. Physiological and pathophysiological significance of UEFA in the human adherent monocyte modulation of the superoxide anion (O2.-) production has been examined by our group, leading to a possible mechanism of modulation of LDL oxidative modification. The O2.- production-modulating effect of a 30-min UEFA preincubation has been studied in intact human adherent monocytes (HAM) after stimulation by a direct effector of protein kinase C (PKC). It has been established that UEFA alone (in the absence of PKC effectors) were not able to modulate the O2.- production of HAM whereas they had such a capacity in the presence of PKC effectors, phorbol myristate acetate (PMA) or diacylglycerol (DAG). In this case inhibitors of PKC such as GF 109203 X suppressed the modulating effect. UEFA have also been shown to possess a bimodal action in the presence of PKC effectors: they depressed or enhanced O2.- production at micromolar or nanomolar concentrations, respectively. All these results contrasted with others obtained in neutrophils or nonadherent monocytes, suggesting an absolute requirement of PKC for the phagocyte-NADPH oxydase (PHOX) activation especially in the case of HAM. In HAM, the maximal enhancing effects were obtained with monomethyl ramified saturated (MMRS) and linear unsaturated (LU) FAs such as

  12. Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

    Science.gov (United States)

    Lewandowski, Daniel; Linassier, Claude; Iochmann, Sophie; Degenne, Michel; Domenech, Jorge; Colombat, Philippe; Binet, Christian; Hérault, Olivier

    2002-08-01

    All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

  13. Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kuwahara, Ayuko; Arisaka, Satomi; Takeya, Masahiro; Iijima, Hiroko; Hirai, Masami Yokota; Osanai, Takashi

    2015-01-01

    Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Respiratory activity was down-regulated under nitrogen-replete conditions. Photosynthetic activity was slightly lower in the hik8-overexpressing strain than in the wild-type after nitrogen depletion, and the values of photosynthetic parameters were altered by hik8 overexpression under nitrogen-replete and nitrogen-depleted conditions. Transcripts of genes encoding Photosystem I and II were increased by hik8 overexpression under nitrogen-replete conditions. Nitrogen starvation triggers increase in amino acids but the magnitude of the increase in several amino acids was diminished by hik8 overexpression. These genetic data indicate that Hik8 regulates the photosynthetic electron transport, which in turn alters primary metabolism during nitrogen starvation in this cyanobacterium.

  14. Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Ayuko eKuwahara

    2015-10-01

    Full Text Available Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Respiratory activity was down-regulated under nitrogen-replete conditions. Photosynthetic activity was slightly lower in the hik8-overexpressing strain than in the wild-type after nitrogen depletion, and the values of photosynthetic parameters were altered by hik8 overexpression under nitrogen-replete and nitrogen-depleted conditions. Transcripts of genes encoding Photosystem I and II were increased by hik8 overexpression under nitrogen-replete conditions. Nitrogen starvation triggers increase in amino acids but the magnitude of the increase in several amino acids was diminished by hik8 overexpression. These genetic data indicate that Hik8 regulates the photosynthetic electron transport, which in turn alters primary metabolism during nitrogen starvation in this cyanobacterium.

  15. LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huiling; Li, Ridong [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China); Li, Li [Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing (China); Ge, Zemei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China); Zhou, Rouli, E-mail: rlzhou@bjmu.edu.cn [Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing (China); Li, Runtao, E-mail: lirt@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China)

    2015-02-27

    In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{sub 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.

  16. A GEF-to-phospholipase molecular switch caused by phosphatidic acid, Rac and JAK tyrosine kinase that explains leukocyte cell migration.

    Science.gov (United States)

    Mahankali, Madhu; Henkels, Karen M; Gomez-Cambronero, Julian

    2013-03-15

    Phospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ∼3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at >15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF-lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

  17. The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

    Science.gov (United States)

    You, Jae-Sung; Lincoln, Hannah C; Kim, Chan-Ran; Frey, John W; Goodman, Craig A; Zhong, Xiao-Ping; Hornberger, Troy A

    2014-01-17

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

  18. The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy*

    Science.gov (United States)

    You, Jae-Sung; Lincoln, Hannah C.; Kim, Chan-Ran; Frey, John W.; Goodman, Craig A.; Zhong, Xiao-Ping; Hornberger, Troy A.

    2014-01-01

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass. PMID:24302719

  19. Down-regulation of sphingosine kinase 2 (SphK2) increases the effects of all-trans-retinoic acid (ATRA) on colon cancer cells.

    Science.gov (United States)

    Chu, Jia-Hui; Gao, Zu-Hua; Qu, Xian-Jun

    2014-10-01

    Sphingosine kinase 2 (SphK2) is a type of sphingosine kinase, which express highly in most of cancers. SphK2 produce sphingosine-1-phosphate (S1P) and then accumulate in cancer cells. Our previous study showed that S1P antagonized the effects of all-trans-retinoic acid (ATRA) via the receptor-dependent and independent pathway. In this study, we aimed to investigate the roles of SphK2 in affecting ATRA's activity in human colon cancer cells. Cell proliferation was estimated by the clonogenic assay. The distribution of cell cycle was analyzed by flow cytometry assay. The apoptotic cells were determined by Annexin V-FITC/PI staining method. Western blotting assay was performed to analyze the levels of the proteins related to apoptosis and cell cycle. The mRNA levels of SphK2 and RARβ were evaluated by real-time PCR assay. RNA interference assay was performed to evaluate SphK2 activity. S1P antagonized the effect of ATRA on HT-29 cell proliferation, the ATRA-induced RARβ expression, the arrest of cell cycle in G1-phase, and induction of apoptosis. Down-regulation of SphK2 resulted in the reverse actions on the S1P-induced antagonistic effects on ATRA. Western blotting analysis indicated that down-regulation of SphK2 might activate apoptotic proteins, regulation of p53/p21(Waf1/Cip1) and EGFR and PI3K/AKT signaling pathways. In conclusion, down-regulation of SphK2 increased the effects of ATRA on colon cancer cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

    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. PMID:24843152

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

    Science.gov (United States)

    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.

  2. Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae [Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Hwang, Jae-Kwan, E-mail: jkhwang@yonsei.ac.kr [Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Translational Research Center for Protein Function Control, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2010-10-08

    Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepatic triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.

  3. Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

    In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CSL), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPCR), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPCR (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CSL (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CSL mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD660) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

  4. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  5. The p21-activated kinase (PAK) family member PakD is required for chemorepulsion and proliferation inhibition by autocrine signals in Dictyostelium discoideum.

    Science.gov (United States)

    Phillips, Jonathan E; Gomer, Richard H

    2014-01-01

    In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK) family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass) on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.

  6. The p21-activated kinase (PAK family member PakD is required for chemorepulsion and proliferation inhibition by autocrine signals in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Jonathan E Phillips

    Full Text Available In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.

  7. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    Science.gov (United States)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  8. Correlation analysis for protein evolutionary family based on amino acid position mutations and application in PDZ domain.

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    Qi-Shi Du

    Full Text Available BACKGROUND: It has been widely recognized that the mutations at specific directions are caused by the functional constraints in protein family and the directional mutations at certain positions control the evolutionary direction of the protein family. The mutations at different positions, even distantly separated, are mutually coupled and form an evolutionary network. Finding the controlling mutative positions and the mutative network among residues are firstly important for protein rational design and enzyme engineering. METHODOLOGY: A computational approach, namely amino acid position conservation-mutation correlation analysis (CMCA, is developed to predict mutually mutative positions and find the evolutionary network in protein family. The amino acid position mutative function, which is the foundational equation of CMCA measuring the mutation of a residue at a position, is derived from the MSA (multiple structure alignment database of protein evolutionary family. Then the position conservation correlation matrix and position mutation correlation matrix is constructed from the amino acid position mutative equation. Unlike traditional SCA (statistical coupling analysis approach, which is based on the statistical analysis of position conservations, the CMCA focuses on the correlation analysis of position mutations. CONCLUSIONS: As an example the CMCA approach is used to study the PDZ domain of protein family, and the results well illustrate the distantly allosteric mechanism in PDZ protein family, and find the functional mutative network among residues. We expect that the CMCA approach may find applications in protein engineering study, and suggest new strategy to improve bioactivities and physicochemical properties of enzymes.

  9. Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib

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    Svenja Beckmann

    2014-12-01

    Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA and alpha-1 acid glycoprotein (AGP or orosomucoid negated Imatinib’s deleterious effects on adult S. mansoni and schistosomula (post-infective larvae in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6–8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments.

  10. A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Belousova, Lubov; Plesner, Igor

    1993-01-01

    is is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (View the MathML source at ionic strength View the MathML source and 5°C); after protonation its affinity increased many times (View the MathML source). Determination...... on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine...

  11. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    Science.gov (United States)

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  12. Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway.

    Science.gov (United States)

    Lim, Hyung-Kyu; Choi, Young-Ae; Park, Wan; Lee, Taehoon; Ryu, Sung Ho; Kim, Seong-Yong; Kim, Jae-Ryong; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-11-14

    Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and the production of nitric oxide, prostaglandin E2, which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.

  13. Kinetic basis for the conjugation of auxin by a GH3 family indole-acetic acid-amido synthetase.

    Science.gov (United States)

    Chen, Qingfeng; Westfall, Corey S; Hicks, Leslie M; Wang, Shiping; Jez, Joseph M

    2010-09-24

    The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg(2+) or Mn(2+) for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4-9-fold reductions in k(cat)/K(m) relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.

  14. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

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

  15. Structural Evolution of the Protein Kinase-Like Superfamily.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available The protein kinase family is large and important, but it is only one family in a larger superfamily of homologous kinases that phosphorylate a variety of substrates and play important roles in all three superkingdoms of life. We used a carefully constructed structural alignment of selected kinases as the basis for a study of the structural evolution of the protein kinase-like superfamily. The comparison of structures revealed a "universal core" domain consisting only of regions required for ATP binding and the phosphotransfer reaction. Remarkably, even within the universal core some kinase structures display notable changes, while still retaining essential activity. Hence, the protein kinase-like superfamily has undergone substantial structural and sequence revision over long evolutionary timescales. We constructed a phylogenetic tree for the superfamily using a novel approach that allowed for the combination of sequence and structure information into a unified quantitative analysis. When considered against the backdrop of species distribution and other metrics, our tree provides a compelling scenario for the development of the various kinase families from a shared common ancestor. We propose that most of the so-called "atypical kinases" are not intermittently derived from protein kinases, but rather diverged early in evolution to form a distinct phyletic group. Within the atypical kinases, the aminoglycoside and choline kinase families appear to share the closest relationship. These two families in turn appear to be the most closely related to the protein kinase family. In addition, our analysis suggests that the actin-fragmin kinase, an atypical protein kinase, is more closely related to the phosphoinositide-3 kinase family than to the protein kinase family. The two most divergent families, alpha-kinases and phosphatidylinositol phosphate kinases (PIPKs, appear to have distinct evolutionary histories. While the PIPKs probably have an

  16. EGFR kinase inhibitors and gastric acid suppressants in EGFR-mutant NSCLC: a retrospective database analysis of potential drug interaction.

    Science.gov (United States)

    Kumarakulasinghe, Nesaretnam Barr; Syn, Nicholas; Soon, Yu Yang; Asmat, Atasha; Zheng, Huili; Loy, En Yun; Pang, Brendan; Soo, Ross Andrew

    2016-12-20

    Erlotinib and gefitinib are weak base drugs whose absorption and clinical efficacy may be impaired by concomitant gastric acid suppressive (AS) therapy, yet proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H2As) are widely indicated in non-small cell lung cancer (NSCLC) patients for the prevention and treatment of erlotinib-induced gastrointestinal injury and corticosteroid-associated gastric irritation. We assessed the clinical relevance of this potential drug-drug interaction (DDI) in a retrospective cohort of EGFR-mutant NSCLC patients. The AS usage rate was 35%. In the overall cohort, AS users did not experience poorer OS (HR: 1.47, 95% CI: 0.92 - 2.35, P = 0.10; median, 11.4 versus 17.5 months) or PFS (HR = 1.37, 95% CI: 0.89 - 2.12, P = 0.16; median, 7.6 versus 8.7 months) compared with non-users in multivariate Cox regression analysis. However, subgroup analyses indicated that AS usage was associated with significantly poorer OS and PFS in patients who had fewer or milder comorbidities (Charlson comorbidity index ≤ 2), those with Karnofsky performance status < 90, and never-smokers. A retrospective database analysis of 157 patients given erlotinib or gefitinib for EGFR-mutant advanced NSCLC from two institutions was conducted. Patients were classified as AS-users if the periods of AS and anti-EGFR therapy overlapped by ≥ 30%. Overall survival (OS) and progression-free survival (PFS) were assessed according to AS usage. Concomitant AS therapy did not have an adverse impact on OS and/or PFS in the overall cohort. Our subgroup findings should be regarded exploratory and require replication in a large prospective cohort.

  17. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    Science.gov (United States)

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  18. [Effect of mitogen-activated protein kinase signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells].

    Science.gov (United States)

    Meng, T; Yang, M; Li, Y X; Jia, Q; Yu, G C; Dai, Y F

    2017-05-20

    Objective: The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells. Methods: 16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h. Results: The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by

  19. JAK protein kinase inhibitors.

    Science.gov (United States)

    Thompson, James E

    2005-06-01

    In humans, the Janus protein tyrosine kinase family (JAKs) contains four members: JAK1, JAK2, JAK3 and TYK2. JAKs phosphorylate signal transducers and activators of transcription (STATs) simultaneously with other phosphorylations required for activation, and there are several cellular mechanisms in place to inhibit JAK/STAT signaling. That one might be able to modulate selected JAK/STAT-mediated cellular signals by inhibiting JAK kinase activity to effect a positive therapeutic outcome is a tantalizing prospect, as yet incompletely realized. While current data suggest no therapeutic use for JAK1 and TYK2 inhibition, JAK2 inhibition seems a promising but not definitively tested mechanism for treatment of leukemia. More promising, however, are data indicating a possible therapeutic use of JAK3 inhibition. The restriction of the JAK3-deficient phenotype to the hematopoietic system and the resulting profound immune suppression suggest that JAK3 could be a target for immunosuppressive therapies used to prevent organ transplant rejection.

  20. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

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    Ngai John

    2006-12-01

    Full Text Available Abstract Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs: the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s, these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

  1. the Effect of six weeks of high intensity interval training and zinc ‎supplement on serum ‎creatine kinase and uric acid levels in ‎futsal players ‎

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    Malihe Saeedy

    2017-01-01

    Full Text Available Background: strenuous training‎-induced reactive oxygen species is associated with several ‎chronic diseases‎ by damaging cell proteins and membrane lipids; it seems uric acid as a major ‎intracellular antioxidant could lower membranous lipid peroxidation and muscle damage. The aim ‎of this study was to examine the effect of six weeks of high-intensity interval training with and ‎‎without zinc ‎on serum Creatine Kinase and uric acid in female futsal players.‎ Methods: Thirty-two female futsal players were randomly divided into four groups: placebo, ‎Zinc, ‎HIT ‎and Zinc+HIT. All subjects had to attend futsal-specific training three sessions per ‎week. Zinc and ‎Placebo groups took ‎30 mg ‎day−1 of zinc gluconate or dextrose, respectively; ‎HIT groups accomplished high-intensity interval training contained 6 to 10 repetitions of a 30-‎second ‎running at 100% of VO2peak with a 4-minutes rest between efforts, during six weeks.‎ Results: After six weeks, Creatine Kinase ‎levels augmented insignificantly from 83.98 to 120.19‎ ‎‎(P=0.101 in ‎placebo, from 99.58 to 150.1(P=0.167 in HIT and from 81.07 to 107.90‎ ‎‎(P=0.152 ‎in HIT+Zinc group; while Creatine Kinase ‎levels increased significantly from 66.86 to ‎‎‎124.81(P=0.004 only in Zinc group. Uric acid levels increased in all groups (Placebo (P=1, Zinc ‎‎(P=‎0.317‎, HIT (P=‎0.157‎, ‎Zinc+HIT (P=1 insignificantly Conclusions: The findings indicated that ‎after six weeks, serum Creatine Kinase and uric acid ‎levels increased insignificantly in all groups; Creatine Kinase ‎levels augmented significantly, only ‎in Zinc group. Zinc as an antioxidant supplement could not decrease the muscle damage, and even ‎increased the serum Creatine Kinase as a marker of muscle damage, significantly

  2. Lysosomal acid lipase deficiency: A hidden disease among cohorts of familial hypercholesterolemia?

    Science.gov (United States)

    Chora, Joana Rita; Alves, Ana Catarina; Medeiros, Ana Margarida; Mariano, Cibelle; Lobarinhas, Goreti; Guerra, António; Mansilha, Helena; Cortez-Pinto, Helena; Bourbon, Mafalda

    Lysosomal acid lipase deficiency (LALD) is an autosomal recessive disorder and an unrecognized cause of dyslipidemia. Patients usually present with dyslipidemia and altered liver function and mutations in LIPA gene are the underlying cause of LALD. The aim of this study was to investigate LALD in individuals with severe dyslipidemia and/or liver steatosis. Coding, splice regions, and promoter region of LIPA were sequenced by Sanger sequencing in a cohort of mutation-negative familial hypercholesterolemia (FH) patients (n = 492) and in a population sample comprising individuals with several types of dyslipidemia and/or liver steatosis (n = 258). This study led to the identification of LALD in 4 children referred to the Portuguese FH Study, all with a clinical diagnosis of FH. Mild liver dysfunction was present at the age of FH diagnosis; however, a diagnosis of LALD was not considered. No adults at the time of referral have been identified with LALD. LALD is a life-threatening disorder, and early identification is crucial for the implementation of specific treatment to avoid premature mortality. FH cohorts should be investigated to identify possible LALD patients, who will need appropriate treatment. These results highlight the importance of correctly identifying the etiology of the dyslipidemia. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  3. Phosphate recovery through struvite-family crystals precipitated in the presence of citric acid: mineralogical phase and morphology evaluation.

    Science.gov (United States)

    Perwitasari, D S; Edahwati, L; Sutiyono, S; Muryanto, S; Jamari, J; Bayuseno, A P

    2017-11-01

    Precipitation strategy of struvite-family crystals is presented in this paper to recover phosphate and potassium from a synthetic wastewater in the presence of citric acid at elevated temperature. The crystal-forming solutions were prepared from crystals of MgCl2 and NH4H2PO4 with a molar ratio of 1:1:1 for Mg(+2), [Formula: see text], and [Formula: see text], and the citric acid (C6H8O7) was prepared (1.00 and 20.00 ppm) from citric acid crystals. The Rietveld analysis of X-ray powder diffraction pattern confirmed a mixed product of struvite, struvite-(K), and newberyite crystallized at 30°C in the absence of citric acid. In the presence of citric acid at 30° and 40°C, an abundance of struvite and struvite-(K) were observed. A minute impurity of sylvite and potassium peroxide was unexpectedly found in certain precipitates. The crystal solids have irregular flake-shaped morphology, as shown by scanning electron microscopy micrograph. All parameters (citric acid, temperature, pH, Mg/P, and N/P) were deliberately arranged to control struvite-family crystals precipitation.

  4. Dietary linolenic acid is inversely associated with plasma triacylglycerol: the National Heart, Lung, and Blood Institute Family Heart Study.

    Science.gov (United States)

    Djoussé, Luc; Hunt, Steven C; Arnett, Donna K; Province, Michael A; Eckfeldt, John H; Ellison, R Curtis

    2003-12-01

    Dietary intake of linolenic acid is associated with a decreased risk of cardiovascular disease mortality. However, the mechanisms by which dietary linolenic acid affects cardiovascular disease risk are not clearly understood. We examined the association between dietary linolenic acid and plasma triacylglycerol concentrations. In a cross-sectional design, we studied 4440 white subjects (2036 men and 2404 women) aged 25-93 y who participated in the National Heart, Lung, and Blood Institute Family Heart Study. We used generalized linear models to estimate adjusted mean triacylglycerol concentrations according to categories of total dietary linolenic acid (alpha- and gamma-linolenic acid) intake. The mean dietary linolenic acid intakes were 0.81 and 0.69 g/d for the men and the women, respectively. High consumption of dietary linolenic acid was associated with young age; high intakes of energy, fat, carbohydrates, fruit, vegetables, and fish; low HDL cholesterol; current smoking; and frequent consumption of creamy salad dressing. High consumption of dietary linolenic acid was also associated with low plasma triacylglycerol concentrations. From the lowest to the highest quintile of linolenic acid intake, the multivariate-adjusted mean triacylglycerol concentrations were 1.75 (95% CI: 1.65, 1.85), 1.74 (1.66, 1.82), 1.69 (1.61, 1.77), 1.66 (1.58, 1.74), and 1.54 (1.44, 1.64) mmol/L, respectively (P for linear trend = 0.007). When linolenic acid was used as a continuous variable, the corresponding regression coefficient was -0.2811 (-0.4922, -0.07001). Consumption of total linolenic acid is inversely related to plasma triacylglycerol concentrations in both white men and white women. This suggests a pathway by which dietary linolenic acid might reduce cardiovascular disease risk.

  5. Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

    Directory of Open Access Journals (Sweden)

    Travis W Bainbridge

    Full Text Available Receptor tyrosine kinase-like orphan receptors (ROR 1 and 2 are atypical members of the receptor tyrosine kinase (RTK family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

  6. n-3 Polyunsaturated Fatty Acids Improve Inflammation via Inhibiting Sphingosine Kinase 1 in a Rat Model of Parenteral Nutrition and CLP-Induced Sepsis.

    Science.gov (United States)

    Tian, Tao; Zhao, Yunzhao; Huang, Qian; Li, Jieshou

    2016-03-01

    The sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) pathway plays a key role in inflammation. Parenteral nutrition containing n-3 polyunsaturated fatty acids (n-3 PUFA) may regulate inflammatory reactions. The aim of this study is to determine whether n-3 PUFA may improve inflammatory responses by neutralizing SphK1 signaling. Rat models of parenteral nutrition, cecal ligation and puncture (CLP)-induced sepsis were generated. Male Sprague-Dawley rats were operated for CLP on day 2 after venous catheterization. The rats were randomized to receive normal saline (NS; n = 20), parenteral nutrition (PN; n = 20), or PN + fish oil (FO; n = 20) for 5 days. The daily intake of fish oil (1.25-2.82 g EPA and 1.44-3.09 g DHA per 100 ml) in the FO group was approximately 1.8 g/kg body weight/day. Rats in the control group (n = 10) were subjected to sham operation and received a chow diet. Spleen tissues were collected for SphK1 and S1P receptor expression analysis. Our data showed that n-3 PUFA ameliorated the survival rate. SphK1 expression and its enzymatic activity were significantly upregulated in sepsis rats. Furthermore, mRNA and protein levels of S1PR3, but not S1PR1, were also facilitated after CLP. However, PN + FO dramatically decreased SphK1 mRNA level and its enzymatic activity. S1PR3 expression was also attenuated by FO addition. In conclusion, the anti-inflammatory effect of n-3 PUFA may be linked to the inhibition of the SphK1/S1P pathway in a rat model of parenteral nutrition and CLP-induced sepsis.

  7. Multiple roles of protein kinase a in arachidonic acid-mediated Ca2+ entry and tumor-derived human endothelial cell migration.

    Science.gov (United States)

    Fiorio Pla, Alessandra; Genova, Tullio; Pupo, Emanuela; Tomatis, Cristiana; Genazzani, Armando; Zaninetti, Roberta; Munaron, Luca

    2010-11-01

    We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca(2+) entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca(2+) signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser(1177), and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI(14-22) completely prevented both AA- and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N(G)-nitro-l-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs. ©2010 AACR.

  8. Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Audrey Schlernitzauer

    Full Text Available Chicoric acid (CA is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans. In L6 myotubes, CA was a potent reactive oxygen species (ROS scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK pathway and displayed various features associated with AMPK activation: CA (a enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx and superoxide dismutase (SOD activities, (b favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 μM. These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

  9. Household fear of deportation in Mexican-origin families: Relation to body mass index percentiles and salivary uric acid.

    Science.gov (United States)

    Martínez, Airín D; Ruelas, Lillian; Granger, Douglas A

    2017-11-01

    Fear of deportation (FOD) is a prevalent concern among mixed-status families. Yet, our understanding of how FOD shapes human health and development is in its infancy. To begin to address this knowledge gap, we examined the relationship between household FOD, body mass index (BMI) percentiles and salivary uric acid (sUA), a biomarker related to oxidative stress/hypertension/metabolic syndrome, among 111 individuals living in Mexican-origin families. Participants were 65 children (2 months-17 years, 49% female) and 46 adults (20-58 years, 71% female) living in 30 Mexican-origin families with at least one immigrant parent in Phoenix, AZ. We recruited families using cluster probability sampling of 30 randomly selected census tracts with a high proportion of Hispanic/Latino immigrants. The head of household completed a survey containing demographic, FOD, and psychosocial measures. All family members provided saliva (later assayed for sUA) and anthropometric measures. Relationships between household FOD, BMI percentile, and sUA levels were estimated using multilevel models. Higher levels of household FOD were associated with lower BMI percentiles and lower sUA levels between families, after controlling for social support and socioeconomic proxies. Key features of the social ecology in which mixed-status families are embedded are associated with individual differences in biological processes linked to increased risk for chronic disease. © 2017 Wiley Periodicals, Inc.

  10. MST kinases in development and disease

    National Research Council Canada - National Science Library

    Thompson, Barry J; Sahai, Erik

    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...

  11. HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

    Science.gov (United States)

    Roy, Ajit; Ranjan, Akash

    2016-02-23

    Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family.

  12. Global trophic position comparison of two dominant mesopelagic fish families (Myctophidae, Stomiidae using amino acid nitrogen isotopic analyses.

    Directory of Open Access Journals (Sweden)

    C Anela Choy

    Full Text Available The δ(15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ(15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ(15N values. Regional differences in the δ(15N values of phenylalanine confirmed that bulk tissue δ(15N values reflect region-specific water mass biogeochemistry controlling δ(15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP for lanternfishes (family Myctophidae (AA-TP ∼2.9 largely align with expectations from stomach content studies (TP ∼3.2, while AA-TPs for dragonfishes (family Stomiidae (AA-TP ∼3.2 were lower than TPs derived from stomach content studies (TP∼4.1. We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure.

  13. The maize (Zea mays ssp. mays var. B73 genome encodes 33 members of the purple acid phosphatase gene family

    Directory of Open Access Journals (Sweden)

    Eliécer eGonzález Muñoz

    2015-05-01

    Full Text Available Purple acid phosphatases (PAPs play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73 reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members.

  14. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    KAUST Repository

    Yunta, Cristina

    2011-11-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  15. Principal transcriptional regulation and genome-wide system interactions of the Asp-family and aromatic amino acid networks of amino acid metabolism in plants.

    Science.gov (United States)

    Less, Hadar; Angelovici, Ruthie; Tzin, Vered; Galili, Gad

    2010-10-01

    Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energy-associated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058-2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156-167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain

  16. The Gibberellic Acid Stimulated-Like gene family in maize and its role in lateral root development.

    Science.gov (United States)

    Zimmermann, Roman; Sakai, Hajime; Hochholdinger, Frank

    2010-01-01

    In an approach to study lateral root development in monocots, genome-wide searches for homologs of the Gibberellic Acid Stimulated Transcript-like (GAST-like) gene family in rice (Oryza sativa) and maize (Zea mays) were carried out. Six novel GAST-like genes in rice and 10 members of the gene family in maize, which were designated ZmGSL (for Z. mays Gibberellic Acid Stimulated-Like), were identified. The ZmGSL family encodes small proteins of 75 to 128 amino acids, which are characterized by a conserved 59 to 64 amino acid C-terminal domain. Within this domain, 17 amino acids, including 12 cysteines, are perfectly conserved. The transcript of the ZmGSL1 gene is differentially spliced into the alternative variants ZmGSL1a and ZmGSL1b, the latter of which is translated into a premature protein that lacks the C-terminal domain. The presence of an additional N-terminal cleavable signal sequence in eight of the 10 ZmGSL proteins suggests that they are secreted into the extracellular matrix. In-depth root-specific gene expression analyses carried out in the wild type and the lateral root mutants lrt1 and rum1 suggest a role for ZmGSL genes in early lateral root development, which is likely regulated by gibberellic acid. Expression patterns of ZmGSL1a and ZmGSL1b propose antagonistic functions of these splice variants during early lateral root formation.

  17. Carbon isotopes in otolith amino acids identify residency of juvenile snapper (Family: Lutjanidae) in coastal nurseries

    Science.gov (United States)

    McMahon, K. W.; Berumen, M. L.; Mateo, I.; Elsdon, T. S.; Thorrold, S. R.

    2011-12-01

    This study explored the potential for otolith geochemistry in snapper (Family: Lutjanidae) to identify residency in juvenile nursery habitats with distinctive carbon isotope values. Conventional bulk otolith and muscle stable isotope analyses (SIA) and essential amino acid (AA) SIA were conducted on snapper collected from seagrass beds, mangroves, and coral reefs in the Red Sea, Caribbean Sea, and Pacific coast of Panama. While bulk stable isotope values in otoliths showed regional differences, they failed to distinguish nursery residence on local scales. Essential AA δ13C values in otoliths, on the other hand, varied as a function of habitat type and provided a better tracer of residence in different juvenile nursery habitats than conventional bulk otolith SIA alone. A strong linear relationship was found between paired otolith and muscle essential AA δ13C values regardless of species, geographic region, or habitat type, indicating that otolith AAs recorded the same dietary information as muscle AAs. Juvenile snapper in the Red Sea sheltered in mangroves but fed in seagrass beds, while snapper from the Caribbean Sea and Pacific coast of Panama showed greater reliance on mangrove-derived carbon. Furthermore, compound-specific SIA revealed that microbially recycled detrital carbon, not water-column-based new phytoplankton carbon, was the primary carbon source supporting snapper production on coastal reefs of the Red Sea. This study presented robust tracers of juvenile nursery residence that will be crucial for reconstructing ontogenetic migration patterns of fishes among coastal wetlands and coral reefs. This information is key to determining the importance of nursery habitats to coral reef fish populations and will provide valuable scientific support for the design of networked marine-protected areas.

  18. Circulating levels of fatty acid-binding protein family and metabolic phenotype in the general population.

    Directory of Open Access Journals (Sweden)

    Shutaro Ishimura

    Full Text Available OBJECTIVE: Fatty acid-binding proteins (FABPs are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population. METHODS: From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1~5 in their serum samples were assayed. RESULTS: Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR, but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT, blood pressure (BP, and brain natriuretic peptide (BNP for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC, BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP, and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP. CONCLUSIONS: Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.

  19. Carbon isotopes in otolith amino acids identify residency of juvenile snapper (Family: Lutjanidae) in coastal nurseries

    KAUST Repository

    McMahon, Kelton

    2011-08-26

    This study explored the potential for otolith geochemistry in snapper (Family: Lutjanidae) to identify residency in juvenile nursery habitats with distinctive carbon isotope values. Conventional bulk otolith and muscle stable isotope analyses (SIA) and essential amino acid (AA) SIA were conducted on snapper collected from seagrass beds, mangroves, and coral reefs in the Red Sea, Caribbean Sea, and Pacific coast of Panama. While bulk stable isotope values in otoliths showed regional differences, they failed to distinguish nursery residence on local scales. Essential AA δ13C values in otoliths, on the other hand, varied as a function of habitat type and provided a better tracer of residence in different juvenile nursery habitats than conventional bulk otolith SIA alone. A strong linear relationship was found between paired otolith and muscle essential AA δ13C values regardless of species, geographic region, or habitat type, indicating that otolith AAs recorded the same dietary information as muscle AAs. Juvenile snapper in the Red Sea sheltered in mangroves but fed in seagrass beds, while snapper from the Caribbean Sea and Pacific coast of Panama showed greater reliance on mangrove-derived carbon. Furthermore, compound-specific SIA revealed that microbially recycled detrital carbon, not water-column-based new phytoplankton carbon, was the primary carbon source supporting snapper production on coastal reefs of the Red Sea. This study presented robust tracers of juvenile nursery residence that will be crucial for reconstructing ontogenetic migration patterns of fishes among coastal wetlands and coral reefs. This information is key to determining the importance of nursery habitats to coral reef fish populations and will provide valuable scientific support for the design of networked marine-protected areas. © 2011 Springer-Verlag.

  20. Exploring some of the physico-chemical properties of the LAMMER protein kinase DOA of Drosophila.

    Science.gov (United States)

    Farkas, Robert; Kováciková, Michaela; Liszeková, Denisa; Beno, Milan; Danis, Peter; Rabinow, Leonard; Chase, Bruce A; Raska, Ivan

    2009-01-01

    Members of the highly conserved LAMMER family of protein kinases have been described in all eukaryotes. LAMMER kinases possess markedly similar peptide motifs in their kinase catalytic subdomains that are responsible for phosphotransfer and substrate interaction, suggesting that family members serve similar functions in widely diverged species. This hypothesis is supported by their phosphorylation of SR and SR-related proteins in diverged species. Here we describe a 3-dimensional homology model of the catalytic domain of DOA, a representative LAMMER kinase, encoded by the Drosophila locus Darkener of apricot (Doa). Homology modeling of DOA based on a Sky1p template revealed a highly conserved structural framework within conserved core regions. These adopt typical kinase folding like that of other protein kinases. However, in contrast to Sky1p, some structural features, such as those in helix alphaC suggest that the DOA kinase is not a constitutively active enzyme but requires activation. This may occur by phosphorylation within an activation loop that forms a broad turn and in which interactions between the side chains occur across the loop. The fold of the activation loop is stabilized through interactions with residues in the C-terminal tail, which is not part of the conserved kinase core and is variable among protein kinases. Immediately following the activation loop in the segment between the beta9 sheet and helix alphaF is a P + 1 loop. The electrostatic surface potential of the DOA substrate-binding groove is largely negative, as it is in other known SR protein kinases, suggesting that DOA substrates must be basic. All differences between D. melanogaster and other Drosophila species are single amino acid changes situated in regions outside of any alpha-helices or beta-sheets, and after modeling these had absolutely no visible effect on protein structure. The absence of evolved amino acid changes among 12 Drosophila species that would cause at least

  1. MAP kinases nomenclature: Time for curation.

    Science.gov (United States)

    Moustafa, Khaled

    2017-12-02

    The nomenclature of Mitogen Activated Protein Kinases (MAPKs) takes different formats composed of symbols, prefixes, suffixes, or descriptive acronyms of their functions that sometimes lead to confusion and make the indexed information redundant and inconsistent. To avoid such redundancy and reduce confusion, a curation of the terminology of MAP kinase families, and that of other protein families that present similar nomenclature issues, is required. Some arguable suggestions are presented here toward this goal.

  2. MST kinases in development and disease.

    Science.gov (United States)

    Thompson, Barry J; Sahai, Erik

    2015-09-14

    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. © 2015 Thompson and Sahai.

  3. Control of cerebellar long-term potentiation by P-Rex-family guanine-nucleotide exchange factors and phosphoinositide 3-kinase.

    Directory of Open Access Journals (Sweden)

    Claire Jackson

    2010-08-01

    Full Text Available Long-term potentiation (LTP at the parallel fibre-Purkinje cell synapse in the cerebellum is a recently described and poorly characterized form of synaptic plasticity. The induction mechanism for LTP at this synapse is considered reciprocal to "classical" LTP at hippocampal CA1 pyramidal neurons: kinases promote increased trafficking of AMPA receptors into the postsynaptic density in the hippocampus, whereas phosphatases decrease internalization of AMPA receptors in the cerebellum. In the hippocampus, LTP occurs in overlapping phases, with the transition from early to late phases requiring the consolidation of initial induction processes by structural re-arrangements at the synapse. Many signalling pathways have been implicated in this process, including PI3 kinases and Rho GTPases.We hypothesized that analogous phases are present in cerebellar LTP, and took as the starting point for investigation our recent discovery that P-Rex--a Rac guanine nucleotide exchange factor which is activated by PtdIns(3,4,5P(3--is highly expressed in mouse cerebellar Purkinje neurons and plays a role in motor coordination. We found that LTP evoked at parallel fibre synapses by 1 Hz stimulation or by NO donors was not sustained beyond 30 min when P-Rex was eliminated or Rac inhibited, suggesting that cerebellar LTP exhibits a late phase analogous to hippocampal LTP. In contrast, inhibition of PI3 kinase activity eliminated LTP at the induction stage.Our data suggest that a PI3K/P-Rex/Rac pathway is required for late phase LTP in the mouse cerebellum, and that other PI3K targets, which remain to be discovered, control LTP induction.

  4. Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis

    Directory of Open Access Journals (Sweden)

    Rawat Richa

    2011-05-01

    Full Text Available Abstract Background Cyclopropane fatty acids (CPA have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory. Results Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. Conclusions In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model

  5. The aspartate-family pathway of plants: linking production of essential amino acids with energy and stress regulation.

    Science.gov (United States)

    Galili, Gad

    2011-02-01

    The Asp family pathway of plants is highly important from a nutritional standpoint because it leads to the synthesis of the four essential amino acids Lys, Thr, Met and Ile. These amino acids are not synthesized by human and its monogastric livestock and should be supplemented in their diets. Among the Asp-family amino acids, Lys is considered as the nutritionally most important essential amino acid because its level is most limiting in cereal grains, representing the largest source of plant foods and feeds worldwide. Metabolic engineering approaches led to significant increase in Lys level in seeds by enhancing its synthesis and reducing its catabolism. However, results from the model plant Arabidopsis showed that this approach may retard seed germination due to a major negative effect on the levels of a number of TCA cycle metabolites that associate with cellular energy. In the present review, we discuss the regulatory metabolic link of the Asp-family pathway with the TCA cycle and its biological significance upon exposure to stress conditions that cause energy deprivation. In addition, we also discuss how deep understanding of the regulatory metabolic link of the Asp-family pathway with energy and stress regulation can be used to improve Lys level in seeds of important crop species, minimizing the interference with the cellular energy status and plant-stress interaction. This review thus provides an example showing how deep understanding the inter-regulation of metabolism with plant stress physiology can lead to successful nutritional improvements with minimal negative effect on plant growth and response to stressful environments.

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

    Indian Academy of Sciences (India)

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

  7. Relationship between Fecal Content of Fatty Acids and Cyclooxygenase mRNA Expression and Fatty Acid Composition in Duodenal Biopsies, Serum Lipoproteins, and Dietary Fat in Colectomized Familial Adenomatous Polyposis Patients

    Directory of Open Access Journals (Sweden)

    K. Almendingen

    2010-01-01

    Full Text Available A few familial adenomatous polyposis studies have focused upon faecal sterols and bile acids but none has analysed the fecal content of fatty acids. We report here findings of an observational study on 29 colectomized familial adenomatous polyposis patients that describe the fecal content of fatty acids, and relate this to the proportions of fatty acids and levels of cyclooxygenase mRNA expression in duodenal biopsies, levels of serum lipoproteins, and diet. In the ileostomy group separately (n=12, the fecal content of arachidonic acid was correlated negatively to the proportions of eicosapentaenoic acid and docosahexaenoic acid in duodenal biopsies. Total serum-cholesterol was negatively correlated to the fecal content of saturates and monounsaturates. The fecal palmitoleic acid/palmitic acid ratio was positively correlated to the levels of cyclooxygease-2 expression in duodenal biopsies.In the ileal-pouch-anal anastomosis group separately (n=17, significant correlations were found between the fecal contents of oleic acid, linoleic acid, and alpha-linolenic acid, and the proportions of myristic acid, oleic acid and eicosaenoic acid in duodenal biopsies. Dietary monounsaturates were positively correlated to different fecal fatty acids. Future studies should focus on molecular mechanisms relevant to fatty acid metabolism, inflammation, and angiogenesis, in addition to nutrition.

  8. [Effects of treating with concentrated sulfuric acid on the seed germination of ten Hibiscus hamabo provenance families].

    Science.gov (United States)

    Wang, Xiao-Xue; Sun, Hai-Jing; Liu, Yun; Chen, Yi-Tai; Feng, Da-Lan; Li, Sha

    2012-11-01

    The seeds from ten Hibiscus hamabo provenance families were treated with concentrated sulfuric acid for different durations (0, 10, 15, and 20 min) , and the seed germination rate, germination energy, and germination index, as well as the seed relative water adsorption rate, soluble sugar and starch contents, and alpha-amylase activity during the germination, were determined, aimed to study the effects of treating with concentrated sulfuric acid on the seed germination of H. hamabo and the differences of the seed germination among different H. hamabo provenance families. After treated with concentrated sulfuric acid, the seed germination rate, germination energy, and germination index increased significantly, and the germination time shortened remarkably. Treating with concentrated sulfuric acid for 15 minutes had the best effect, i. e., the germination rate, germination energy, and germination index were up to 95.7%, 91.3%, and 13.28, respectively, and the germination time was the shortest. The seed germination rate, germination energy, and germination index differed significantly with different provenance families, the highest germination index (15.13) being 2.12 times of the lowest germination index (7.15), and the highest germination energy (98.0%) being 1.77 times of the lowest one (55.5%). Treating with concentrated sulfuric acid accelerated the physiological and biochemical processes of seed germination, and the relative water absorption rate, soluble sugar content, and a-amylase activity were decreased after an initial increase, with the maximum at the early stage of germination. There was a significant negative relationship between the seed starch content and the seed germination index.

  9. Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

    Science.gov (United States)

    Alcántara, Cristina; Bäuerl, Christine; Revilla-Guarinos, Ainhoa; Pérez-Martínez, Gaspar; Monedero, Vicente; Zúñiga, Manuel

    2016-04-01

    A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups. © 2015 John Wiley & Sons Ltd.

  10. Lyn, a tyrosine kinase closely linked to the differentiation status of primary acute myeloid leukemia blasts, associates with negative regulation of all-trans retinoic acid (ATRA) and dihydroxyvitamin D3 (VD3)-induced HL-60 cells differentiation.

    Science.gov (United States)

    Iriyama, Noriyoshi; Yuan, Bo; Hatta, Yoshihiro; Takagi, Norio; Takei, Masami

    2016-01-01

    Lyn, an import member of Src family kinases (SFKs), is supposed to be implicated in acute myeloid leukemia (AML) pathogenesis and development by participation in AML differentiation, yet the details still remain incompletely understood. The expression status of Lyn and its correlation with multiple clinical parameters including cell differentiation degree, different cytogenetic risk classification, and the activity of myeloperoxidase (MPO) were thus investigated. To address the mechanisms underlying the involvement of Lyn in differentiation induction, the effects of dasatinib, an inhibitor for SFKs including Lyn, on the alterations of all-trans retinoic acid (ATRA)- or dihydroxyvitamin D3 (VD3)-induced differentiation, and c-Myc protein expression were investigated. Primary AML blasts were obtained from 31 newly diagnosed AML patients with different French-American-British (FAB) subtypes. The expression of phosphorylated and total Lyn, c-Myc, and CD11b, CD11c and CD15 was analyzed by flow cytometry. The activation of Akt and Erk known to be involved in the regulation of c-Myc expression was investigated using western blotting. Significant higher expression levels of total Lyn were observed in AML patients with favorable cytogenetics, higher MPO activity and FAB M2 subtype. A clear positive correlation between the expression levels of Lyn and differentiation status of primary AML blasts was observed. Dasatinib inhibited the expression of phosphorylated Lyn, and further enhanced the differentiation-inducing activity of ATRA and VD3 in HL-60 cells. Augmented downregulation of c-Myc protein expression was observed in the combination treatment with ATRA, VD3 and dasatinib compared to treatment with each reagent alone in HL-60 cells. The suppression of the activation of Akt and Erk was also observed concomitantly. The expression level of total Lyn is closely linked to the differentiation status of AML blasts. The enhancement of differentiation-inducing activity of ATRA

  11. Salt induction and the partial purification/characterization of phosphoenolpyruvate carboxylase protein-serine kinase from an inducible crassulacean-acid-metabolism (CAM) plant, Mesembryanthemum crystallinum L.

    Science.gov (United States)

    Li, B; Chollet, R

    1994-10-01

    Treatment of the common ice plant (Mesembryanthemum crystallinum) with high salinity caused the well-documented increase in phosphoenolpyruvate carboxylase (PEPC) protein and a concomitant rise in the activity of a Ca(2+)-independent PEPC-kinase (PEPC-PK). When the plants were irrigated with 0.5 M NaCl, PEPC protein level and PEPC-PK activity started to increase after 2 days of treatment and continued to rise for the next 8 days, attaining about a 14- and 8-fold total increase, respectively. This salt-induced PEPC-kinase activity was detected only in leaves harvested from the stressed plants at night. This highly regulated protein kinase was partially purified about 3500-fold from these darkened, salt-stressed plants by sequential fast-protein liquid chromatography on phenyl-Sepharose, blue dextran-agarose, and Superdex 75. The gel-filtration data indicated that the native PEPC-kinase has a molecular weight around 33,000. Complementary analysis by denaturing electrophoresis and subsequent in situ renaturation and assay of PEPC-kinase activity revealed two major PEPC-PK polypeptides with approximate molecular masses of 39 and 32 kDa. The partially purified M. crystallinum PEPC-kinase readily phosphorylated PEPCs purified from maize, M. crystallinum, and tobacco leaves and a recombinant sorghum enzyme. In contrast, this Ca(2+)-independent protein kinase phosphorylated neither a recombinant sorghum mutant PEPC in which the target residue (Ser-8) was changed by site-directed mutagenesis to Asp nor histone III-S, casein, and bovine serum albumin. The optimal pH for PEPC-PK activity was pH 8.0 and this activity was affected by both the substrate (phosphoenolpyruvate) and the negative allosteric effector (L-malate) of PEPC in a pH-dependent manner. Overall, the molecular properties of this highly regulated PEPC-kinase from M. crystallinum are strikingly similar to those reported recently by this laboratory for the reversibly light-activated C4 enzyme from maize (Arch

  12. FAK/src-family dependent activation of the Ste20-like kinase SLK is required for microtubule-dependent focal adhesion turnover and cell migration.

    Directory of Open Access Journals (Sweden)

    Simona Wagner

    2008-04-01

    Full Text Available Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.

  13. Differential control of the NIMA-related kinases, Nek6 and Nek7, by serum stimulation.

    Science.gov (United States)

    Minoguchi, Shigeru; Minoguchi, Mayu; Yoshimura, Akihiko

    2003-02-21

    Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling. Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive. In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions. Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6. Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect. However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased. This is well consistent with the lower activity of Nek7 in cells under normal growth conditions. In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not. These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells.

  14. Effects of doxepin on gene expressions of Bcl-2 family, TNF-α, MAP kinase 14, and Akt1 in the hippocampus of rats exposed to stress.

    Science.gov (United States)

    Reisi, Parham; Eidelkhani, Nastaran; Rafiee, Laleh; Kazemi, Mohammad; Radahmadi, Maryam; Alaei, Hojjatallah

    2017-02-01

    Stress is one of the effective factors in the development of depressive disorders that performs some parts of its effects by affecting hippocampus. Since doxepin has been shown to have neuroprotective effects, in this study, we focused on the effects of doxepin on the expression of involved genes in neuronal survival and plasticity in the rat hippocampus following chronic stress. Male Wistar rats were divided into four groups, the control, the stress, the stress-doxepin 1 mg/kg and the stress-doxepin 5 mg/kg, respectively. To induce stress, the rats were placed within adjustable restraint chambers for 6 h/day, for 21 days. Before daily induction of the stress, rats received an i.p. injection of doxepin. At the end of experiments, expression of Bax, Bad, Bcl-2, tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14) and serine-threonine protein kinase AKT1 genes were detected by reverse transcription polymerase chain reaction (RT-PCR) in the hippocampus. Results showed significant enhancements in expression of Bax, Bad and Bcl-2 genes in the stressed rats, whereas expression of TNF-α, MAPK14, and AKT1 genes didn't show significant differences. Doxepin could decrease the expression of Bax and Bad genes in the stress group, but had no significant effects on the expression of other genes. The present findings indicated that doxepin can probably change the pattern of gene expression in the hippocampus to maintain neurons against destructive effects of stress.

  15. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Promotes Phenotypic Switching from Dedifferentiated to Differentiated Phenotype via Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase in Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Dan, Juhua; Sun, Xin; Li, Wanlu; Zhang, Yun; Li, Xuesong; Xu, Haobo; Li, Zhitao; Tian, Zhen; Guo, Shuyuan; Yao, Jianting; Gao, Weidong; Tian, Ye

    2015-06-01

    Sonodynamic therapy (SDT) has been found to inhibit in-stent restenosis in animal models. However, the mechanism is not fully elucidated. Here, we investigated the effects of 5-aminolevulinic acid (ALA)-mediated SDT (ALA-SDT) on vascular smooth muscle cells (VSMCs), a cause of restenosis, with a focus on SDT-induced phenotypic switching. Serum-induced dedifferentiated VSMCs were cultured with ALA (1 mm, 24 h) and exposed to ultrasound (0.8 W/cm(2)) for 5 min. Results indicated that ALA-SDT inhibited the migration and proliferation of VSMCs and enhanced the expression of differentiated phenotypic markers in VSMCs. Additionally, ALA-SDT increased intracellular reactive oxygen species accumulation and phosphorylated p38 mitogen-activated protein kinase in VSMCs. Inhibition of reactive oxygen species elevation or p38 mitogen-activated protein kinase activity abolished the expression of smooth muscle 22α (SM22α) in VSMCs induced by ALA-SDT. Taken together, these results suggest that ALA-SDT promotes transformation of the VSMC phenotype from the dedifferentiated to differentiated status via reactive oxygen species and activated p38 mitogen-activated protein kinase. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  16. High Temperature During Rice Grain Filling Enhances Aspartate Metabolism in Grains and Results in Accumulation of Aspartate-Family Amino Acids and Protein Components

    Directory of Open Access Journals (Sweden)

    Cheng-gang LIANG

    2013-09-01

    Full Text Available Global warming causes the exacerbation of rice growing environment, which seriously affects rice growth and reproduction, and finally results in the decrease of rice yield and quality. We investigated the activities of aspartate metabolism enzymes in grains, and the contents of Aspartate-family amino acids and protein components to further understand the effects of high temperature (HT on rice nutritional quality during rice grain filling. Under HT, the average activities of aspartate aminotransferase (AAT and aspartokinase (AK in grains significantly increased, the amino acid contents of aspartate (Asp, lysine (Lys, threonine (Thr, methionine (Met and isoleucine (Ile and the protein contents of albumin, globulin, prolamin and glutelin also significantly increased. The results indicated that HT enhanced Asp metabolism during rice grain filling and the enhancement of Asp metabolism might play an important role in the increase of Asp-family amino acids and protein components in grains. In case of the partial appraisal of the change of Asp-family amino acids and protein components under HT, we introduced eight indicators (amino acid or protein content, ratio of amino acid or protein, amino acid or protein content per grain and amino acid or protein content per panicle to estimate the effects of HT. It is suggested that HT during rice grain filling was benefit for the accumulation of Asp-family amino acids and protein components. Combined with the improvement of Asp-family amino acid ratio in grains under HT, it is suggested that HT during grain filling may improve the rice nutritional quality. However, the yields of parts of Asp-family amino acids and protein components were decreased under HT during rice grain filling.

  17. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

    Science.gov (United States)

    Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

    2009-06-01

    Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

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

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

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

  19. ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Hiba El Hajj

    2007-02-01

    Full Text Available Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243-539 possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence.

  20. Mitotic regulation by NIMA-related kinases

    Directory of Open Access Journals (Sweden)

    Blot Joelle

    2007-08-01

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

  1. Mitotic regulation by NIMA-related kinases.

    Science.gov (United States)

    O'regan, Laura; Blot, Joelle; Fry, Andrew M

    2007-08-29

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

  2. CK2: a protein kinase in need of control

    DEFF Research Database (Denmark)

    Guerra, B; Boldyreff, B; Sarno, S

    1999-01-01

    Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regula...... response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential....

  3. Comparative study of rosmarinic acid content in some plants of Labiatae family

    National Research Council Canada - National Science Library

    Shekarchi, Maryam; Hajimehdipoor, Homa; Saeidnia, Soodabeh; Gohari, Ahmad Reza; Hamedani, Morteza Pirali

    2012-01-01

    Plants of Labiatae are used in traditional medicine and phytotherapy. Rosmarinic acid (RA) is a phenolic compound which is found in many genus of Labiatae and exhibits important biological activities...

  4. Neurosteroid Allopregnanolone Suppresses Median Nerve Injury-induced Mechanical Hypersensitivity and Glial Extracellular Signal-regulated Kinase Activation through γ-Aminobutyric Acid Type A Receptor Modulation in the Rat Cuneate Nucleus.

    Science.gov (United States)

    Huang, Chun-Ta; Chen, Seu-Hwa; Lue, June-Horng; Chang, Chi-Fen; Wen, Wen-Hsin; Tsai, Yi-Ju

    2016-12-01

    Mechanisms underlying neuropathic pain relief by the neurosteroid allopregnanolone remain uncertain. We investigated if allopregnanolone attenuates glial extracellular signal-regulated kinase (ERK) activation in the cuneate nucleus (CN) concomitant with neuropathic pain relief in median nerve chronic constriction injury (CCI) model rats. We examined the time course and cellular localization of phosphorylated ERK (p-ERK) in CN after CCI. We subsequently employed microinjection of a mitogen-activated protein kinase kinase (ERK kinase) inhibitor, PD98059, to clarify the role of ERK phosphorylation in neuropathic pain development. Furthermore, we explored the effects of allopregnanolone (by mouth), intra-CN microinjection of γ-aminobutyric acid type A receptor antagonist (bicuculline) or γ-aminobutyric acid type B receptor antagonist (phaclofen) plus allopregnanolone, and allopregnanolone synthesis inhibitor (medroxyprogesterone; subcutaneous) on ERK activation and CCI-induced behavioral hypersensitivity. At 7 days post-CCI, p-ERK levels in ipsilateral CN were significantly increased and reached a peak. PD98059 microinjection into the CN 1 day after CCI dose-dependently attenuated injury-induced behavioral hypersensitivity (withdrawal threshold [mean ± SD], 7.4 ± 1.1, 8.7 ± 1.0, and 10.3 ± 0.8 g for 2.0, 2.5, and 3.0 mM PD98059, respectively, at 7 days post-CCI; n = 6 for each dose). Double immunofluorescence showed that p-ERK was localized to both astrocytes and microglia. Allopregnanolone significantly diminished CN p-ERK levels, glial activation, proinflammatory cytokines, and behavioral hypersensitivity after CCI. Bicuculline, but not phaclofen, blocked all effects of allopregnanolone. Medroxyprogesterone treatment reduced endogenous CN allopregnanolone and exacerbated nerve injury-induced neuropathic pain. Median nerve injury-induced CN glial ERK activation modulated the development of behavioral hypersensitivity. Allopregnanolone attenuated

  5. A novel family of proline/serine-rich proteins, which are phospho-targets of stress-related mitogen-activated protein kinases, differentially regulates growth and pathogen defense in Arabidopsis thaliana.

    Science.gov (United States)

    Palm-Forster, Mieder Anthony Thomas; Eschen-Lippold, Lennart; Uhrig, Joachim; Scheel, Dierk; Lee, Justin

    2017-09-01

    The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein-protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.

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

    Science.gov (United States)

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

  7. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G

    1994-01-01

    are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  8. Antiepileptic Effect of Uncaria rhynchophylla and Rhynchophylline Involved in the Initiation of c-Jun N-Terminal Kinase Phosphorylation of MAPK Signal Pathways in Acute Seizures of Kainic Acid-Treated Rats

    Directory of Open Access Journals (Sweden)

    Hsin-Cheng Hsu

    2013-01-01

    Full Text Available Seizures cause inflammation of the central nervous system. The extent of the inflammation is related to the severity and recurrence of the seizures. Cell surface receptors are stimulated by stimulators such as kainic acid (KA, which causes intracellular mitogen-activated protein kinase (MAPK signal pathway transmission to coordinate a response. It is known that Uncaria rhynchophylla (UR and rhynchophylline (RP have anticonvulsive effects, although the mechanisms remain unclear. Therefore, the purpose of this study is to develop a novel strategy for treating epilepsy by investigating how UR and RP initiate their anticonvulsive mechanisms. Sprague-Dawley rats were administered KA (12 mg/kg, i.p. to induce seizure before being sacrificed. The brain was removed 3 h after KA administration. The results indicate that pretreatment with UR (1.0 g/kg, RP (0.25 mg/kg, and valproic acid (VA, 250 mg/kg for 3 d could reduce epileptic seizures and could also reduce the expression of c-Jun aminoterminal kinase phosphorylation (JNKp of MAPK signal pathways in the cerebral cortex and hippocampus brain tissues. Proinflammatory cytokines interleukin (IL-1β, IL-6, and tumor necrosis factor-α remain unchanged, indicating that the anticonvulsive effect of UR and RP is initially involved in the JNKp MAPK signal pathway during the KA-induced acute seizure period.

  9. Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment.

    Science.gov (United States)

    Klein, Christian L; Rovelli, Giorgio; Springer, Wolfdieter; Schall, Christoph; Gasser, Thomas; Kahle, Philipp J

    2009-11-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO(LRRK2) self-interaction. Interestingly, ROCO(LRRK2) fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO(LRRK2) reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO(LRRK2) fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.

  10. Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803

    OpenAIRE

    Kuwahara, Ayuko; Arisaka, Satomi; Takeya, Masahiro; Iijima, Hiroko; Hirai, Masami Yokota; Osanai, Takashi

    2015-01-01

    Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the ...

  11. Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

    Science.gov (United States)

    Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

  12. JAK and Src tyrosine kinase signaling in asthma.

    Science.gov (United States)

    Tundwal, Kavita; Alam, Rafeul

    2012-06-01

    Tyrosine kinases play a critical role in transducing intracellular signals from the receptors. Many receptors do not have intrinsic tyrosine kinase activity, so they rely on cytosolic and/or membrane-associated tyrosine kinases for initial signal generation. The Src and JAK family kinases are frequently associated with receptors and generate the initial cytosolic signals. These signals are then transduced to other compartments of the cytosol and to the nucleus to elicit a specific cellular response. In this review we focus on these two families of tyrosine kinases and review their involvement in activation of cells that are involved in the pathogenesis of asthma. A Th2-type immune response dominates the processes that lead to the phenotype of asthma. For this reason we give special attention to the tyrosine kinases that are involved in a Th2 response. Further we examine the involvement of tyrosine kinases in activation of mast cells, eosinophils and other cells.

  13. Genome-Wide Identification and Functional Analysis of the Calcineurin B-like Protein and Calcineurin B-like Protein-Interacting Protein Kinase Gene Families in Turnip (Brassica rapa var. rapa

    Directory of Open Access Journals (Sweden)

    Xin Yin

    2017-07-01

    Full Text Available The calcineurin B-like protein (CBL–CBL-interacting protein kinase (CIPK complex has been identified as a primary component in calcium sensors that perceives various stress signals. Turnip (Brassica rapa var. rapa has been widely cultivated in the Qinghai–Tibet Plateau for a century as a food crop of worldwide economic significance. These CBL–CIPK complexes have been demonstrated to play crucial roles in plant response to various environmental stresses. However, no report is available on the genome-wide characterization of these two gene families in turnip. In the present study, 19 and 51 members of the BrrCBL and BrrCIPK genes, respectively, are first identified in turnip and phylogenetically grouped into three and two distinct clusters, respectively. The expansion of these two gene families is mainly attributable to segmental duplication. Moreover, the differences in expression patterns in quantitative real-time PCR, as well as interaction profiles in the yeast two-hybrid assay, suggest the functional divergence of paralog genes during long-term evolution in turnip. Overexpressing and complement lines in Arabidopsis reveal that BrrCBL9.2 improves, but BrrCBL9.1 does not affect, salt tolerance in Arabidopsis. Thus, the expansion of the BrrCBL and BrrCIPK gene families enables the functional differentiation and evolution of some new gene functions of paralog genes. These paralog genes then play prominent roles in turnip's adaptation to the adverse environment of the Qinghai–Tibet Plateau. Overall, the study results contribute to our understanding of the functions of the CBL–CIPK complex and provide basis for selecting appropriate genes for the in-depth functional studies of BrrCBL–BrrCIPK in turnip.

  14. Incorporation of alpha-Ketoglutaric Acid as a Fixed Bed Scrubber Media for the Neutralization of Hydrazine Family Hypergolic Fuels

    Science.gov (United States)

    DeVor, R. W.; Santiago-Maldonado, E.; Parkerson, J. K.

    2010-01-01

    A candidate scrubber media, alpha-ketoglutaric acid (aKGA) adsorbed onto a silica-based substrate was examined as a potential alternative to the hydrazine-family hypergolic fuel neutralization techniques currently utilized at NASA/Kennedy Space Center (KSC). Helvenson et. al. has indicated that aKGA will react with hydrazines to produce non-hazardous, possibly biodegradable products. Furthermore, the authors have previously tested and demonstrated the use of aKGA aqueous solutions as a replacement neutralizing agent for citric acid, which is currently used as a scrubbing agent in liquid scrubbers at KSC. Specific properties examined include reaction efficiency, the loading capacity of aKGA onto various silica substrates, and the comparison of aKGA media performance to that of the citric acid vapor scrubber systems at KSC and a commercial vapor scrubber media. Preliminary investigations showed hydrophobic aerogel particles to be an ideal substrate for the deposition of the aKGA. Current studies have shown that the laboratory produced aKGA-Aerogel absorbent media are more efficient and cost effective than a commercially available fixed bed scrubber media, although much less cost effective than liquid-based citric acid scrubbers (although possibly safer and less labor intensive). A comparison of all three alternative scrubber technologies (liquid aKGA, solid-phase aKGA, and commercially available sorbent materials) is given considering both hypergolic neutralization capabilities and relative costs (as compared to the current citric acid scrubbing technology in use at NASA/KSC).

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

    Directory of Open Access Journals (Sweden)

    Ramaswamy Rakshambikai

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

  16. Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Yuanli Dong

    2015-01-01

    Full Text Available Background: Hypocretin (HCRT signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R] expression. The signaling pathways involved in these processes were also assessed. Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK pathway activation was performed to further assess the signaling mechanism of XSTQ. Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK. Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

  17. Elevated levels of perfluoroalkyl acids in family members of occupationally exposed workers: the importance of dust transfer.

    Science.gov (United States)

    Fu, Jianjie; Gao, Yan; Wang, Thanh; Liang, Yong; Zhang, Aiqian; Wang, Yawei; Jiang, Guibin

    2015-03-20

    The exposure pathways of perfluoroalkyl acids (PFAAs) to humans are still not clear because of the complex living environment, and few studies have simultaneously investigated the bioaccumulative behaviour of different PFAAs in humans. In this study, serum, dust, duplicate diet, and other matrices were collected around a manufacturing plant in China, and homologous series of PFAAs were analysed. PFAA levels in dust and serum of local residents in this area were considerably higher than those in non-polluted area. Although dietary intake was the major exposure pathway in the present study, dust ingestion played an important role in this case. Serum PFAAs in local residents was significantly correlated with dust PFAAs levels in their living or working microenvironment. Serum PFAAs and dust PFAAs were significantly higher in family members of occupational workers (FM) than in ordinary residents (OR) (p < 0.01). After a careful analysis of the PFAAs exposure pathway, a potential pathway in addition to direct dust ingestion was suggested: PFAAs might transferred from occupational worker's clothes to dinners via cooking processes. The bioaccumulative potential of PFHxS and PFOS were higher than other PFAAs, which suggested a substantial difference between the bioaccumulative ability of perfluorinated sulfonic acids and perfluorinated carboxylic acids.

  18. [Cytoplasmic kinase inhibitors].

    Science.gov (United States)

    Mano, Hiroyuki

    2010-10-01

    Protein kinases play essential roles in the regulation of cell proliferation. Point mutations or/and fusions of protein kinases are frequently identified in human cancers, and targeting such activated kinases provides us with a chance to eradicate tumor cells. This was first proved by imatinib mesylate that inhibits ABL tyrosine kinase and, thereby, efficiently kills malignant cells in chronic myeloid leukemia. In addition, other clinical trials are ongoing for kinase inhibitors against EML4--ALK in lung cancer, JAK2 in myeloproliferative disorders and BRAF in malignant melanoma. Early reports indeed reveal that such targeting compounds are promising drugs for human cancers with activated kinases.

  19. Concentrations of the urinary pyrethroid metabolite 3-phenoxybenzoic acid in farm worker families in the MICASA study

    Energy Technology Data Exchange (ETDEWEB)

    Trunnelle, Kelly J., E-mail: kjtrunnelle@ucdavis.edu [Department of Environmental Toxicology, University of California, Davis 1 Shields Avenue, Davis, CA 95616 (United States); Bennett, Deborah H. [Department of Public Health Sciences, University of California, Davis, CA 95616 (United States); Ahn, Ki Chang [Department of Entomology and Cancer Center, University of California, Davis 1 Shields Avenue, Davis, CA 95616 (United States); Schenker, Marc B. [Department of Public Health Sciences, University of California, Davis, CA 95616 (United States); Tancredi, Daniel J. [Department of Pediatrics, University of California, Davis School of Medicine, 4610 X Street Sacramento, CA 95817 (United States); Gee, Shirley J. [Department of Entomology and Cancer Center, University of California, Davis 1 Shields Avenue, Davis, CA 95616 (United States); Stoecklin-Marois, Maria T. [Department of Public Health Sciences, University of California, Davis, CA 95616 (United States); Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis 1 Shields Avenue, Davis, CA 95616 (United States)

    2014-05-01

    Indoor pesticide exposure is a growing concern, particularly from pyrethroids, a commonly used class of pesticides. Pyrethroid concentrations may be especially high in homes of immigrant farm worker families who often live in close proximity to agricultural fields, and are faced with poor housing conditions, causing higher pest infestation and more pesticide use. We investigate exposure of farm worker families to pyrethroids in a study of mothers and children living in Mendota, CA within the population-based Mexican Immigration to California: Agricultural Safety and Acculturation (MICASA) Study. We present pyrethroid exposure based on an ELISA analysis of urinary metabolite 3-phenoxybenzoic acid (3PBA) levels among 105 women and 103 children. The median urinary 3PBA levels (children=2.56 ug/g creatinine, mothers=1.46 ug/g creatinine) were higher than those reported in population based studies for the United States general population, but similar to or lower than studies with known high levels of pyrethroid exposure. A positive association was evident between poor housing conditions and the urinary metabolite levels, showing that poor housing conditions are a contributing factor to the higher levels of 3PBA seen in the urine of these farm worker families. Further research is warranted to fully investigate sources of exposure. - Highlights: • We investigate exposure of farm worker families to pyrethroids. • We present pyrethroid exposure based on an ELISA analysis of urinary 3PBA levels. • 3PBA levels were higher than those reported for the U.S. general population. • Poor housing conditions may be associated with pyrethroid exposure.

  20. Comparative study of rosmarinic acid content in some plants of Labiatae family

    OpenAIRE

    Shekarchi, Maryam; Hajimehdipoor, Homa; Saeidnia, Soodabeh; Gohari, Ahmad Reza; Hamedani, Morteza Pirali

    2012-01-01

    Background: Plants of Labiatae are used in traditional medicine and phytotherapy. Rosmarinic acid (RA) is a phenolic compound which is found in many genus of Labiatae and exhibits important biological activities. Materials and Methods: In this investigation, RA contents of 29 species of Labiatae named Salvia officinalis, Salvia limbata, Salvia virgata, Salvia hypoleuca, Salvia macrosiphon, Salvia choloroleuca, Melissa officinalis, Origanum vulgare, Lavandula angustifolia, Rosmarinus officinal...

  1. QUALITATIVE COMPOSITION AND ORGANI C ACIDS CONTENT IN THE ABOVEGROUN D PART OF PLANTS FRO M FAMILIES LAMIACEAE, ASTERACEAE, APIACEAE AND CHENOPODIACEAE

    Directory of Open Access Journals (Sweden)

    S. M. Marchyshyn

    2016-05-01

    Full Text Available Background. Organic acids are the compounds of aliphatic or aromatic orders, which are widespread in flora and have a wide range of biological activity. We studied the qualitative composition and quantitative contents of organic acids in the aboveground part of some unofficial medicinal plants from families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae is relevant. Objective. The objects of the research are the aboveground part of unofficial medicinal plants from families Lamiaceae, Asteraceae, Apiaceae and Chenopodiaceae. Methods. Identification of organic acids was performed by means of thin-layer and paper chromatography, their content was determined by means of gas chromatography, the quantitative amount of organic acids was defined by titrimetric analysis. Results. In the studied raw plants the quality of organic acids and their total contents were determined (in terms of malic acid. It is established that the maximum content of organic acids is accumulated in the grass Hyssopus officinalis L. (Lamiaceae, and the minimal is in the leaves of Chrysánthemum xhortorum L. variety Apro (Asteraceae. In all studied raw plants the dominance of aliphatic acids (citric, malic, oxalic and malonic was determined by means of gas chromatography. Benzoic is predominant among the aromatic acids. Conclusions. In the studied raw plants the quality of organic acids and their total content were determined. The following results can be used in developing the methods of quality control of the studied raw plants and during the study of new bioactive substances.

  2. MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis

    National Research Council Canada - National Science Library

    Sebastian Bartels; Jeffrey C. Anderson; Manna A. González Besteiro; Alessandro Carreri; Heribert Hirt; Antony Buchala; Jean-Pierre Métraux; Scott C. Peck; Roman Ulm

    2009-01-01

    ...) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae...

  3. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Cho, Hyun-Ji, E-mail: hjcho.dr@gmail.com [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  4. Strong and weak hydrogen bonds in protein-ligand complexes of kinases: a comparative study.

    Science.gov (United States)

    Panigrahi, Sunil K

    2008-05-01

    Strong and weak hydrogen bonds between protein and ligand are analyzed in a group of 233 X-ray crystal structures of the kinase family. These kinases are from both eukaryotic and prokaryotic organisms. The dataset comprises of 44 sub-families, out of which 35 are of human origin and the rest belong to other organisms. Interaction analysis was carried out in the active sites, defined here as a sphere of 10 A radius around the ligand. A majority of the interactions are observed between the main chain of the protein and the ligand atoms. As a donor, the ligand frequently interacts with amino acid residues like Leu, Glu and His. As an acceptor, the ligand interacts often with Gly, and Leu. Strong hydrogen bonds N-H...O, O-H...O, N-H...N and weak bonds C-H...O, C-H...N are common between the protein and ligand. The hydrogen bond donor capacity of Gly in N-H...O and C-H...O interactions is noteworthy. Similarly, the acceptor capacity of main chain Glu is ubiquitous in several kinase sub-families. Hydrogen bonds between protein and ligand form characteristic hydrogen bond patterns (supramolecular synthons). These synthon patterns are unique to each sub-family. The synthon locations are conserved across sub-families due to a higher percentage of conserved sequences in the active sites. The nature of active site water molecules was studied through a novel classification scheme, based on the extent of exposure of water molecules. Water which is least exposed usually participates in hydrogen bond formation with the ligand. These findings will help structural biologists, crystallographers and medicinal chemists to design better kinase inhibitors.

  5. The JAK kinases: not just another kinase drug discovery target.

    Science.gov (United States)

    Wilks, Andrew F

    2008-08-01

    There are four members of the JAK family of protein tyrosine kinases (PTKs) in the human genome. Since their discovery in 1989, great strides have been made in the understanding of their role in normal intracellular signalling. Importantly, their roles in pathologies ranging from cancer to immune deficiencies have placed them front and centre as potential drug targets. The recent discovery of the role of activating mutations in the kinase-like domain (KLD) of JAK2 in the development of polycythemia rubra vera, and the elaboration of KLD mutation as a broader mechanism by which cells might become hyperproliferative has sparked enormous interest in the development of JAK selective drug candidates. I review herein the progress that has been made in the discovery of JAK-targeted inhibitors, and discuss the challenges that face the development of these drugs for use in the clinic.

  6. Phlebotomy treatment for elimination of perfluoroalkyl acids in a highly exposed family: a retrospective case-series.

    Directory of Open Access Journals (Sweden)

    Stephen J Genuis

    Full Text Available BACKGROUND: Perfluoroalkyl acids (PFAAs are a family of commonly used synthetic chemicals that have become widespread environmental contaminants. In human serum, perfluorohexane sulfonate (PFHxS, perflurooctane sulfonate (PFOS, and perfluorooctanoate (PFOA are most frequently detected, in part owing to their long elimination half-lives of between 3.8 yrs (PFOA and 8.5 yrs (PFHxS. These PFAAs also cross the placenta and have been associated with developmental toxicity, and some are considered likely human carcinogens. Interventions to eliminate PFAAs in highly contaminated individuals would reduce future health risks, but minimal research has been conducted on methods to facilitate accelerated human clearance of these persistent substances. METHODS: Six patients with elevated serum concentrations from a single family were treated by intermittent phlebotomy over a 4-5 year period at intervals similar to, or less frequent than what is done for routine blood donation at Canadian Blood Services. The apparent elimination half-life (HLapp for PFHxS, PFOS, and PFOA in this treated population was calculated in each patient and compared to the intrinsic elimination half-lives (HLin from a literature reference population of untreated fluorochemical manufacturing plant retirees (n = 26, age >55 yrs. RESULTS: For all three PFAAs monitored during phlebotomy, HLapp in each of the family members (except the mother, who had a low rate of venesection was significantly shorter than the geometric mean HL measured in the reference population, and in some cases were even shorter compared to the fastest eliminator in the reference population. CONCLUSION: This study suggests significantly accelerated PFAA clearance with regular phlebotomy treatment, but the small sample size and the lack of controls in this clinical intervention precludes drawing firm conclusions. Given the minimal risks of intermittent phlebotomy, this may be an effective and safe clinical

  7. Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.

    Science.gov (United States)

    Tobiason, D M; Buchner, J M; Thiel, W H; Gernert, K M; Karls, A C

    2001-02-01

    Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

  8. 3D-QSAR and virtual screening studies of thiazolidine-2,4-dione analogs: Validation of experimental inhibitory potencies towards PIM-1 kinase

    Science.gov (United States)

    Asati, Vivek; Bharti, Sanjay Kumar; Budhwani, Ashok Kumar

    2017-04-01

    The proviral insertion site in moloney murine leukemia virus (PIM) is a family of serine/threonine kinase of Ca2+-calmodulin-dependent protein kinase (CAMK) group which is responsible for the activation and regulation of cellular transcription and translation. The three isoforms of PIM kinase (PIM-1, PIM-2 and PIM-3) share high homology and functional idleness are widely expressed and involved in a variety of biological processes including cell survival, proliferation, differentiation and apoptosis. Altered expression of PIM-1 kinase correlated with hematologic malignancies and solid tumors. In the present study, atom-based 3D-QSAR, docking and virtual screening studies have been performed on a series of thiazolidine-2,4-dione derivatives as PIM-1 kinase inhibitors. 3D-QSAR and docking approach has shortlisted the most active thiazolidine-2,4-dione derivatives such as 28, 31, 33 and 35 with the incorporation of more than one structural feature in a single molecule. External validations by various parameters and molecular docking studies at the active site of PIM-1 kinase have proved the reliability of the developed 3D-QSAR model. The generated pharmacophore (AADHR.33) from 3D-QSAR study was used for screening of drug like compounds from ZINC database, where ZINC15056464 and ZINC83292944 showed potential binding affinities at the active site amino acid residues (LYS67, GLU171, ASP128 and ASP186) of PIM-1 kinase (PDB ID: "pdb:4DTK").

  9. 90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1

    DEFF Research Database (Denmark)

    Jensen, Claus Antonio Juel; Buch, M B; Krag, T O

    1999-01-01

    90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of th...... of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.......90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation...... involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation...

  10. iHSP-PseRAAAC: Identifying the heat shock protein families using pseudo reduced amino acid alphabet composition.

    Science.gov (United States)

    Feng, Peng-Mian; Chen, Wei; Lin, Hao; Chou, Kuo-Chen

    2013-11-01

    Heat shock proteins (HSPs) are a type of functionally related proteins present in all living organisms, both prokaryotes and eukaryotes. They play essential roles in protein-protein interactions such as folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. Their dysfunction may cause various life-threatening disorders, such as Parkinson's, Alzheimer's, and cardiovascular diseases. Based on their functions, HSPs are usually classified into six families: (i) HSP20 or sHSP, (ii) HSP40 or J-class proteins, (iii) HSP60 or GroEL/ES, (iv) HSP70, (v) HSP90, and (vi) HSP100. Although considerable progress has been achieved in discriminating HSPs from other proteins, it is still a big challenge to identify HSPs among their six different functional types according to their sequence information alone. With the avalanche of protein sequences generated in the post-genomic age, it is highly desirable to develop a high-throughput computational tool in this regard. To take up such a challenge, a predictor called iHSP-PseRAAAC has been developed by incorporating the reduced amino acid alphabet information into the general form of pseudo amino acid composition. One of the remarkable advantages of introducing the reduced amino acid alphabet is being able to avoid the notorious dimension disaster or overfitting problem in statistical prediction. It was observed that the overall success rate achieved by iHSP-PseRAAAC in identifying the functional types of HSPs among the aforementioned six types was more than 87%, which was derived by the jackknife test on a stringent benchmark dataset in which none of HSPs included has ≥40% pairwise sequence identity to any other in the same subset. It has not escaped our notice that the reduced amino acid alphabet approach can also be used to investigate other protein classification problems. As a user-friendly web server, iHSP-PseRAAAC is accessible to the public at http

  11. Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1.

    Science.gov (United States)

    Perrais, Michaël; Pigny, Pascal; Copin, Marie-Christine; Aubert, Jean-Pierre; Van Seuningen, Isabelle

    2002-08-30

    The 11p15 mucin genes (MUC2, MUC5AC, MUC5B and MUC6) possess a cell-specific pattern of expression in normal lung that is altered during carcinogenesis. Growth factors of the epidermal growth factor family are known to target key genes that in turn may affect the homeostasis of lung mucosae. Our aim was to study the regulation of the 11p15 mucin genes both at the promoter and protein levels to assess whether their altered expression may represent a key event during lung carcinogenesis. Studies were performed in the mucoepidermoid NCI-H292 lung cancer cell line. Cell treatment with epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or tumor necrosis factor alpha (TNF-alpha) resulted in a dramatic increase of MUC2 and MUC5AC mRNAs levels, promoter activity, and apomucin expression, whereas those of MUC5B and MUC6 were unchanged. pGL3 deletion mutants of MUC2, MUC5AC, and MUC5B promoters were constructed and used in transient transfection assays to characterize EGF- and TGF-alpha-responsive regulatory regions within the promoters. They were located in the -2627/-2097 and -202/-1 regions of MUC2 and MUC5AC promoters, respectively. Finally, we demonstrate that transcription factor Sp1 not only binds and activates MUC2 and MUC5AC promoters but also participates to their EGF- and TGF-alpha-mediated up-regulation. We also show that Sp3 is a strong inhibitor of 11p15 mucin gene transcription. In conclusion, MUC2 and MUC5AC are two target genes of EGFR ligands in lung cancer cells, and up-regulation of these two genes goes through concomitant activation of the EGFR/Ras/Raf/Extracellular Signal-regulated Kinase-signaling pathway and Sp1 binding to their promoters.

  12. MAP kinases in inflammatory bowel disease

    DEFF Research Database (Denmark)

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

    2011-01-01

    The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...

  13. Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide

    Energy Technology Data Exchange (ETDEWEB)

    Jobson, Andrew G.; Lountos, George T.; Lorenzi, Philip L.; Llamas, Jenny; Connelly, John; Cerna, David; Tropea, Joseph E.; Onda, Akikazu; Zoppoli, Gabriele; Kondapaka, Sudhir; Zhang, Guangtao; Caplen, Natasha J.; Cardellina, II, John H.; Yoo, Stephen S.; Monks, Anne; Self, Christopher; Waugh, David S.; Shoemaker, Robert H.; Pommier, Yves; (NIH)

    2010-04-05

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {l_brace}4-[1-(guanidinohydrazone)-ethyl]-phenyl{r_brace}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

  14. Genome-Wide Survey and Characterization of Fatty Acid Desaturase Gene Family in Brassica napus and Its Parental Species.

    Science.gov (United States)

    Xue, Yufei; Chen, Baojun; Wang, Rui; Win, Aung Naing; Li, Jiana; Chai, Yourong

    2018-02-01

    Rapeseed (Brassica napus) is an important oilseed crop worldwide, and fatty acid (FA) compositions determine the nutritional and economic value of its seed oil. Fatty acid desaturases (FADs) play a pivotal role in regulating FA compositions, but to date, no comprehensive genome-wide analysis of FAD gene family in rapeseed and its parent species has been reported. In this study, using homology searches, 84, 45, and 44 FAD genes were identified in rapeseed, Brassica rapa, and Brassica oleracea genomes, respectively. These FAD genes were unevenly located in 17 chromosomes and 2 scaffolds of rapeseed, 9 chromosomes and 1 scaffold of B. rapa, and all the chromosomes of B. oleracea. Phylogenetic analysis showed that the soluble and membrane-bound FADs in the three Brassica species were divided into four and six subfamilies, respectively. Generally, the soluble FADs contained two conserved histidine boxes, while three highly conserved histidine boxes were harbored in membrane-bound FADs. Exon-intron structure, intron phase, and motif composition and position were highly conserved in each FAD subfamily. Putative subcellular locations of FAD proteins in three Brassica species were consistent with those of corresponding known FADs. In total, 25 of simple sequence repeat (SSR) loci were found in FAD genes of the three Brassica species. Transcripts of selected FAD genes in the three species were examined in various organs/tissues or stress treatments from NCBI expressed sequence tag (EST) database. This study provides a critical molecular basis for quality improvement of rapeseed oil and facilitates our understanding of key roles of FAD genes in plant growth and development and stress response.

  15. Theoretical studies of the role of C-terminal cysteines in the process of S-nitrosylation of human Src kinases.

    Science.gov (United States)

    Andre, Fernanda R; dos Santos, Paloma Freire; Rando, Daniela G

    2016-01-01

    Src tyrosine kinases are a family of non-receptor proteins that are responsible for the growth process, cellular proliferation, differentiation and survival. Lack of Src kinase control has been associated with the development of certain human cancers. This family of proteins is constituted of four domains, with SH1 being the kinase or catalytic domain. SH1 also presents three important regulatory sites. Two residues, Tyr416 and Tyr527, are responsible for important phosphorylation processes that lead to, respectively, activation and deactivation of these kinases. More recently, however, a set of four cysteine residues located near the C-terminus-Cys483, Cys487, Cys496 and Cys498-has been associated with the activation of the Src kinases through S-nitrosylation reactions. Particularly, the Cys498 has been specified as a fundamental residue when considering this regulatory mechanism. Aiming to understand the role of these four cysteines in S-nitrosylation, theoretical studies of electrostatic, steric and hydrophobic properties were performed with a sequence of 20 amino acids, enclosing the four cysteine residues under study, extracted from the PDB coordinates of the crystal obtained from the inactive state of Src kinase. Results indicate that Cys498 is buried deeply in the protein, in hydrophobic surroundings in which NO is more likely to suffer decomposition into the electrophilic intermediates known to be responsible for S-nitrosylation reactions. Electronic calculated properties, such as punctual atomic charges, electrostatic potentials and molecular orbital energy, also demonstrated the good nucleophilic potential of Cys498. Graphical Abstract Structure of Src kinase with zoomed area representing the 20 amino acids comprising the CC motif extracted from the whole protein structure. Right upper panel Electrostatic potential map, right lower panel hydrophilic map in anterior view.

  16. [Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].

    Science.gov (United States)

    Chakravorty, S; Sarkar, S; Gachhui, R

    2015-01-01

    The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.

  17. Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth: Roles of cyclic AMP and p38 mitogen-activated protein kinase

    OpenAIRE

    Liang, Yu-Chih; Wu, Chih-Hsiung; Chu, Jan-Show; Wang, Chung-Kwe; Hung, Ling-Fang; Wang, Ying-Jan; Ho, Yuan-Soon; Chang, Jan-Gowth; Lin, Shyr-Yi

    2005-01-01

    AIM: Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process.

  18. Diacylglycerol kinases in T cell tolerance and effector function

    Directory of Open Access Journals (Sweden)

    Shelley S Chen

    2016-11-01

    Full Text Available Diacylglycerol kinases (DGKs are a family of enzymes that regulate the relative levels of diacylglycerol (DAG and phosphatidic acid (PA in cells by phosphorylating DAG to produce PA. Both DAG and PA are important second messengers cascading T cell receptor (TCR signal by recruiting multiple effector molecules such as RasGRP1, PKC, and mTOR. Studies have revealed important physiological functions of DGKs in the regulation of receptor signaling and the development and activation of immune cells. In this review, we will focus on recent progresses in our understanding of two DGK isoforms,  and , in CD8 T effector and memory cell differentiation, regulatory T cell development and function, and invariant NKT cell development and effector lineage differentiation.

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  20. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

  1. Myotonia congenita and myotonic dystrophy in the same family: coexistence of a CLCN1 mutation and expansion in the CNBP (ZNF9) gene

    DEFF Research Database (Denmark)

    Sun, C; Van Ghelue, M; Tranebjaerg, L

    2010-01-01

    channel genes SCN4A and CLCN1, the myotonic dystrophy protein kinase (DMPK) gene, and the CCHC-type zinc finger, nucleic acid binding protein gene CNBP. Additional gene(s) and/or modifying factor(s) remain to be identified. In this study, we investigated a large Norwegian family with clinically different...

  2. Ursolic acid and luteolin-7-glucoside improves rat plasma lipid profile and increases liver glycogen content through glycogen synthase kinase-3

    OpenAIRE

    Azevedo, Marisa; Camsari, Çagri; Sá, Carla M.; Lima, Cristóvão F.; Ferreira, Manuel Fernandes; Wilson, Cristina Pereira

    2010-01-01

    Documento submetido para revisão pelos pares. A publicar em Phytotherapy Research. ISSN 0951-418X In the present study, two phytochemicals – ursolic acid (UA) and luteolin-7-glucoside (L7G) – were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profi le (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucos...

  3. Caenorhabditis elegans unc-82 encodes a serine/threonine kinase important for myosin filament organization in muscle during growth.

    Science.gov (United States)

    Hoppe, Pamela E; Chau, Johnnie; Flanagan, Kelly A; Reedy, April R; Schriefer, Lawrence A

    2010-01-01

    Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.

  4. Effects of Salicylic Acid on Some Morphophysiological Characteristics of Border Flowers from Asteraceae Family under Water Deficit

    Directory of Open Access Journals (Sweden)

    majid zargarian

    2017-02-01

    Full Text Available Introduction: In order to improve physical and mental human healthy, development of the urban area landscape is necessary. Water deficit is one of the most important problems in arid and semi- arid regions. Water deficit causes to reduce growth of different plants parts, including roots, aerial parts, leaf area, height, dry weight, proteins, amino acids and chlorophyll. Using drought tolerance plantsisone solution. Moreover, one physiological method to decrease the hazardous effects of environmental stresses on plants is to applyplant growth regulators such as salicylic acid (SA. SA is one of the plant phenolic compounds in low amounts (mg/g wet weight or lower. This hormone can perform an important role in resistance to stresses. Asteraceae is one of the biggest plant families with over 20,000 species which is distributed worldwide in moderate and tropical regions. A lot of border flowers of this family are widely used in landscaping. Toselect drought tolerance flowers and application of materials that reducing stress effects, this experiment was conducted to investigate the effects of salicylic acid on some morphological and physiological characteristics of five border flowers of this family. Materials and Methods: The field experiment was conducted as split plot on randomized complete blocks design with four replications in which factors included two levels of water deficit (50, 100% FC as main plot and two levels of SA (0, 1mmol/L-1 as sub plot in the horticulturedepartment of faculty of Agriculture atFerdowsi University of Mashhad. In this investigation five border flowers (Ageratum houstonianum‘Blue Danube’, Tagetserecta ‘Discovery Yellow’, Tagetspatula‘Hero Harmony’, Zinnia elegans ‘Lilliput rose’andCallistephuschinensis ‘Milady Mix’ of Asteraceae family were chosen for study. The soil mixture was analyzed before starting the test. According to the result of physicochemical analysis of the soil, it had loamy texture

  5. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  6. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

    Science.gov (United States)

    Gavi, Shai; Shumay, Elena; Wang, Hsien-yu; Malbon, Craig C

    2006-03-01

    G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

  7. Fyn kinase regulates translation in mammalian mitochondria.

    Science.gov (United States)

    Koc, Emine C; Miller-Lee, Jennifer L; Koc, Hasan

    2017-03-01

    Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known. Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches. Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes. Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. The Multiple DSF-family QS Signals are Synthesized from Carbohydrate and Branched-chain Amino Acids via the FAS Elongation Cycle

    Science.gov (United States)

    Zhou, Lian; Yu, Yonghong; Chen, Xiping; Diab, Abdelgader Abdeen; Ruan, Lifang; He, Jin; Wang, Haihong; He, Ya-Wen

    2015-01-01

    Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have identified RpfF as a key enzyme for the biosynthesis of DSF family signals, many questions in their biosynthesis remain to be addressed. In this study with the phytopathogen Xanthomonas campestris pv. campestris (Xcc), we show that Xcc produces four DSF-family signals (DSF, BDSF, CDSF and IDSF) during cell culture, and that IDSF is a new functional signal characterized as cis-10-methyl-2-dodecenoic acid. Using a range of defined media, we further demonstrate that Xcc mainly produces BDSF in the presence of carbohydrates; leucine and valine are the primary precursor for DSF biosynthesis; isoleucine is the primary precursor for IDSF biosynthesis. Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF. Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals. Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals. PMID:26289160

  9. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa

    Directory of Open Access Journals (Sweden)

    Talevich Eric

    2011-11-01

    Full Text Available Abstract Background The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. Results We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK, calcium-dependent protein kinase (CDPK and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. Conclusions We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and

  10. Nintedanib, a triple tyrosine kinase inhibitor, attenuates renal fibrosis in chronic kidney disease.

    Science.gov (United States)

    Liu, Feng; Wang, Li; Qi, Hualin; Wang, Jun; Wang, Yi; Jiang, Wei; Xu, Liuqing; Liu, Na; Zhuang, Shougang

    2017-08-15

    Nintedanib (BIBF1120) is a triple kinase inhibitor of platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptors (FGFR), vascular endothelial growth factor receptor (VEGFR), and Src family kinase, which has recently been approved by FDA to treat idiopathic pulmonary fibrosis. Whether it affects renal fibrosis remains unknown. Here, we demonstrated that administration of nintedanib immediately or 3 days after unilateral ureteral obstruction (UUO) injury and with folic acid (FA) injection attenuated renal fibrosis and inhibited activation of renal interstitial fibroblasts. Delayed administration of nintedanib also partially reversed established renal fibrosis. Treatment with nintedanib blocked UUO-induced phosphorylation of PDGFRβ, FGFR1, FGFR2, VEGFR2, and several Src family kinases including Src, Lck, Lyn as well as activation of signal transducer and activator of transcription-3 (STAT3), nuclear factor-κB (NF-κB), and Smad-3 in the kidney. Furthermore, nintedanib inhibited UUO-elicited renal proinflammatory cytokine expression and macrophage infiltration. These data indicate that nintedanib is a potent anti-fibrotic agent in the kidney and may hold therapeutic potential as a treatment of chronic fibrotic kidney disease. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-29

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

  12. RAF protein-serine/threonine kinases: Structure and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Roskoski, Robert, E-mail: rrj@brimr.org [Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742 (United States)

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  13. Structure of LP2179, the first representative of Pfam family PF08866, suggests a new fold with a role in amino-acid metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Bakolitsa, Constantina; Kumar, Abhinav; Carlton, Dennis; Miller, Mitchell D.; Krishna, S.Sri; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K.; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Tien, Henry J.; Trout, Christina V.; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A. (Burnham)

    2011-08-17

    The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel {alpha} + {beta} fold comprising two {beta}-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.

  14. Structure of LP2179, the first representative of Pfam family PF08866, suggests a new fold with a role in amino-acid metabolism.

    Science.gov (United States)

    Bakolitsa, Constantina; Kumar, Abhinav; Carlton, Dennis; Miller, Mitchell D; Krishna, S Sri; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elsliger, Marc André; Feuerhelm, Julie; Grzechnik, Slawomir K; Grant, Joanna C; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Marciano, David; McMullan, Daniel; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L; Tien, Henry J; Trout, Christina V; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2010-10-01

    The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+β fold comprising two β-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.

  15. The Gibberellic Acid Stimulated-Like Gene Family in Maize and Its Role in Lateral Root Development1[C][W][OA

    Science.gov (United States)

    Zimmermann, Roman; Sakai, Hajime; Hochholdinger, Frank

    2010-01-01

    In an approach to study lateral root development in monocots, genome-wide searches for homologs of the Gibberellic Acid Stimulated Transcript-like (GAST-like) gene family in rice (Oryza sativa) and maize (Zea mays) were carried out. Six novel GAST-like genes in rice and 10 members of the gene family in maize, which were designated ZmGSL (for Z. mays Gibberellic Acid Stimulated-Like), were identified. The ZmGSL family encodes small proteins of 75 to 128 amino acids, which are characterized by a conserved 59 to 64 amino acid C-terminal domain. Within this domain, 17 amino acids, including 12 cysteines, are perfectly conserved. The transcript of the ZmGSL1 gene is differentially spliced into the alternative variants ZmGSL1a and ZmGSL1b, the latter of which is translated into a premature protein that lacks the C-terminal domain. The presence of an additional N-terminal cleavable signal sequence in eight of the 10 ZmGSL proteins suggests that they are secreted into the extracellular matrix. In-depth root-specific gene expression analyses carried out in the wild type and the lateral root mutants lrt1 and rum1 suggest a role for ZmGSL genes in early lateral root development, which is likely regulated by gibberellic acid. Expression patterns of ZmGSL1a and ZmGSL1b propose antagonistic functions of these splice variants during early lateral root formation. PMID:19926801

  16. The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis☆

    Science.gov (United States)

    Elashry, Abdelnaser; Okumoto, Sakiko; Siddique, Shahid; Koch, Wolfgang; Kreil, David P.; Bohlmann, Holger

    2013-01-01

    The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes. PMID:23831821

  17. Molecular genetic responses to lysergic acid diethylamide include transcriptional activation of MAP kinase phosphatase-1, C/EBP-beta and ILAD-1, a novel gene with homology to arrestins.

    Science.gov (United States)

    Nichols, Charles D; Sanders-Bush, Elaine

    2004-08-01

    We recently demonstrated that the potent hallucinogenic drug lysergic acid diethylamide (LSD) dynamically influences the expression of a small collection of genes within the mammalian prefrontal cortex. Towards generating a greater understanding of the molecular genetic effects of hallucinogens and how they may relate to alterations in behavior, we have identified and characterized expression patterns of a new collection of three genes increased in expression by acute LSD administration. These genes were identified through additional screens of Affymetrix DNA microarrays and examined in experiments to assess dose-response, time course and the receptor mediating the expression changes. The first induced gene, C/EBP-beta, is a transcription factor. The second gene, MKP-1, suggests that LSD activates the MAP (mitogen activated protein) kinase pathway. The third gene, ILAD-1, demonstrates sequence similarity to the arrestins. The increase in expression of each gene was partially mediated through LSD interactions at 5-HT2A (serotonin) receptors. There is evidence of alternative splicing at the ILAD-1 locus. Furthermore, data suggests that various splice isoforms of ILAD-1 respond differently at the transcriptional level to LSD. The genes thus far found to be responsive to LSD are beginning to give a more complete picture of the complex intracellular events initiated by hallucinogens.

  18. Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses.

    Science.gov (United States)

    Pöntinen, Anna; Lindström, Miia; Skurnik, Mikael; Korkeala, Hannu

    2017-08-01

    To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HK deletion mutant strains under heat (42.5 °C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H2O2) stresses. The growth of ΔliaS (Δlmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The ΔvirS (Δlmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of ΔagrC (Δlmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HK mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L. monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L. monocytogenes EGD-e. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Systemic concentrations of short chain fatty acids are elevated in salmonellosis and exacerbation of familial Mediterranean fever

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    Zhanna eKtsoyan

    2016-05-01

    Full Text Available Gut microbiota-produced short chain fatty acids (SCFAs play an important role in the normal human metabolism and physiology. Although the gradients of SCFAs from the large intestine, where they are largely produced, to the peripheral blood as well as the main routes of SCFA metabolism by different organs are known well for the healthy state, there is a paucity of information regarding how these are affected in disease. In particular, how the inflammation caused by infection or autoinflammatory disease affect the concentration of SCFAs in the peripheral venous blood. In this work, we revealed that diseases caused either by infectious agents (two Salmonella enterica serovars, S. Enteritidis and S. Typhimurium or by the exacerbation of an autoinflammatory disease, familial Mediterranean fever (FMF, both result in a significantly elevated systemic concentration of SCFAs. In the case of salmonellosis the concentration of SCFAs in peripheral blood was significantly and consistently higher, from five- to 20-fold, compared to control. In the case of FMF, however, a significant increase of SCFAs in the peripheral venous blood was detected only in the acute phase of the disease, with a lesser impact in remission. It seems counterintuitive that the dysbiotic conditions, with a reduced number of gut microorganisms, produce such an effect. This phenomenon, however, must be appraised within the context of how the inflammatory diseases affect the normal physiology. We discuss a number of factors that may contribute to the leak and persistence of gut-produced SCFAs into the systemic circulation in infectious and autoinflammatory diseases.

  20. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2016-06-01

    Full Text Available This study investigated the potential effects of dehydroeburicoic acid (TT, a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom on membrane glucose transporter 4 (GLUT4 and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4 and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels, fenofibrate (Feno (at 0.25 g/kg body weight, metformin (Metf (at 0.3 g/kg body weight or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%. TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase, an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  1. Two SNF1-Related Protein Kinases from Spinach Leaf Phosphorylate and Inactivate 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, Nitrate Reductase, and Sucrose Phosphate Synthase in Vitro1

    Science.gov (United States)

    Sugden, Christopher; Donaghy, Paul G.; Halford, Nigel G.; Hardie, D. Grahame

    1999-01-01

    We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides. PMID:10318703

  2. Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree.

    Science.gov (United States)

    Guo, Dong; Zhou, Ying; Li, Hui-Liang; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2017-03-23

    Abscisic acid (ABA) is an essential phytohormone involved in diverse physiological processes. Although genome-wide analyses of the ABA receptor PYR/PYL/RCAR (PYL) protein/gene family have been performed in certain plant species, little is known about the ABA receptor protein/gene family in the rubber tree (Hevea brasiliensis). In this study, we identified 14 ABA receptor PYL proteins/genes (designated HbPYL1 through HbPYL14) in the most recent rubber tree genome. A phylogenetic tree was constructed, which demonstrated that HbPYLs can be divided into three subfamilies that correlate well with the corresponding Arabidopsis subfamilies. Eight HbPYLs are highly expressed in laticifers. Five of the eight genes are simultaneously regulated by ABA, jasmonic acid (JA) and ethylene (ET). The identification and characterization of HbPYLs should enable us to further understand the role of ABA signal in the rubber tree.

  3. Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth: roles of cyclic AMP and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Liang, Yu-Chih; Wu, Chih-Hsiung; Chu, Jan-Show; Wang, Chung-Kwe; Hung, Ling-Fang; Wang, Ying-Jan; Ho, Yuan-Soon; Chang, Jan-Gowth; Lin, Shyr-Yi

    2005-05-07

    Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process. We investigated the expression and regulation of FACL4 in HCC, adjacent non-tumorous liver tissues, and cell lines. In HCC patients, we demonstrated that FACL4 gene expression was markedly elevated in the cancerous tissues than in the adjacent non-cancerous liver tissues. In addition, several human hepatoma cell lines, including Hep3B and HepG2, expressed high levels of FACL4. Stable overexpression of FACL4 knockdown plasmids (small interfering RNA, siRNA) to Hep3B cells significantly decreased FACL4 expression and subsequently limited the cell proliferation. Treatment of Hep3B cells with 8-bromo-cAMP and SB203508 (p38 MAPK inhibitor) significantly suppressed the FACL4 expression. FACL4 is involved in the HCC tumorigenesis and both cAMP and p38 MAPK pathways are associated with the regulation of FACL4 in HCC.

  4. A framework for classification of prokaryotic protein kinases.

    Science.gov (United States)

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-05-26

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

  5. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

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

  6. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

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    Shuilin eHe

    2015-09-01

    Full Text Available The tripartite mitogen-activated protein kinase (MAPK signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK were performed in pepper. A total of 19 pepper MAPK (CaMAPKs genes and five MAPKK (CaMAPKKs genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

  7. How a microbial drug transporter became essential for crop cultivation on acid soils: aluminium tolerance conferred by the multidrug and toxic compound extrusion (MATE) family.

    Science.gov (United States)

    Magalhaes, Jurandir V

    2010-07-01

    Aluminium (Al) toxicity is a major agricultural constraint for crop cultivation on acid soils, which comprise a large portion of the world's arable land. One of the most widely accepted mechanisms of Al tolerance in plants is based on Al-activated organic acid release into the rhizosphere, with organic acids forming stable, non-toxic complexes with Al. This mechanism has recently been validated by the isolation of bona-fide Al-tolerance genes in crop species, which encode membrane transporters that mediate Al-activated organic acid release leading to Al exclusion from root apices. In crop species such as sorghum and barley, members in the multidrug and toxic compound extrusion (MATE) family underlie Al tolerance by a mechanism based on Al-activated citrate release. The study of Al tolerance in plants as conferred by MATE family members is in its infancy. Therefore, much is yet to be discovered about the functional diversity and evolutionary dynamics that led MATE proteins to acquire transport properties conducive to Al tolerance in plants. In this paper we review the major characteristics of transporters in the MATE family and will relate this knowledge to Al tolerance in plants. The MATE family is clearly extremely flexible with respect to substrate specificity, which raises the possibility that Al tolerance as encoded by MATE proteins may not be restricted to Al-activated citrate release in plant species. There are also indications that regulatory loci may be of pivotal importance to fully explore the potential for Al-tolerance improvement based on MATE genes.

  8. Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR

    Science.gov (United States)

    Schierhorn, Kristina L.; Jolmes, Fabian; Bespalowa, Julia; Saenger, Sandra; Peteranderl, Christin; Dzieciolowski, Julia; Mielke, Maja; Budt, Matthias; Pleschka, Stephan; Herrmann, Andreas; Herold, Susanne

    2017-01-01

    ABSTRACT The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR−/− mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional

  9. Unravelling the diversity of the cyclopiazonic acid family of mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS

    Science.gov (United States)

    Cyclopiazonic acid (a-cyclopiazonic acid, a-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures, approximately 40 years ago, its contribution to the...

  10. Biological Functions of ilvC in Branched-Chain Fatty Acid Synthesis and Diffusible Signal Factor Family Production in Xanthomonas campestris

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    Kai-Huai Li

    2017-12-01

    Full Text Available In bacteria, the metabolism of branched-chain amino acids (BCAAs is tightly associated with branched-chain fatty acids (BCFAs synthetic pathways. Although previous studies have reported on BCFAs biosynthesis, more detailed associations between BCAAs metabolism and BCFAs biosynthesis remain to be addressed. In this study, we deleted the ilvC gene, which encodes ketol-acid reductoisomerase in the BCAAs synthetic pathway, from the Xanthomonas campestris pv. campestris (Xcc genome. We characterized gene functions in BCFAs biosynthesis and production of the diffusible signal factor (DSF family signals. Disruption of ilvC caused Xcc to become auxotrophic for valine and isoleucine, and lose the ability to synthesize BCFAs via carbohydrate metabolism. Furthermore, ilvC mutant reduced the ability to produce DSF-family signals, especially branched-chain DSF-family signals, which might be the main reason for Xcc reduction of pathogenesis toward host plants. In this report, we confirmed that BCFAs do not have major functions in acclimatizing Xcc cells to low temperatures.

  11. Comparative analysis of P450 signature motifs EXXR and CXG in the large and diverse kingdom of fungi: identification of evolutionarily conserved amino acid patterns characteristic of P450 family.

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    Khajamohiddin Syed

    Full Text Available Cytochrome P450 monooxygenases (P450s are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research.

  12. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  13. Pyruvate kinase blood test

    Science.gov (United States)

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

  14. Transcriptional profiling of the PDR gene family in rice roots in response to plant growth regulators, redox perturbations and weak organic acid stresses.

    Science.gov (United States)

    Moons, Ann

    2008-12-01

    The role of plant pleiotropic drug resistance (PDR) type ATP-binding cassette (ABC) transporters remains poorly understood. We characterized the expression of the rice pleiotropic drug resistance (PDR) gene family in roots, where PDR transporters are believed to have major functions. A prototypical oligonucleotide array was developed containing 70-mers chosen in the gene-specific 3' untranslated regions of the rice PDR genes, other full-molecule rice ABC transporter genes and relevant marker genes. Jasmonates, which are involved in plant defense and secondary metabolism, proved major inducers of PDR gene expression. Over half of the PDR genes were JA-induced in roots of rice; OsPDR9 to the highest level. Salicylic acid, involved in plant pathogen defense, markedly induced the expression of OsPDR20. OsPDR20 was cDNA cloned and characterized. Abscisic acid, typically involved in water deficit responses, particularly induced OsPDR3 in roots and shoot and OsPDR6 in rice leaves. OsPDR9 and OsPDR20 were furthermore up-regulated in response to dithiothreitol- or glutathione-induced redox perturbations. Exogenous application of the weak organic acids lactic acid, malic acid, and citric acid differentially induced the expression of OsPDR3, OsPDR8, OsPDR9 and OsPDR20 in rice seedling roots. This transcriptional survey represents a guide for the further functional analysis of individual PDR transporters in roots of rice.

  15. Hantzsch-type three-component approach to a new family of carbon-linked glycosyl amino acids. Synthesis of C-glycosylmethyl pyridylalanines.

    Science.gov (United States)

    Dondoni, Alessandro; Massi, Alessandro; Aldhoun, Mohammad

    2007-09-28

    C-Glycosylmethyl pyridylalanines reported in this paper constitute a novel family of glycosyl amino acids that contain a pyridine ring linking the carbohydrate and amino acid residues. These amino acids may serve to prepare nonnatural glycopeptides displaying firmly bound carbohydrate fragments through a rigid and highly stable tether. A viable route to these new hybrid molecules has been opened via thermally induced Hantzsch-type cyclocondensation using an aldehyde-ketoester-enamino ester system. To one of these reagents was attached a C-glycosyl residue, while to another was bound an amino acid fragment. In a one-pot optimized methodology, the dihydropyridine was not isolated while its purification was carried out by removal of unreacted material and side products using polymer-supported scavengers. Then the dihydropyridine (mixture of diastereoisomers) was oxidized by a polymer-bound oxidant to give the target pyridine bearing the two bioactive residues. In this way a range of eight compounds (58-68% yield) was prepared in which the elements of diversity were (i) the gluco and galacto configurations of the pyranose ring, (ii) the alpha- and beta-configurations at the anomeric center, and (iii) the positions of the carbohydrate and amino acid sectors in the pyridine ring. The orthogonal functional group protection in these amino acids allowed their easy incorporation into oligopeptides via sequential amino and carboxylic group coupling.

  16. The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Klopffleisch, Karsten; Issinger, Olaf-Georg

    2008-01-01

    The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2alpha belongs to the CMGC family of...

  17. Venus Kinase Receptors: prospects in signalling and biological functions of these invertebrate receptors

    Directory of Open Access Journals (Sweden)

    Colette eDissous

    2014-05-01

    Full Text Available Venus Kinase Receptors (VKRs form a family of invertebrate receptor tyrosine kinases (RTKs initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors which contain an extracellular Venus Flytrap (VFT structure similar to the ligand binding domain of G Protein Coupled Receptors of class C, and an intracellular Tyrosine Kinase domain close to that of Insulin Receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms, and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. Vkr gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in Schistosoma .mansoni. VKRs are activated by amino-acids, and highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates, nor in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/ control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  18. Sevoflurane Inhibits Glutamate-Aspartate Transporter and Glial Fibrillary Acidic Protein Expression in Hippocampal Astrocytes of Neonatal Rats Through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) Pathway.

    Science.gov (United States)

    Wang, Wei; Lu, Rui; Feng, Da-Yun; Zhang, Hui

    2016-07-01

    The mechanisms underlying general anesthesia-induced neurotoxicity are unclear. Astrocytes have been recognized as important contributors to neuronal development. Until now, the response of the astrocytes to neonatal general anesthetic exposure has been unreported. Postnatal day 7 rats received 2.5% sevoflurane for 6 hours. Expressions of glial fibrillary acidic protein (GFAP) and glutamate-aspartate transporter (GLAST) and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway were detected on days 1, 3, 7, and 14 after sevoflurane inhalation. In addition, cultured astrocytes were exposed to 2.5% sevoflurane for 2 hours and GFAP, GLAST expressions, and JAK/STAT phosphorylation were evaluated. Furthermore, we pharmacologically disrupted JAK/STAT signaling in vivo by treatment with the JAK/STAT inhibitor AG490 and in vitro by treatment with JAK inhibitor I to detect the consequent expression of GFAP and GLAST. Sevoflurane induced a robust decrease of GFAP and GLAST expression in hippocampal tissue compared with sham control groups at 1 to 14 days after sevoflurane exposure. Immunohistochemistry showed colocalization of GFAP, GLAST, and pSTAT3 in the hippocampal CA1 region. Western blot analysis also revealed a significant decrease of pJAK1, pJAK2, and pSTAT3 in the sevoflurane group. In vitro study showed that GFAP, GLAST, pJAK1, pJAK2, and pSTAT3 expressions in cultured astrocytes were remarkably decreased at 24 to 48 hours after sevoflurane treatment. Either AG490 or JAK inhibitor I significantly decreased expressions of GFAP and GLAST in hippocampus or cultured astrocytes. Astrocytic GLAST was inhibited by sevoflurane in the hippocampus of neonatal rats. Inactivation of the JAK/STAT pathway possibly contributes to this effect of sevoflurane. Astrocytic dysfunction induced by sevoflurane may contribute to its neurotoxicity in the developing brain.

  19. Cytoplasmic Localization of Proline, Glutamic Acid, Leucine-rich Protein 1 (PELP1) Induces Breast Epithelial Cell Migration through Up-regulation of Inhibitor of κB Kinase ϵ and Inflammatory Cross-talk with Macrophages.

    Science.gov (United States)

    Girard, Brian J; Knutson, Todd P; Kuker, Bethanie; McDowell, Laura; Schwertfeger, Kathryn L; Ostrander, Julie H

    2017-01-06

    Cytoplasmic localization of proline, glutamic acid, leucine-rich protein 1 (PELP1) is observed in ∼40% of women with invasive breast cancer. In mouse models, PELP1 overexpression in the mammary gland leads to premalignant lesions and eventually mammary tumors. In preliminary clinical studies, cytoplasmic localization of PELP1 was seen in 36% of women at high risk of developing breast cancer. Here, we investigated whether cytoplasmic PELP1 signaling promotes breast cancer initiation in models of immortalized human mammary epithelial cells (HMECs). Global gene expression analysis was performed on HMEC lines expressing vector control, PELP1-wt, or mutant PELP1 in which the nuclear localization sequence was altered, resulting in cytoplasmic localization of PELP1 (PELP1-cyto). Global gene expression analysis identified that PELP1-cyto expression in HMECs induced NF-κB signaling pathways. Western blotting analysis of PELP1-cyto HMECs showed up-regulation of inhibitor of κB kinase ϵ (IKKϵ) and increased phosphorylation of the NF-κB subunit RelB. To determine whether secreted factors produced by PELP1-cyto HMECs promote macrophage activation, THP-1 macrophages were treated with HMEC-conditioned medium (CM). PELP1-cyto CM induced changes in THP-1 gene expression as compared with control cell CM. Double conditioned medium (DCM) from the activated THP-1 cells was then applied to HMECs to determine whether paracrine signaling from PELP1-cyto-activated macrophages could in turn promote migration of HMECs. PELP1-cyto DCM induced robust HMEC migration, which was reduced in DCM from PELP1-cyto HMECs expressing IKKϵ shRNA. Our findings suggest that cytoplasmic localization of PELP1 up-regulates pro-tumorigenic IKKϵ and secreted inflammatory signals, which through paracrine macrophage activation regulates the migratory phenotype associated with breast cancer initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Targeting the HER-kinase axis in cancer.

    Science.gov (United States)

    Gross, Mitchell E; Shazer, Ronald L; Agus, David B

    2004-02-01

    The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases controls critical pathways involved in the differentiation, growth, division, and motility of normal epithelial cells. Most human solid tumors are of epithelial origin. The process of malignant transformation and progression in many cancers may depend on activation of ligands and receptors that function as part of the HER-kinase pathway. This signaling axis has earned increased attention because of the development of antibodies and small-molecule tyrosine kinase inhibitors that specifically target components of the HER-kinase axis for cancer therapy. This review focuses on the basic biology underlying HER-kinase pathway activation and the current state of development for agents that target this axis. In particular, the importance of pan-HER inhibitors is discussed.

  1. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction

    DEFF Research Database (Denmark)

    Frödin, M; Gammeltoft, S

    1999-01-01

    Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), which is activated...... by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1......), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK...

  2. KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily.

    Science.gov (United States)

    Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa; Brunak, Søren; Valencia, Alfonso; Izarzugaza, Jose Mg

    2016-06-23

    The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional annotations from UniProt, Phospho.ELM and FireDB. KinMutRF identifies disease-associated variants satisfactorily (Acc: 0.88, Prec:0.82, Rec:0.75, F-score:0.78, MCC:0.68) when trained and cross-validated with the 3689 human kinase variants from UniProt that have been annotated as neutral or pathogenic. All unclassified variants were excluded from the training set. Furthermore, KinMutRF is discussed with respect to two independent kinase-specific sets of mutations no included in the training and testing, Kin-Driver (643 variants) and Pon-BTK (1495 variants). Moreover, we provide predictions for the 848 protein kinase variants in UniProt that remained unclassified. A public implementation of KinMutRF, including documentation and examples, is available online ( http://kinmut2.bioinfo.cnio.es ). The source code for local installation is released under a GPL version 3 license, and can be downloaded from https://github.com/Rbbt-Workflows/KinMut2 . KinMutRF is capable of classifying kinase variation with good performance. Predictions by KinMutRF compare favorably in a benchmark with other state

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2005-03-18

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