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Sample records for acid kinase family

  1. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

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    Enrique Marcos

    2011-09-01

    Full Text Available Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  2. On the conservation of the slow conformational dynamics within the amino acid kinase family: NAGK the paradigm.

    Science.gov (United States)

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2010-04-08

    N-acetyl-L-glutamate kinase (NAGK) is the structural paradigm for examining the catalytic mechanisms and dynamics of amino acid kinase family members. Given that the slow conformational dynamics of the NAGK (at the microseconds time scale or slower) may be rate-limiting, it is of importance to assess the mechanisms of the most cooperative modes of motion intrinsically accessible to this enzyme. Here, we present the results from normal mode analysis using an elastic network model representation, which shows that the conformational mechanisms for substrate binding by NAGK strongly correlate with the intrinsic dynamics of the enzyme in the unbound form. We further analyzed the potential mechanisms of allosteric signalling within NAGK using a Markov model for network communication. Comparative analysis of the dynamics of family members strongly suggests that the low-frequency modes of motion and the associated intramolecular couplings that establish signal transduction are highly conserved among family members, in support of the paradigm sequence-->structure-->dynamics-->function.

  3. The Src family kinases: distinct functions of c-Src, Yes, and Fyn in the liver.

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    Reinehr, Roland; Sommerfeld, Annika; Häussinger, Dieter

    2013-04-01

    The Src family kinases Yes, Fyn, and c-Src play a pivotal role in regulating diverse liver functions such as bile flow, proteolysis, apoptosis, and proliferation and are regulated by anisoosmotic cell volume changes, death receptor ligands, and bile acids. For example, cell swelling leads to an integrin-sensed and focal adhesion kinase-mediated activation of c-Src-triggering choleresis, proteolysis inhibition, regulatory volume decrease via p38MAPK and proliferation via the activation of the epidermal growth factor receptor and extracellular regulated kinases 1 and 2. In contrast, hepatocyte shrinkage generates an almost instantaneous oxidative stress response that triggers the activation of c-Jun N-terminal kinase and the Src family kinases Fyn and Yes. Whereas Fyn activation mediates cholestasis, Yes triggers CD95 activation and apoptosis. This review will discuss the role of Src family kinases in the regulation of liver function with emphasis on their role in osmo-signaling and bile acid signaling.

  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. Protein Kinase D family kinases: roads start to segregate.

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    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue.

  6. 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......, 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....

  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......, 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....

  8. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

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    Wu, Qiuling; Lv, Tingting; Chen, Yan; Wen, Lu; Zhang, Junli; Jiang, Xudong; Liu, Fang

    2015-07-01

    The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

  9. Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

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    Falk, Matthew D.; Liu, Wei; Bolaños, Ben; Unsal-Kacmaz, Keziban; Klippel, Anke; Grant, Stephan; Brooun, Alexei; Timofeevski, Sergei

    2014-01-01

    The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors. PMID:27919031

  10. p21-activated kinase family: promising new drug targets

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    Huynh N

    2015-05-01

    Full Text Available Nhi Huynh, Hong He Department of Surgery, University of Melbourne, Austin Health, Melbourne, VIC, Australia Abstract: The p21-activated kinase (PAK family of serine/threonine protein kinases are downstream effectors of the Rho family of GTPases. PAKs are frequently upregulated in human diseases, including various cancers, and their overexpression correlates with disease progression. Current research findings have validated important roles for PAKs in cell proliferation, survival, gene transcription, transformation, and cytoskeletal remodeling. PAKs are shown to act as a converging node for many signaling pathways that regulate these cellular processes. Therefore, PAKs have emerged as attractive targets for treatment of disease. This review discusses the physiological and pathological roles of PAKs, validation of PAKs as new promising drug targets, and current challenges and advances in the development of PAK-targeted anticancer therapy, with a focus on PAKs and human cancers. Keywords: p21-activated kinase, cancer, inhibitor

  11. 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 o...

  12. Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

    OpenAIRE

    Falk, Matthew D.; Liu, Wei; Bolaños, Ben; Unsal-Kacmaz, Keziban; Klippel, Anke; Grant, Stephan; Brooun, Alexei; Timofeevski, Sergei

    2014-01-01

    The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipi...

  13. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.

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    Kögel, D; Prehn, J H; Scheidtmann, K H

    2001-04-01

    The DAP (Death Associated Protein) kinase family is a novel subfamily of pro-apoptotic serine/threonine kinases. All five DAP kinase family members identified to date are ubiquitously expressed in various tissues and are capable of inducing apoptosis. The sequence homology of the five kinases is largely restricted to the N-terminal kinase domain. In contrast, the adjacent C-terminal regions are very diverse and link individual family members to specific signal transduction pathways. There is increasing evidence that DAP kinase family members are involved in both extrinsic and intrinsic pathways of apoptosis and may play a role in tumor progression. This review will focus on structural composition and subcellular localization of DAP kinase family members and on signal transduction pathways leading to their activation. Potential mechanisms of DAP kinase family-mediated apoptosis will be discussed. BioEssays 23:352-358, 2001. Copyright 2001 John Wiley & Sons, Inc.

  14. Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases

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    Laughlin, J. D.; Nwachukwu, J. C.; Figuera-Losada, M.; Cherry, L.; Nettles, K. W.; LoGrasso, P. V.

    2012-12-05

    c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations.

  15. Evolutionary history of the vertebrate mitogen activated protein kinases family.

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    Meng Li

    Full Text Available BACKGROUND: The mitogen activated protein kinases (MAPK family pathway is implicated in diverse cellular processes and pathways essential to most organisms. Its evolution is conserved throughout the eukaryotic kingdoms. However, the detailed evolutionary history of the vertebrate MAPK family is largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: The MAPK family members were collected from literatures or by searching the genomes of several vertebrates and invertebrates with the known MAPK sequences as queries. We found that vertebrates had significantly more MAPK family members than invertebrates, and the vertebrate MAPK family originated from 3 progenitors, suggesting that a burst of gene duplication events had occurred after the divergence of vertebrates from invertebrates. Conservation of evolutionary synteny was observed in the vertebrate MAPK subfamilies 4, 6, 7, and 11 to 14. Based on synteny and phylogenetic relationships, MAPK12 appeared to have arisen from a tandem duplication of MAPK11 and the MAPK13-MAPK14 gene unit was from a segmental duplication of the MAPK11-MAPK12 gene unit. Adaptive evolution analyses reveal that purifying selection drove the evolution of MAPK family, implying strong functional constraints of MAPK genes. Intriguingly, however, intron losses were specifically observed in the MAPK4 and MAPK7 genes, but not in their flanking genes, during the evolution from teleosts to amphibians and mammals. The specific occurrence of intron losses in the MAPK4 and MAPK7 subfamilies might be associated with adaptive evolution of the vertebrates by enhancing the gene expression level of both MAPK genes. CONCLUSIONS/SIGNIFICANCE: These results provide valuable insight into the evolutionary history of the vertebrate MAPK family.

  16. Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

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    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

    Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

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

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    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.

  18. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

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    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.

  19. The Cbl Proto-Oncogene Product Negatively Regulates the Src-Family Tyrosine Kinase Fyn by Enhancing Its Degradation

    OpenAIRE

    2000-01-01

    Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism...

  20. Activation of BTK by a Phosphorylation Mechanism Initiated by SRC Family Kinases

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    Rawlings, David J.; Scharenberg, Andrew M.; Park, Hyunsun; Wahl, Matthew I.; Lin, Siqi; Kato, Roberta M.; Fluckiger, Anne-Catherine; Witte, Owen N.; Kinet, Jean-Pierre

    1996-02-01

    Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was pre-dominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

  1. Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3.

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    Brand, Toni M; Iida, Mari; Luthar, Neha; Wleklinski, Matthew J; Starr, Megan M; Wheeler, Deric L

    2013-01-01

    Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁) and 27 (B₂) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

  2. Terreic Acid, a Quinone Epoxide Inhibitor of Bruton's Tyrosine Kinase

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    Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

    1999-03-01

    Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

  3. Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk.

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    Tomlinson, M G; Kurosaki, T; Berson, A E; Fujii, G H; Johnston, J A; Bolen, J B

    1999-05-07

    Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.

  4. Incorporation of extracellular fatty acids by a fatty acid kinase-dependent pathway in Staphylococcus aureus.

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    Parsons, Joshua B; Frank, Matthew W; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O

    2014-04-01

    Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4 ) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

  5. Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells.

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    Šrámek, Jan; Němcová-Fürstová, Vlasta; Kovář, Jan

    2016-09-12

    Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.

  6. A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

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    Ota, Hiroko; Sakasegawa, Shin-Ichi; Yasuda, Yuko; Imamura, Shigeyuki; Tamura, Tomohiro

    2008-12-01

    The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

  7. Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.

    Science.gov (United States)

    Serina, L; Blondin, C; Krin, E; Sismeiro, O; Danchin, A; Sakamoto, H; Gilles, A M; Bârzu, O

    1995-04-18

    The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Science.gov (United States)

    2005-02-01

    of the prodrug, kaempferol 3-O-(2",3",4"-tri-O-acetyl-a-L-rhamnopyranoside)(3Ac- SL0101), a novel inhibitor of the Ser/Thr protein kinase, RSK...Y, Holman NJ, Hecht SM, Lannigan DA. Synthesis of the prodrug, kaempferol 3-O-(2", 3", 4"-tri-O-acetyl-a-L-rhamnopyranoside) (3Ac-SLO1 01), a novel

  9. Src Family Kinases and Receptors: Analysis of Three Activation Mechanisms by Dynamic Systems Modeling

    OpenAIRE

    Fuß, Hendrik; Dubitzky, Werner; Downes, C. Stephen; Kurth, Mary Jo

    2007-01-01

    Src family kinases (SFKs) interact with a number of cellular receptors. They participate in diverse signaling pathways and cellular functions. Most of the receptors involved in SFK signaling are characterized by similar modes of regulation. This computational study discusses a general kinetic model of SFK-receptor interaction. The analysis of the model reveals three major ways of SFK activation: release of inhibition by C-terminal Src kinase, weakening of the inhibitory intramolecular phospho...

  10. Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

    Science.gov (United States)

    Nguyen, Hien-Anh; El Khoury, Takla; Guiral, Sébastien; Laaberki, Maria-Halima; Candusso, Marie-Pierre; Galisson, Frédéric; Foucher, Anne-Emmanuelle; Kesraoui, Salsabil; Ballut, Lionel; Vallet, Sylvain; Orelle, Cédric; Zucchini, Laure; Martin, Juliette; Page, Adeline; Attieh, Jihad; Aghajari, Nushin; Grangeasse, Christophe; Jault, Jean-Michel

    2017-10-13

    Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Mutual Regulation of Src Family Kinases and the Neurotrophin Receptor TrkB*

    OpenAIRE

    Huang, Yang Z.; McNamara, James O.

    2010-01-01

    The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Us...

  12. Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors

    Science.gov (United States)

    2013-01-01

    Background The widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood. Results In this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each. Conclusions Our work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features

  13. Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates.

    Science.gov (United States)

    Nesić, Dragana; Miller, Marshall C; Quinkert, Zachary T; Stein, Markus; Chait, Brian T; Stebbins, C Erec

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

    Science.gov (United States)

    Neišić, Dragana; Miller, Marshall C.; Quinkert, Zachary T.; Stein, Markus; Chait, Brian T.; Stebbins, C. Erec

    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 co-crystal 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. PMID:19966800

  15. 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.

  16. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F. (Sunesis); (Biogen)

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. The rice diacylglycerol kinase family: functional analysis using transient RNA interference

    Directory of Open Access Journals (Sweden)

    Hongliang eGe

    2012-03-01

    Full Text Available Diacylglycerol kinase (DGK is a pivotal enzyme that phosphorylates diacylglycerol (DAG to form phosphatidic acid (PA. The production of PA from phospholipase D (PLD and the coupled phospholipase C (PLC/DGK route is an important signaling process in animal and plant cells. In this study, we report a genomic analysis of eight putative rice DGKs encoded by a gene family (OsDGKs grouped into three clusters. To further investigate the functions of the OsDGKs, a double-stranded RNA (dsRNA-induced RNA silencing method was established. Introduction of in vitro-synthesized dsRNAs corresponding to a unique or conserved region of OsDGKs into rice protoplasts abolished or diminished the expression of individual or multiple OsDGK genes. Suppressing the expression of OsDGKs resulted in a distinct depletion of the transcripts of the defense gene OsNPR1 and the salt-responsive gene OsCIPK15. Our primary results suggest that OsDGKs are involved in the signaling of stress responses.

  3. Abl tyrosine kinases modulate cadherin-dependent adhesion upstream and downstream of Rho family GTPases.

    Science.gov (United States)

    Zandy, Nicole L; Pendergast, Ann Marie

    2008-02-15

    Formation and dissolution of intercellular adhesions are processes of paramount importance during tissue morphogenesis and for pathological conditions such as tumor metastasis. Cadherin-mediated intercellular adhesion requires dynamic regulation of the actin cytoskeleton. The pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. We have recently uncovered a novel role for the Abl family of tyrosine kinases linking cadherin-mediated adhesion to actin dynamics via the regulation of Rho family GTPases. Abl kinases are activated by cadherin engagement, localize to cell-cell junctions and are required for the formation of adherens junctions. Notably, we showed that Abl kinases are required for Rac activation during formation of adherens junctions, and also regulate a Rho-ROCK-myosin signaling pathway that is required for the maintenance of intercellular adhesion. Here we show that Abl kinases regulate the formation and strengthening of adherens junctions downstream of active Rac, and that Abl tyrosine kinases are components of a positive feed-back loop that employs the Crk/CrkL adaptor proteins to promote the formation and maturation of adherens junctions.

  4. Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn

    Directory of Open Access Journals (Sweden)

    Bird Ian N

    2011-01-01

    Full Text Available Abstract Background Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease. Results In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others. Conclusions Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.

  5. Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

    Science.gov (United States)

    Maroney, A C; Finn, J P; Connors, T J; Durkin, J T; Angeles, T; Gessner, G; Xu, Z; Meyer, S L; Savage, M J; Greene, L A; Scott, R W; Vaught, J L

    2001-07-06

    CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  7. 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

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

    Science.gov (United States)

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

    1997-08-15

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

  9. Structural propensities of kinase family proteins from a Potts model of residue co-variation.

    Science.gov (United States)

    Haldane, Allan; Flynn, William F; He, Peng; Vijayan, R S K; Levy, Ronald M

    2016-08-01

    Understanding the conformational propensities of proteins is key to solving many problems in structural biology and biophysics. The co-variation of pairs of mutations contained in multiple sequence alignments of protein families can be used to build a Potts Hamiltonian model of the sequence patterns which accurately predicts structural contacts. This observation paves the way to develop deeper connections between evolutionary fitness landscapes of entire protein families and the corresponding free energy landscapes which determine the conformational propensities of individual proteins. Using statistical energies determined from the Potts model and an alignment of 2896 PDB structures, we predict the propensity for particular kinase family proteins to assume a "DFG-out" conformation implicated in the susceptibility of some kinases to type-II inhibitors, and validate the predictions by comparison with the observed structural propensities of the corresponding proteins and experimental binding affinity data. We decompose the statistical energies to investigate which interactions contribute the most to the conformational preference for particular sequences and the corresponding proteins. We find that interactions involving the activation loop and the C-helix and HRD motif are primarily responsible for stabilizing the DFG-in state. This work illustrates how structural free energy landscapes and fitness landscapes of proteins can be used in an integrated way, and in the context of kinase family proteins, can potentially impact therapeutic design strategies. © 2016 The Protein Society.

  10. Function and regulation of Aurora/Ipl1p kinase family in cell division

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p fanily,a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer.

  11. Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

    Science.gov (United States)

    Prudnikova, Tatiana Y; Rawat, Sonali J; Chernoff, Jonathan

    2015-01-01

    RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

  12. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity.

    Science.gov (United States)

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-07-19

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

  13. The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis.

    Science.gov (United States)

    Xu, Z; Maroney, A C; Dobrzanski, P; Kukekov, N V; Greene, L A

    2001-07-01

    Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.

  14. 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.

  15. 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; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

    2017-02-20

    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.

  16. 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

  17. 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......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...

  18. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer.

    Science.gov (United States)

    Graham, Douglas K; DeRyckere, Deborah; Davies, Kurtis D; Earp, H Shelton

    2014-12-01

    The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

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

    Science.gov (United States)

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

    2016-12-16

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

  20. Genomics, evolution, and crystal structure of a new family of bacterial spore kinases

    OpenAIRE

    2009-01-01

    Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionar...

  1. Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish

    OpenAIRE

    Yoo, Sa Kan; Freisinger, Christina M.; LeBert, Danny C.; Huttenlocher, Anna

    2012-01-01

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H2O2 at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immedi...

  2. Genomics, evolution, and crystal structure of a new family of bacterial spore kinases.

    Science.gov (United States)

    Scheeff, Eric D; Axelrod, Herbert L; Miller, Mitchell D; Chiu, Hsiu-Ju; Deacon, Ashley M; Wilson, Ian A; Manning, Gerard

    2010-05-01

    Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionary history. Sequence and structure analyses indicate that the BSKs are CAKs, a prevalent group of small molecule kinases in bacteria that is distantly related to the eukaryotic protein kinases. YtaA has substantial structural similarity to CAKs, but also displays distinctive features that broaden our understanding of the CAK group. Evolutionary constraint analysis of the protein surfaces indicates that members of the BSK family have distinct clade-conserved patterns in the substrate binding region, and probably bind and phosphorylate distinct targets. Several classes of BSKs have apparently independently lost catalytic activity to become pseudokinases, indicating that the family also has a major noncatalytic function.

  3. Ror family receptor tyrosine kinases regulate the maintenance of neural progenitor cells in the developing neocortex.

    Science.gov (United States)

    Endo, Mitsuharu; Doi, Ryosuke; Nishita, Michiru; Minami, Yasuhiro

    2012-04-15

    The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of βIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

  4. Role of ErbB family receptor tyrosine kinases in intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aberrant expression and signaling of epidermal growth factor receptor (ErbB) family receptor tyrosine kinases, most notably that of ErbB2 and ErbB1, have been implicated in the molecular pathogenesis of intrahepatic cholangiocarcinoma. Constitutive overexpression of ErbB2 and/or ErbB1 in malignant cholangiocytes has raised interest in the possibility that agents which selectively target these receptors could potentially be effective in cholangiocarcinoma therapy. However, current experience with such ErbB-directed therapies have at best produced only modest responses in patients with biliary tract cancers. This review provides a comprehensive and critical analysis of both preclinical and clinical studies aimed at assessing the role of altered ErbB2 and/or ErbB1 expression, genetic modifications, and dysregulated signaling on cholangiocarcinoma development and progression. Specific limitations in experimental approaches that have been used to assess human cholangiocarcinoma specimens for ErbB2 and/or ErbB1 overexpression and gene amplification are discussed. In addition, current rodent models of intrahepatic cholangiocarcinogenesis associated with constitutive ErbB2 overexpression are reviewed. Select interactive relationships between ErbB2 or ErbB1 with other relevant molecular signaling pathways associated with intrahepatic cholangiocarcinoma development and progression are also detailed, including those linking ErbB receptors to bile acid, cyclooxygenase-2, interleukin-6/gp130, transmembrane mucins, hepatocyte growth factor/Met, and vascular endothelial growth factor signaling. Lastly, various factors that can limit therapeutic efficacy of ErbB-targeted agents against cholangiocarcinoma are considered.

  5. 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. PMID:27536870

  6. COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

    Science.gov (United States)

    Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.Ward KW, Rogers EH, Hunter ES 3rd.Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.Haloacetic acids ...

  7. Dynamics of the Tec-family tyrosine kinase SH3 domains.

    Science.gov (United States)

    Roberts, Justin M; Tarafdar, Sreya; Joseph, Raji E; Andreotti, Amy H; Smithgall, Thomas E; Engen, John R; Wales, Thomas E

    2016-04-01

    The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.

  8. 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.

  9. 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

    2009-05-21

    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. 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. The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.

  10. 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.

  11. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    Science.gov (United States)

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  12. Genome-Wide Identification and Expression Analysis of the Mitogen-Activated Protein Kinase Gene Family in Cassava

    Science.gov (United States)

    Yan, Yan; Wang, Lianzhe; Ding, Zehong; Tie, Weiwei; Ding, Xupo; Zeng, Changying; Wei, Yunxie; Zhao, Hongliang; Peng, Ming; Hu, Wei

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA) signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars. PMID:27625666

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

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

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

  14. 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...

  15. VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

    Science.gov (United States)

    Lampropoulou, Anastasia; Senatore, Valentina; Brash, James T.; Liyanage, Sidath E.; Raimondi, Claudio; Bainbridge, James W.

    2017-01-01

    The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth. PMID:28289053

  16. Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer.

    Science.gov (United States)

    Robinson, Dan R; Kalyana-Sundaram, Shanker; Wu, Yi-Mi; Shankar, Sunita; Cao, Xuhong; Ateeq, Bushra; Asangani, Irfan A; Iyer, Matthew; Maher, Christopher A; Grasso, Catherine S; Lonigro, Robert J; Quist, Michael; Siddiqui, Javed; Mehra, Rohit; Jing, Xiaojun; Giordano, Thomas J; Sabel, Michael S; Kleer, Celina G; Palanisamy, Nallasivam; Natrajan, Rachael; Lambros, Maryou B; Reis-Filho, Jorge S; Kumar-Sinha, Chandan; Chinnaiyan, Arul M

    2011-11-20

    Breast cancer is a heterogeneous disease that has a wide range of molecular aberrations and clinical outcomes. Here we used paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers have a variety of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving genes encoding microtubule-associated serine-threonine kinase (MAST) and members of the Notch family. Both MAST and Notch-family gene fusions have substantial phenotypic effects in breast epithelial cells. Breast cancer cell lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and overexpression of MAST1 or MAST2 gene fusions has a proliferative effect both in vitro and in vivo. These findings show that recurrent gene rearrangements have key roles in subsets of carcinomas and suggest that transcriptome sequencing could identify individuals with rare, targetable gene fusions.

  17. Odin (ANKS1A is a Src family kinase target in colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Feller Stephan M

    2008-10-01

    Full Text Available Abstract Background Src family kinases (SFK are implicated in the development of some colorectal cancers (CRC. One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised. Results 64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM, GIT1, vimentin and AFAP1L2 (XB130. Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells. Conclusion Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.

  18. Haemophilus ducreyi targets Src family protein tyrosine kinases to inhibit phagocytic signaling.

    Science.gov (United States)

    Mock, Jason R; Vakevainen, Merja; Deng, Kaiping; Latimer, Jo L; Young, Jennifer A; van Oers, Nicolai S C; Greenberg, Steven; Hansen, Eric J

    2005-12-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

  19. 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.

  20. 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.

  1. 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), c

  2. Molecular dynamics analysis of conserved hydrophobic and hydrophilic bond-interaction networks in ErbB family kinases.

    Science.gov (United States)

    Shih, Andrew J; Telesco, Shannon E; Choi, Sung-Hee; Lemmon, Mark A; Radhakrishnan, Ravi

    2011-06-01

    The EGFR (epidermal growth factor receptor)/ErbB/HER (human EGFR) family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signalling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer against dimer and wild-type against mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdomain motions are co-ordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating bond network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating bond network. The present molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck.

  3. 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.

  4. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell–cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated. PMID:22117215

  5. Emerging roles of protein kinase CK2 in abscisic acid (ABA signaling

    Directory of Open Access Journals (Sweden)

    Belmiro eVilela

    2015-11-01

    Full Text Available The phytohormone abscisic acid (ABA regulates many aspects of plant growth and development as well as responses to multiple stresses. Post-translational modifications such as phosphorylation or ubiquitination have pivotal roles in the regulation of ABA signaling. In addition to the positive regulator sucrose non-fermenting-1 related protein kinase 2 (SnRK2, the relevance of the role of other protein kinases, such as CK2, has been recently highlighted. We have recently established that CK2 phosphorylates the maize ortholog of open stomata 1 OST1, ZmOST1, suggesting a role of CK2 phosphorylation in the control of ZmOST1 protein degradation (Vilela et al., 2015. CK2 is a pleiotropic enzyme involved in multiple developmental and stress-responsive pathways. This review summarizes recent advances that taken together suggest a prominent role of protein kinase CK2 in ABA signaling and related processes.

  6. Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

    Science.gov (United States)

    Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

    2012-01-01

    Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

  7. Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish.

    Science.gov (United States)

    Yoo, Sa Kan; Freisinger, Christina M; LeBert, Danny C; Huttenlocher, Anna

    2012-10-15

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.

  8. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

    NARCIS (Netherlands)

    Zwieten, R. van; Oirschot, B.A.J.A. van; Veldthuis, M.; Dobbe, J.G.; Streekstra, G.J.; Solinge, W.W. van; Schutgens, R.E.; Wijk, R. Gerth van

    2015-01-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK defic

  9. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis

    NARCIS (Netherlands)

    van Zwieten, Rob; van Oirschot, Brigitte A; Veldthuis, Martijn; Dobbe, Johannes G; Streekstra, Geert J; van Solinge, Wouter W; Schutgens, Roger E G; van Wijk, Richard

    2015-01-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK defic

  10. 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

    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...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... 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...

  11. Entamoeba histolytica Phagocytosis of Human Erythrocytes Involves PATMK, a Member of the Transmembrane Kinase Family

    Science.gov (United States)

    Boettner, Douglas R; Huston, Christopher D; Linford, Alicia S; Buss, Sarah N; Houpt, Eric; Sherman, Nicholas E; Petri, William A

    2008-01-01

    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 (PATMKΔ932). Expression of the carboxy-truncation of PATMKΔ932 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. PMID:18208324

  12. 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.

  13. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

    Institute of Scientific and Technical Information of China (English)

    Heping Yang; Dapeng Wu; Xiaojie Zhang; Xiang Wang; Yi Peng; Zhiping Hu

    2012-01-01

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU.In this study,we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process.Western blot analysis demonstrated that telencephalin,phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid,while they were expressed in PAJU cells transfected with a telencephalin expression plasmid.After treatment with 1.0 nM amyloid beta protein 42,expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished,while levels of phosphorylated ezrin/radixin/moesin increased.In addition,the high levels of telencephalin,phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002.These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  14. 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.

  15. 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.

  16. 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. PMID:24324463

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

    Directory of Open Access Journals (Sweden)

    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.

  18. “Stop Ne(c)king around”: How interactomics contributes to functionally characterize Nek family kinases

    Institute of Scientific and Technical Information of China (English)

    Gabriela; Vaz; Meirelles; Arina; Marina; Perez; Edmárcia; Elisa; de; Souza; Ferna; Luisa; Basei; Priscila; Ferreira; Papa; Talita; Diniz; Melo; Hanchuk; Vanessa; Bomfim; Cardoso; Jrg; Kobarg

    2014-01-01

    Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A(NIMA)-related kinases(Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals:(1) centrioles/mitosis;(2) primary ciliary function/ciliopathies; and(3) DNA damage response(DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.

  19. Arachidonic acid modulates hippocampal calcium current via protein kinase C and oxygen radicals.

    Science.gov (United States)

    Keyser, D O; Alger, B E

    1990-10-01

    Arachidonic acid (AA) is a second messenger liberated via receptor activation of phospholipase A2 or diacylglycerol-lipase. We used whole-cell voltage clamp of acutely isolated hippocampal CA1 pyramidal cells to investigate the hypothesis that AA modulates Ca2+ channel current (ICa) via activation of protein kinase C (PKC) and generation of free radicals. AA depressed ICa in a dose- and time-dependent manner similar to that previously reported for the action of phorbol esters on ICa. A similar depression was seen with a xanthine-based free radical generating system. The specific PKC inhibitor PKCI (19-36), the protein kinase inhibitor H-7, and the superoxide free radical scavenger SOD each blocked ICa depression by 70%-80%. Complete block of the AA response occurred when SOD was used simultaneously with a PKC inhibitor. These data suggest that PKC and free radicals play a role in AA-induced suppression of ICa.

  20. Hierarchical Disabled-1 Tyrosine Phosphorylation in Src family Kinase Activation and Neurite Formation

    Science.gov (United States)

    Katyal, Sachin; Gao, Zhihua; Monckton, Elizabeth; Glubrecht, Darryl; Godbout, Roseline

    2013-01-01

    There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons. PMID:17350651

  1. 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.

  2. 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.

  3. Differences in c-Jun N-terminal kinase recognition and phosphorylation of closely related stathmin-family members.

    Science.gov (United States)

    Yip, Yan Y; Yeap, Yvonne Y C; Bogoyevitch, Marie A; Ng, Dominic C H

    2014-03-28

    The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif ((41)KKKDLSL(47)) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP.

  4. 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.

  5. Opa+ Neisseria gonorrhoeae exhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes.

    Science.gov (United States)

    Johnson, M Brittany; Ball, Louise M; Daily, Kylene P; Martin, Jennifer N; Columbus, Linda; Criss, Alison K

    2015-05-01

    During gonorrhoeal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial contents. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signalling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signalling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils' full antimicrobial arsenal.

  6. 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 does...

  7. Implications of the role of reactive cystein in arginine kinase: reactivation kinetics of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase reactivated by dithiothreitol.

    Science.gov (United States)

    Pan, Ji-Cheng; Cheng, Yuan; Hui, En-Fu; Zhou, Hai-Meng

    2004-04-30

    The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.

  8. Pyruvate kinase is a dosage-dependent regulator of cellular amino acid homeostasis

    Science.gov (United States)

    Grüning, Nana-Maria; Feichtinger, René; Krüger, Antje; Wamelink, Mirjam; Lehrach, Hans; Tate, Stephen; Neureiter, Daniel; Kofler, Barbara

    2012-01-01

    The glycolytic enzyme pyruvate kinase (PK) is required for cancer development, and has been implicated in the metabolic transition from oxidative to fermentative metabolism, the Warburg effect. However, the global metabolic response that follows changes in PK activity is not yet fully understood. Using shotgun proteomics, we identified 31 yeast proteins that were regulated in a PK-dependent manner. Selective reaction monitoring confirmed that their expression was dependent on PK isoform, level and activity. Most of the PK targets were amino acid metabolizing enzymes or factors of protein translation, indicating that PK plays a global regulatory role in biosynthethic amino acid metabolism. Indeed, we found strongly altered amino acid profiles when PK levels were changed. Low PK levels increased the cellular glutamine and glutamate concentrations, but decreased the levels of seven amino acids including serine and histidine. To test for evolutionary conservation of this PK function, we quantified orthologues of the identified PK targets in thyroid follicular adenoma, a tumor characterized by high PK levels and low respiratory activity. Aminopeptidase AAP-1 and serine hydroxymethyltransferase SHMT1 both showed PKM2- concentration dependence, and were upregulated in the tumor. Thus, PK expression levels and activity were important for maintaining cellular amino acid homeostasis. Mediating between energy production, ROS clearance and amino acid biosynthesis, PK thus plays a central regulatory role in the metabolism of proliferating cells. PMID:23154538

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

    Science.gov (United States)

    Hong, Jie; Li, Dan; Cao, Weibiao

    2016-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Role of a hippocampal SRC-family kinase-mediated glutamatergic mechanism in drug context-induced cocaine seeking.

    Science.gov (United States)

    Xie, Xiaohu; Arguello, Amy A; Wells, Audrey M; Reittinger, Andrew M; Fuchs, Rita A

    2013-12-01

    Glutamatergic neurotransmission in the dorsal hippocampus (DH) is necessary for drug context-induced reinstatement of cocaine-seeking behavior in an animal model of drug relapse. Furthermore, in vitro studies suggest that the Src family of tyrosine kinases critically regulates glutamatergic cellular functions within the DH. Thus, Src-family kinases in the DH may similarly control contextual cocaine-seeking behavior. To test this hypothesis, rats were trained to lever press for un-signaled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behavior (non-reinforced active lever pressing) was then assessed in the previously cocaine-paired and extinction contexts after AP5 (N-methyl-D-aspartate glutamate (NMDA) receptor (NMDAR) antagonist; 0.25 or 2.5 μg/0.5 μl/hemisphere), PP2 (Src-family kinase inhibitor; 6.25 or 62.5 ng/0.5 μl/hemisphere), Ro25-6981 (NR2B subunit-containing NMDAR antagonist; 0.2 or 2 μg/0.5 μl/hemisphere), or vehicle administration into the DH. Administration of AP5, PP2, or Ro25-6981 into the DH dose-dependently impaired drug context-induced reinstatement of cocaine-seeking behavior relative to vehicle, without altering instrumental behavior in the extinction context or food-reinforced instrumental responding and general motor activity in control experiments. Cocaine-seeking behavior during the first 20 min of the test session in the cocaine-paired context was associated with an increase in NR2B subunit activation, and intra-DH PP2 pretreatment disrupted this relationship. Together, these findings suggest that Src-family kinase activation, NMDAR stimulation, and likely Src-family kinase-mediated NR2B subunit-containing NMDAR activation in the DH are necessary for incentive motivational and/or memory processes that promote contextual cocaine-seeking behavior.

  13. Knockout of the PKN family of Rho effector kinases reveals a non-redundant role for PKN2 in developmental mesoderm expansion

    OpenAIRE

    Ivan Quétier; Jacqueline J.T. Marshall; Bradley Spencer-Dene; Sylvie Lachmann; Adele Casamassima; Claudio Franco; Sarah Escuin; Joseph T. Worrall; Priththivika Baskaran; Vinothini Rajeeve; Michael Howell; Andrew J. Copp; Gordon Stamp; Ian Rosewell; Pedro Cutillas

    2016-01-01

    Summary 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 s...

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

    OpenAIRE

    Quétier, I.; Marshall, J. J.; Spencer-Dene, B.; Lachmann, S.; Casamassima, A.; Franco, C.; Escuin, S.; Worrall, J. T.; Baskaran, P.; Rajeeve, V.; Howell, M.; Copp, A. J.; Stamp, G.; Rosewell, I.; Cutillas, P.

    2016-01-01

    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 ...

  15. 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....

  16. Rice Mitogen-activated Protein Kinase Gene Family and Its Role in Biotic and Abiotic Stress Response

    Institute of Scientific and Technical Information of China (English)

    Jai S. Rohila; Yinong Yang

    2007-01-01

    The mitogen-activated protein kinase (MARK) cascade is an important signaling module that transduces extracellular stimuli into intracellular responses in eukaryotic organisms. An increasing body of evidence has shown that the MAPK-mediated cellular signaling is crucial to plant growth and development, as well as biotic and abiotic stress responses. To date, a total of 17 MARK genes have been identified from the rice genome. Expression profiling, biochemical characterization and/or functional analysis were carried out with many members of the rice MARK gene family, especially those associated with biotic and abiotic stress responses. In this review, the phylogenetic relationship and classification of rice MARK genes are discussed to facilitate a simple nomenclature and standard annotation of the rice MARK gene family. Functional data relating to biotic and abiotic stress responses are reviewed for each MARK group and show that despite overlapping in functionality, there is a certain level of functional specificity among different rice MAP kinases. The future challenges are to functionally characterize each MARK, to identify their downstream substrates and upstream kinases, and to genetically manipulate the MARK signaling pathway in rice crops for the improvement of agronomically important traits.

  17. Design, synthesis and evaluation of 3-quinoline carboxylic acids as new inhibitors of protein kinase CK2.

    Science.gov (United States)

    Syniugin, Anatolii R; Ostrynska, Olga V; Chekanov, Maksym O; Volynets, Galyna P; Starosyla, Sergiy A; Bdzhola, Volodymyr G; Yarmoluk, Sergiy M

    2016-01-01

    In this article, the derivatives of 3-quinoline carboxylic acid were studied as inhibitors of protein kinase CK2. Forty-three new compounds were synthesized. Among them 22 compounds inhibiting CK2 with IC50 in the range from 0.65 to 18.2 μM were identified. The most active inhibitors were found among tetrazolo-quinoline-4-carboxylic acid and 2-aminoquinoline-3-carboxylic acid derivatives.

  18. Activation of AMP-activated protein kinase and phosphorylation of glycogen synthase kinase3 β mediate ursolic acid induced apoptosis in HepG2 liver cancer cells.

    Science.gov (United States)

    Son, Hyun-Soo; Kwon, Hee Young; Sohn, Eun Jung; Lee, Jang-Hoon; Woo, Hong-Jung; Yun, Miyong; Kim, Sung-Hoon; Kim, Young-Chul

    2013-11-01

    Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3.

    Science.gov (United States)

    Hamamura, Kazunori; Tsuji, Momoko; Hotta, Hiroshi; Ohkawa, Yuki; Takahashi, Masataka; Shibuya, Hidenobu; Nakashima, Hideyuki; Yamauchi, Yoshio; Hashimoto, Noboru; Hattori, Hisashi; Ueda, Minoru; Furukawa, Keiko; Furukawa, Koichi

    2011-05-27

    The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

  20. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins.

  1. Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium.

    Science.gov (United States)

    Shahidullah, M; Mandal, A; Delamere, N A

    2015-11-01

    The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to

  2. Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.

    OpenAIRE

    Anderberg, R J; Walker-Simmons, M K

    1992-01-01

    Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA...

  3. 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.

  4. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  5. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  6. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

    Science.gov (United States)

    van Zwieten, Rob; van Oirschot, Brigitte A; Veldthuis, Martijn; Dobbe, Johannes G; Streekstra, Geert J; van Solinge, Wouter W; Schutgens, Roger E G; van Wijk, Richard

    2015-03-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency.

  7. PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J.; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A.; Witze, Eric S.

    2012-01-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought. PMID:22493067

  8. PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation.

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Pytel, Dariusz; Riese, Matthew J; Vaites, Laura Pontano; Singh, Nickpreet; Koretzky, Gary A; Witze, Eric S; Diehl, J Alan

    2012-06-01

    The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Pöhlmann, Jennifer; Risse, Carmen; Seidel, Constanze; Pohlmann, Thomas; Jakopec, Visnja; Walla, Eva; Ramrath, Pascal; Takeshita, Norio; Baumann, Sebastian; Feldbrügge, Michael; Fischer, Reinhard; Fleig, Ursula

    2014-09-01

    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.

  13. Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

    Science.gov (United States)

    Sancier, Florence; Dumont, Aurélie; Sirvent, Audrey; Paquay de Plater, Ludmilla; Edmonds, Thomas; David, Géraldine; Jan, Michel; de Montrion, Catherine; Cogé, Francis; Léonce, Stéphane; Burbridge, Michael; Bruno, Alain; Boutin, Jean A; Lockhart, Brian; Roche, Serge; Cruzalegui, Francisco

    2011-02-24

    c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

  14. AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis.

    Directory of Open Access Journals (Sweden)

    Theresa S Moser

    Full Text Available The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV, an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism.

  15. NPM-ALK: The Prototypic Member of a Family of Oncogenic Fusion Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Joel D. Pearson

    2012-01-01

    Full Text Available Anaplastic lymphoma kinase (ALK was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5(p23;q35 chromosomal translocation observed in a subset of anaplastic large cell lymphoma (ALCL. The NPM-ALK fusion protein generated by this translocation is a constitutively active tyrosine kinase, and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in ALCL. We now know about the existence of nearly 20 distinct ALK translocation partners, and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large B-cell lymphomas, nonsmall cell lung carcinomas, and inflammatory myofibroblastic tumours. Moreover, the inhibition of ALK has been shown to be an effective treatment strategy in some of these malignancies. In this paper we will highlight malignancies where ALK translocations have been identified and discuss why ALK fusion proteins are constitutively active tyrosine kinases. Finally, using ALCL as an example, we will examine three key signalling pathways activated by NPM-ALK that contribute to proliferation and survival in ALCL.

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

    Science.gov (United States)

    Brodersen, Peter; Petersen, Morten; Bjørn Nielsen, Henrik; Zhu, Shijiang; Newman, Mari-Anne; Shokat, Kevan M; Rietz, Steffen; Parker, Jane; Mundy, John

    2006-08-01

    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 in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA-ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4-EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

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

    Institute of Scientific and Technical Information of China (English)

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

    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

  18. 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.

  19. Abscisic acid activates a Ca2+-calmodulin-stimulated protein kinase involved in antioxidant defense in maize leaves

    Institute of Scientific and Technical Information of China (English)

    Shucheng Xu

    2010-01-01

     The role of a calcium-dependent and calmodulin(CaM)stimulated protein kinase in abscisic acid(ABA)-induced antioxidant defense was determined in leaves of maize (Zea mays).In-gel kinase assays showed that treatments with ABA or H2O2 induced the activation of a 49-kDa protein kinase and a 52-kDa protein kinase significantly.Furthermore,we showed that the 52-kDa protein kinase has the characteristics of CaM-stimulating activity and is sensitive to calcium-CaM-dependent protein kinase Ⅱ (CaMK Ⅱ)inhibitor KN-93 or CaM antagonist W-7.Treatments with ABA or H2O2 not only induced the acti vation of the 52-kDa protein kinase,but also enhanced the total activities of the antioxidant enzymes,including catalase,ascorbate peroxidase,glutathione reductase,and superoxide dismutase.Such enhancements were blocked by pretreatment with a CaMK inhibitor and a reactive oxygen species(ROS)inhibitor or scavenger.Pretreatment with the CaMK inhibitor also substantially arrested the ABA-induced H2O2 production.Kinase activity enhancements induced by ABA were attenuated by pretreatment with an ROS inhibitor or scavenger.These results suggest that the 52-kDa CaMK is involved in ABA-induced antioxidant defense and that cross-talk between CaMK and H2O2 plays a pivotal role in ABA signaling.We infer that CaMK acts both upstream and downstream of H2O2,but mainly acts between ABA and H2O2 in ABA-induced antioxidant-defensive signaling.

  20. 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

  1. Novel Role and Regulation of the Interleukin-1 Receptor Associated Kinase (IRAK) Family Proteins

    Institute of Scientific and Technical Information of China (English)

    Yingsu Huang; Anna Misior; Liwu Li

    2005-01-01

    The interleukin-1 receptor associated kinases (IRAKs) sit at the bottle neck for the Toll-like-receptor (TLR) mediated signal transduction process controlling host innate immune response. However, the exact role and regulation of IRAKs are still in the early stage and not fully understood. This review intends to summarize the recent advancement in this important topic and points out areas that need further intensive investigation.

  2. Novel Role and Regulation of the Interleukin-1 Receptor Associated Kinase (IRAK) Family Proteins

    Institute of Scientific and Technical Information of China (English)

    YingsuHuang; AnnaMisior

    2005-01-01

    The interleukin-1 receptor associated kinases (IRAKs) sit at the bottle neck for the Toll-like-receptor (TLR) mediated signal transduction process controlling host innate immune response. However, the exact role and regulation of IRAKs are still in the early stage and not fully understood. This review intends to summarize the recent advancement in this important topic and points out areas that need further intensive investigation. Cellular & Molecular Immunology. 2005;2(1):36-39.

  3. Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes.

    Science.gov (United States)

    Zhou, Jun; Wang, Qilong; Ding, Ye; Zou, Ming-Hui

    2015-02-01

    We recently reported that genetic deletion of myeloperoxidase (MPO) alleviates obesity-related insulin resistance in mice in vivo. How MPO impairs insulin sensitivity in adipocytes is poorly characterized. As hypochlorous acid (HOCl) is a principal oxidant product generated by MPO, we evaluated the effects of HOCl on insulin signaling in adipocytes differentiated from 3T3-L1 cells. Exposure of 3T3-L1 adipocytes to exogenous HOCl (200 μmol/l) attenuated insulin-stimulated 2-deoxyglucose uptake, GLUT4 translocation, and insulin signals, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and phosphorylation of Akt. Furthermore, treatment with HOCl induced phosphorylation of IRS1 at serine 307, inhibitor κB kinase (IKK), c-Jun NH2-terminal kinase (JNK), and phosphorylation of PKCθ (PKCθ). In addition, genetic and pharmacological inhibition of IKK and JNK abolished serine phosphorylation of IRS1 and impairment of insulin signaling by HOCl. Furthermore, knockdown of PKCθ using siRNA transfection suppressed phosphorylation of IKK and JNK and consequently attenuated the HOCl-impaired insulin signaling pathway. Moreover, activation of PKCθ by peroxynitrite was accompanied by increased phosphorylation of IKK, JNK, and IRS1-serine 307. In contrast, ONOO(-) inhibitors abolished HOCl-induced phosphorylation of PKCθ, IKK, JNK, and IRS1-serine 307, as well as insulin resistance. Finally, high-fat diet (HFD)-induced insulin resistance was associated with enhanced phosphorylation of PKCθ, IKK, JNK, and IRS1 at serine 307 in white adipose tissues from WT mice, all of which were not found in Mpo knockout mice fed HFDs. We conclude that HOCl impairs insulin signaling pathway by increasing ONOO(-) mediated phosphorylation of PKCθ, resulting in phosphorylation of IKK/JNK and consequent serine phosphorylation of IRS1 in adipocytes.

  4. Tomato ABSCISIC ACID STRESS RIPENING (ASR) gene family revisited.

    Science.gov (United States)

    Golan, Ido; Dominguez, Pia Guadalupe; Konrad, Zvia; Shkolnik-Inbar, Doron; Carrari, Fernando; Bar-Zvi, Dudy

    2014-01-01

    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.

  5. 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.

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  8. Structural propensities of kinase family proteins from a Potts model of residue co‐variation

    National Research Council Canada - National Science Library

    Haldane, Allan; Flynn, William F; He, Peng; Vijayan, R.S.K; Levy, Ronald M

    2016-01-01

    ...‐variation of pairs of mutations contained in multiple sequence alignments of protein families can be used to build a Potts Hamiltonian model of the sequence patterns which accurately predicts structural contacts...

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Nuclear expression of Lyn, a Src family kinase member, is associated with poor prognosis in renal cancer patients.

    Science.gov (United States)

    Roseweir, Antonia K; Qayyum, Tahir; Lim, Zhi; Hammond, Rachel; MacDonald, Alasdair I; Fraser, Sioban; Oades, Grenville M; Aitchison, Michael; Jones, Robert J; Edwards, Joanne

    2016-03-16

    8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50%. Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC). SFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound healing. High nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y(419)) and FAK (Y(861)) phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein. Our data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

  11. Urolithin as a converging scaffold linking ellagic acid and coumarin analogues: design of potent protein kinase CK2 inhibitors.

    Science.gov (United States)

    Cozza, Giorgio; Gianoncelli, Alessandra; Bonvini, Paolo; Zorzi, Elisa; Pasquale, Riccardo; Rosolen, Angelo; Pinna, Lorenzo A; Meggio, Flavio; Zagotto, Giuseppe; Moro, Stefano

    2011-12-09

    Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase; its abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other relevant diseases. Previously, using different in silico screening approaches, two potent and selective CK2 inhibitors were identified by our group: ellagic acid, a naturally occurring tannic acid derivative (K(i)=20 nM) and 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC, K(i)=60 nM). Comparing the crystallographic binding modes of both ellagic acid and DBC, an X-ray structure-driven merging approach was taken to design novel CK2 inhibitors with improved target affinity. A urolithin moiety is proposed as a possible bridging scaffold between the two known CK2 inhibitors, ellagic acid and DBC. Optimization of urolithin A as the bridging moiety led to the identification of 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one as a novel, potent and selective CK2 inhibitor, which shows a K(i) value of 7 nM against the protein kinase, representing a significant improvement in affinity for the target compared with the two parent fragments.

  12. The Choline/Ethanolamine Kinase Family in Arabidopsis: Essential Role of CEK4 in Phospholipid Biosynthesis and Embryo Development.

    Science.gov (United States)

    Lin, Ying-Chen; Liu, Yu-Chi; Nakamura, Yuki

    2015-05-01

    Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylethanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoethanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/ethanolamine kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis. © 2015 American Society of Plant Biologists. All rights reserved.

  13. Retinoic acid increases glucocorticoid receptor phosphorylation via cyclin-dependent kinase 5.

    Science.gov (United States)

    Brossaud, Julie; Roumes, Hélène; Helbling, Jean-Christophe; Moisan, Marie-Pierre; Pallet, Véronique; Ferreira, Guillaume; Biyong, Essi-Fanny; Redonnet, Anabelle; Corcuff, Jean-Benoît

    2017-07-01

    Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long-term glucocorticoid exposure. A 4-day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)-induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter-acted the effect of RA on GR sensitive target proteins such as BDNF or tissue-transglutaminase. These data help understanding the interaction between RA- and glucocorticoid-signalling pathways, both of which have strong influences on the adult brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro.

    Science.gov (United States)

    da Silva, Cleide G; Bueno, Ana Rúbia F; Schuck, Patrícia F; Leipnitz, Guilhian; Ribeiro, César A J; Rosa, Rafael B; Dutra Filho, Carlos S; Wyse, Angela T S; Wannmacher, Clóvis M D; Wajner, Moacir

    2004-01-01

    D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

  15. Anilides of (R)-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Bebernitz, G R; Aicher, T D; Stanton, J L; Gao, J; Shetty, S S; Knorr, D C; Strohschein, R J; Tan, J; Brand, L J; Liu, C; Wang, W H; Vinluan, C C; Kaplan, E L; Dragland, C J; DelGrande, D; Islam, A; Lozito, R J; Liu, X; Maniara, W M; Mann, W R

    2000-06-01

    The optimization of a series of anilide derivatives of (R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC(50) = 35 +/- 1.4 microM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20-40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC(50) of 13 +/- 1.5 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 micromol/kg, and increases the ex vivo activity of PDH in muscle, kidney, liver, and heart tissues. However, in contrast to sodium dichloroacetate (DCA), these PDHK inhibitors did not lower blood glucose levels. Nevertheless, they are effective at increasing the utilization and disposal of lactate and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.

  16. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    Science.gov (United States)

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

  17. Constitutive activation of AtMEK5, a MAPK kinase, induces salicylic acid-independent cell death in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    LIU Hongxia; WANG Ying; ZHOU Tianhong; SUN Yujing; LIU Guoqin; REN Dongtao

    2004-01-01

    AtMEK5DD is an active mutant of AtMEK5, a MAP kinase kinase in Arabidopsis. Induction of AtMEK5DD expression in transgenic plants leads to activation of 44 and 48 kD MAPKs and causes a rapid cell death. To compare the cell death induced by the expression of AtMEK5DD with the HR-cell death induced by avirulence pathogen infection, we analyzed the activation of downstream MAP Kinase and induction of PR genes expression in permanent transgenic Arabidopsis plants. In-gel kinase activity assay revealed that the infection of Pseudomonas syringae DC3000 harboring Avr Rpt2 gene also lead to activation of 44 and 48 kD MAPKs. PAL, PR1 and PR5 were strongly induced in plants undergoing HR-cell death caused by the infection of P. Syringae DC3000, while only the expression of PR5 was strongly induced in transgenic plants expressing AtMEK5DD protein. NahG protein in AtMEK5DD×NahG plants cannot suppress the cell death induced by AtMEK5DD. And AtMEK5DD protein expressed AtMEK5DD×NahG plants showed no significant change in salicylic acid (SA)level.All these suggest that the cell death induced by the activation of AtMEK5 is salicylic acid-independent.

  18. The glypiated neuronal cell adhesion molecule contactin/F11 complexes with src-family protein tyrosine kinase Fyn.

    Science.gov (United States)

    Zisch, A H; D'Alessandri, L; Amrein, K; Ranscht, B; Winterhalter, K H; Vaughan, L

    1995-06-01

    Glycosyl phosphatidylinositol-anchored glycoproteins of the immunoglobulin superfamily play an important role in the formation of neuronal networks during development. The mechanism whereby neuronal GPI-linked molecules transduce recognition signals remains to be established. Analysis of detergent-resistant immune-complexes reveals that the glypiated neuronal cell adhesion molecule contactin/F11 specifically complexes with the cytoplasmic, nonreceptor type src-family tyrosine kinase Fyn. Antibody-mediated cross-linking of contactin/F11 on embryonic chick neuronal cells leads to an increase of the Fyn-activity coprecipitated with contactin/F11, and elevates phosphorylation of an additional 75/80 K component within the contactin/F11-immune-complex. Additionally, binding of ligands, i.e., contactin/F11-specific antibody or tenascin-R, a natural ligand of contactin/F11, to the surface of HeLa transfectants expressing contactin/F11, causes capping of contactin/F11 and a concomitant change in the distribution of the intracellular kinase Fyn, thus confirming their physical association. This indicates that contactin/F11-mediated signaling requires Fyn.

  19. Dysregulation of Src family kinases in mast cells from epilepsy-resistant ASK versus epilepsy-prone EL mice.

    Science.gov (United States)

    Kitaura, Jiro; Kawakami, Yuko; Maeda-Yamamoto, Mari; Horejsi, Vaclav; Kawakami, Toshiaki

    2007-01-01

    EL mice have been used as a model of epilepsy, whereas ASK mice are an epilepsy-resistant variant originating from a colony of EL mice. Mast cell-dependent anaphylaxis is easily inducible by stimulation with IgE and Ag in ASK mice, whereas EL mice are resistant to such stimuli. In this study we have characterized mast cells derived from these two strains. ASK mast cells proliferated more vigorously than EL cells in response to IL-3 and stem cell factor. Although ASK mast cells degranulated less vigorously than EL mast cells upon stimulation with IgE and Ag, ASK cells produced and secreted several-fold more TNF-alpha and IL-2 than EL cells. Consistent with the similarities of these ASK and EL mast cell responses with phenotypes of lyn(-/-) and wild-type mast cells, respectively, Lyn activity was reduced in ASK cells. In addition to the impaired Lyn activity, ASK cells just like lyn(-/-) cells exhibited reduced Syk activity, prolonged activation of ERK and JNK, and enhanced activation of Akt. Furthermore, the lipid raft-resident transmembrane adaptor protein Cbp/PAG that associates with Lyn was hypophosphorylated in ASK cells. Importantly, similar to lyn(-/-) cells, Fyn was hyperactivated in ASK cells. Therefore, these results are consistent with the notion that Lyn-dependent phosphorylation of Cbp/PAG negatively regulates Src family kinases. This study also suggests that reduced activity of Lyn, a negative regulator of mast cell activation, underlies the susceptibility of ASK mice to anaphylaxis and implies that dysregulation of Lyn and other Src family kinases contributes to epileptogenesis.

  20. 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

  1. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.

    Science.gov (United States)

    George, Margaret D; Wine, Robert N; Lackford, Brad; Kissling, Grace E; Akiyama, Steven K; Olden, Kenneth; Roberts, John D

    2013-12-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. 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.

  4. 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

    production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......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...

  5. Diagnostic Value of Urinary Mevalonic Acid Excretion in Patients with a Clinical Suspicion of Mevalonate Kinase Deficiency (MKD).

    Science.gov (United States)

    Jeyaratnam, Jerold; Ter Haar, Nienke M; de Sain-van der Velden, Monique G M; Waterham, Hans R; van Gijn, Mariëlle E; Frenkel, Joost

    2016-01-01

    In patients suffering from mevalonate kinase deficiency (MKD), the reduced enzyme activity leads to an accumulation of mevalonic acid which is excreted in the urine. This study aims to evaluate the diagnostic value of urinary mevalonic acid measurement in patients with a clinical suspicion of mevalonate kinase deficiency. In this single-center, retrospective analysis, all patients in whom both measurement of mevalonic acid and genetic testing had been performed in the preceding 17 years have been included. The presence of two pathogenic MVK mutations or demonstration of decreased enzyme activity was considered to be the gold standard for the diagnosis of MKD. Sixty-one patients were included in this study. Thirteen of them harbored two MVK mutations; twelve of them showed elevated levels of mevalonic acid. Forty-eight patients did not harbor any MVK mutations, yet five of them excreted increased amounts of mevalonic acid. This corresponds to a sensitivity of 92%, a specificity of 90%, a positive predictive value of 71%, and a negative predictive value of 98%. The positive likelihood ratio is 10 and the negative likelihood ratio is 0.09. MKD seems very unlikely in patients with a normal mevalonic acid excretion, but it cannot be excluded completely. Further, a positive urinary mevalonic acid excretion still requires MVK analysis to confirm the diagnosis of MKD. Therefore, detection of urinary mevalonic acid should not be mandatory before genetic testing. However, as long as genetic testing is not widely available and affordable, measurement of urinary mevalonic acid is a fair way to select patients for MVK gene analysis or enzyme assay.

  6. Identification of calcium-dependent protein kinase (CDPK): A multi-functional gene family in Rafflesia cantleyi

    Science.gov (United States)

    Amini, Safoora; Goh, Hoe-Han; Wan, Kiew-Lian

    2016-11-01

    Rafflesia, a parasitic plant that belongs to the Rafflesiaceae family, is notable for producing the largest flowers in the world. This study focused on identification of Calcium-dependent protein kinases (CDPKs) due to their vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. RNA-seq data generated from three bud stages of Rafflesia cantleyi ie BS1, BS2, and BS3 and were assembled. Based on the BLAST searches of Rafflesia unique transcripts (UTs) to Arabidopsis TAIR database, a total of 14 unique transcripts (UTs) were identified as CDPK1 to CDPK5, CDPK7 to CDPK11, CDPK16, CDPK18, CDPK19, and CDPK28. These genes are expressed at all three bud stages of R. cantleyi with up-regulation pattern at BS1 vs. BS2 and BS2 vs. BS3. This result shows that the expression of CDPK gene family increases by developmental progress in Rafflesia in order to regulate biochemical and molecular changes at the cellular level in response to exposure to environmental changes. However, CDPKs functions in plants growth and defense process still need more experimental evidence to deeply understand their biological roles in R. cantleyi.

  7. 2-octynoic acid inhibits hepatitis C virus infection through activation of AMP-activated protein kinase.

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    Darong Yang

    Full Text Available Many chronic hepatitis C virus (HCV-infected patients with current therapy do not clear the virus. It is necessary to find novel treatments. The effect of 2-octynoic acid (2-OA on HCV infection in human hepatocytes was examined. The mechanism of 2-OA antiviral activity was explored. Our data showed that 2-OA abrogated lipid accumulation in HCV replicon cells and virus-infected hepatocytes. It suppressed HCV RNA replication and infectious virus production with no cytotoxicity to the host cells. 2-OA did not affect hepatitis B virus replication in HepG2.2.15 cells derived from HepG2 cells transfected with full genome of HBV. Further study demonstrated that 2-OA activated AMP-activated protein kinase (AMPK and inhibited acetyl-CoA carboxylase in viral-infected cells. Compound C, a specific inhibitor of AMPK, inhibited AMPK activity and reversed the reduction of intracellular lipid accumulation and the antiviral effect of 2-OA. Knockdown of AMPK expression by RNA interference abolished the activation of AMPK by 2-OA and blocked 2-OA antiviral activity. Interestingly, 2-OA induced interferon-stimulated genes (ISGs and inhibited microRNA-122 (miR-122 expression in virus-infected hepatocytes. MiR-122 overexpression reversed the antiviral effect of 2-OA. Furthermore, knockdown of AMPK expression reversed both the induction of ISGs and suppression of miR-122 by 2-OA, implying that activated AMPK induces the intracellular innate response through the induction of ISGs and inhibiting miR-122 expression. 2-OA inhibits HCV infection through regulation of innate immune response by activated AMPK. These findings reveal a novel mechanism by which active AMPK inhibits HCV infection. 2-OA and its derivatives hold promise for novel drug development for chronic hepatitis C.

  8. 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.

    2016-01-01

    Purpose 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. Methods 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. Results 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

  9. Upregulation of Na+,Cl--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

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    Ahmad Almilaji

    2013-08-01

    Full Text Available Background/Aims: The serine/threonine kinase Tau-tubulin-kinase 2 (TTBK2 is expressed in various tissues including kidney, liver and brain. Loss of function mutations of TTBK2 lead to autosomal dominant spinocerebellar ataxia type 11 (SCA11. Cell survival is fostered by cellular accumulation of organic osmolytes. Carriers accomplishing cellular accumulation of organic osmolytes include the Na+, Cl--coupled betaine/γ-amino-butyric acid transporter BGT1. The present study explored whether TTBK2 participates in the regulation of BGT1 activity. Methods: Electrogenic transport of GABA was determined in Xenopus oocytes expressing BGT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2- KD and TTBK2[1-450]-KD. Results: Coexpression of wild-type TTBK2, but not of TTBK2[1-450], TTBK2-KD or TTBK2[1-450]-KD, increased electrogenic GABA transport. Wildtype TTBK2 increased the maximal transport rate without significantly modifying affinity of the carrier. Coexpression of wild-type TTBK2 significantly delayed the decline of transport following inhibition of carrier insertion with brefeldin A, indicating that wild-type TTBK2 increased carrier stability in the cell membrane. Conclusion: Tau-tubulin-kinase 2 TTBK2 is a powerful stimulator of the osmolyte and GABA transporter BGT1.

  10. Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner.

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    Purushottam S Narute

    Full Text Available The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts. Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.

  11. Ocular hypotensive efficacy of Src-family tyrosine kinase inhibitors via different cellular actions from Rock inhibitors.

    Science.gov (United States)

    Kirihara, Tomoko; Shimazaki, Atsushi; Nakamura, Masatsugu; Miyawaki, Nobuaki

    2014-02-01

    We investigated the effects of Src-family tyrosine kinase (SFK) inhibitors on intraocular pressure (IOP) and trabecular meshwork (TM) cells. The SFK inhibitors, PP2, PP1, and damnacanthal, significantly lowered IOP from baseline following intracameral injection in ocular normotensive rabbits, and PP2 decreased trans-epithelial electrical resistance (TEER) of TM cell layers in a dose-dependent manner ranging from 0.1 μM to 100 μM. The maximal efficacy of PP2 on TEER was a reduction to 71.7% relative to the vehicle-treated group at 100 μM. PP2 decreased the adhesion of TM cells to culture surfaces either uncoated with specific ECM proteins dose-dependently or coated with extracellular matrix proteins such as laminin I, fibronectin, collagen type I and basement membrane extraction. Tyrosine phosphorylation of focal adhesion kinase and p130(cas) was decreased by PP2. On the other hand, major changes in actin staining of TM cells were not able to be detected after PP2 treatment, although quantitative analysis showed that PP2 induced some morphological changes which were in the different direction to those caused by Y-27632, a Rock inhibitor. Y-27632 at 10 μM increased the permeability of TM cell layers, but did not induce changes in the adhesion of TM cells. These results suggest that SFK inhibitors lower IOP, at least partly, by acting on TM cells in a manner that is distinct from Rock inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum.

    Science.gov (United States)

    Chai, Xin; Shang, Xiuling; Zhang, Yu; Liu, Shuwen; Liang, Yong; Zhang, Yun; Wen, Tingyi

    2016-11-16

    Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role. Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WTΔpyk1Δpyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn(2+) and K(+) and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WTΔpyk1 strain produced 60.27 ± 1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation. Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen

  13. RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis.

    Science.gov (United States)

    Meylan, Etienne; Martinon, Fabio; Thome, Margot; Gschwendt, Michael; Tschopp, Jürg

    2002-12-01

    RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.

  14. RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-κB and is processed during apoptosis

    Science.gov (United States)

    Meylan, Etienne; Martinon, Fabio; Thome, Margot; Gschwendt, Michael; Tschopp, Jürg

    2002-01-01

    RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals. PMID:12446564

  15. 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 and is induced to rotate on its glycosidic bond to the anti conformation as part of the overall mechanism to binding specificity. Using the same theme of using conserved amino acid residues within a group of kinases to suggest mechanistic implications...

  16. 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.

  17. A single amino acid difference between alpha and beta Ca2+/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609.

    Science.gov (United States)

    Tokumitsu, Hiroshi; Inuzuka, Hiroyuki; Ishikawa, Yumi; Kobayashi, Ryoji

    2003-03-28

    We recently developed STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK), and we demonstrated that CaM-KK beta is more sensitive to STO-609 than the CaM-KK alpha isoform (Tokumitsu H., Inuzuka H., Ishikawa Y., Ikeda M., Saji I., and Kobayashi R. (2002) J. Biol. Chem. 277, 15813-15818). By using catalytic chimera and point mutants of both isoforms, we demonstrated that Val(269) in CaM-KK beta/Leu(233) in CaM-KK alpha confers a distinct sensitivity ( approximately 10-fold) to STO-609 on CaM-KK isoforms. Various mutations of Val(269) in CaM-KK beta indicate that substitution by hydrophobic residues with bulky side chains significantly decreases drug sensitivity and that the V269F mutant is the most effective drug-resistant enzyme ( approximately 80-fold higher IC(50) value). These findings are consistent with a result obtained with a full-length mutant expressed in COS-7 cells. Furthermore, suppression of CaM-KK-mediated CaM-KIV activation in transfected HeLa cells by STO-609 treatment was completely abolished by the co-expression of the CaM-KK beta V269F mutant. Based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is because of a single amino acid substitution (Val/Leu) in the ATP-binding pocket, we have generated an STO-609-resistant CaM-KK mutant, which might be useful for validating the pharmacological effects and specificity of STO-609 in vivo.

  18. Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

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    Cathrin Schulz

    Full Text Available BACKGROUND: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. CONCLUSION/SIGNIFICANCE: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

  19. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

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    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  20. YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet β cells.

    Science.gov (United States)

    Yoder, Stephanie M; Dineen, Stacey L; Wang, Zhanxiang; Thurmond, Debbie C

    2014-04-18

    Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

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

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    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.

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

    Science.gov (United States)

    Quétier, Ivan; Marshall, Jacqueline J T; Spencer-Dene, Bradley; Lachmann, Sylvie; Casamassima, Adele; Franco, Claudio; Escuin, Sarah; Worrall, Joseph T; Baskaran, Priththivika; Rajeeve, Vinothini; Howell, Michael; Copp, Andrew J; Stamp, Gordon; Rosewell, Ian; Cutillas, Pedro; Gerhardt, Holger; Parker, Peter J; Cameron, Angus J M

    2016-01-26

    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.

  3. 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.

  4. 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.

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

    OpenAIRE

    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 hereg...

  6. Amino acids involved in polyphosphate synthesis and its mobilization are distinct in polyphosphate kinase-1 from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Payal Mittal

    Full Text Available 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.

  7. 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

  8. 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 proteinaceo

  9. 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

  10. 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 proteinaceo

  11. REVERSE SIGNALING BY GPI-LINKED MANDUCA EPHRIN REQUIRES A SRC FAMILY KINASE TO RESTRICT NEURONAL MIGRATION IN VIVO

    Science.gov (United States)

    Coate, Thomas M.; Swanson, Tracy L.; Copenhaver, Philip F.

    2011-01-01

    Reverse signaling via GPI-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), while adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways. PMID:19295147

  12. 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.

  13. A new solvate of afatinib, a specific inhibitor of the ErbB family of tyrosine kinases

    Directory of Open Access Journals (Sweden)

    Matthias Zeller

    2017-03-01

    Full Text Available Afatinib (systematic name: N-{4-(3-chloro-4-fluoroanilino-7-[(tetrahydrofuran-3-yloxy]quinazolin-6-yl}-4-(dimethylaminobut-2-enamide, is a specific inhibitor of the ErbB family of tyrosine kinases. The free base form crystallizes from acetonitrile as a mixed water–acetonitrile solvent, C24H25ClFN5O3·0.25C2H3N·2H2O. It crystallizes with two independent molecules (A and B in the asymmetric unit of the chiral space group P4212, but exhibits close to perfect pseudo-inversion symmetry, emulating P4/ncc that relates the two molecules to each other. Exact inversion symmetry is however broken by swapping of oxygen and CH2 moieties of the outer tetrahydrofuranyl substituents of the two independent molecules. This can, in turn, be traced back to C—H...N and C—H...O interactions of the acetonitrile solvent molecules with the tetrahydrofuran oxygen and CH2 units. In the crystal, neighboring molecules are connected via N—H...O hydrogen bonds between the secondary amine and the amide keto O atom. Additional hydrogen bonds are formed through the water solvent molecules, which are engaged in O—H...O and O—H...N hydrogen bonds connecting to the dimethylamino N atom, the amide keto O atom, and one of the quinazoline N atoms of a neighboring molecule, leading to an intricate three-dimensional hydrogen-bonded superstructure. There are two types of channels stretching along the direction of the c axis; one along the fourfold rotational axis, occupied by acetonitrile solvent molecules situated on that axis, and parallel channels which are not occupied by any solvent.

  14. Regulation of voltage-gated sodium current by endogenous Src family kinases in cochlear spiral ganglion neurons in culture.

    Science.gov (United States)

    Feng, Shuang; Pflueger, Melissa; Lin, Shuang-Xiu; Groveman, Bradley R; Su, Jiping; Yu, Xian-Min

    2012-04-01

    Voltage-gated sodium (Na+) and potassium (K+)channels have been found to be regulated by Src family kinases(SFKs).However, how these channels are regulated by SFKs in cochlear spiral ganglion neurons (SGNs) remains unknown.Here, we report that altering the activity of endogenous SFKs modulated voltage-gated Na+, but not K+, currents recorded in embryonic SGNs in culture. Voltage-gated Na+ current was suppressed by inhibition of endogenous SFKs or just Src and potentiated by the activation of these enzymes. Detailed investigations showed that under basal conditions, SFK inhibitor application did not significantly affect the voltage-dependent activation, but shifted the steady-state inactivation curves of Na+ currents and delayed the recovery of Na+ currents from inactivation. Application of Src specific inhibitor, Src40–58,not only shifted the inactivation curve but also delayed the recovery of Na+ currents and moved the voltage-dependent activation curve towards the left. The pre-inhibition of SFKs occluded all the effects induced by Src40–58 application, except the left shift of the activation curve. The activation of SFKs did not change either steady-state inactivation or recovery of Na+ currents, but caused the left shift of the activation curve.SFK inhibitor application effectively prevented all the effects induced by SFK activation, suggesting that both the voltage-dependent activation and steady-state inactivation of Na+ current are subjects of SFK regulation. The different effects induced by activation versus inhibition of SFKs implied that under basal conditions, endogenously active and inactive SFKs might be differentially involved in the regulation of voltage-gated Na+ channels in SGNs.

  15. The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Nicolas Dzamko

    Full Text Available Mutations in leucine-rich repeat kinase 2 (LRRK2 are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ and IKK-related (IKKε and TBK1 kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser

  16. 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...

  17. 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...

  18. 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.

  19. Bile Acid Pool Dynamics in Progressive Familial lntrahepatic Cholestasis With Partial External Bile Diversion

    NARCIS (Netherlands)

    Jericho, Hilary S.; Kaurs, Elizabeth; Boverhof, Renze; Knisely, Alex; Shneider, Benjamin L.; Verkade, Henkjan J.; Whitington, Peter F.

    2015-01-01

    Objectives: Partial external bile diversion (PEBD) is an established therapy for low-gamma-glutamyl transferase (GGT) progressive familial intrahepatic cholestasis (PFIC). This study sought to determine whether the dynamics of the cholic acid (CA) and chenodeoxycholic acid (CDCA) pools in subjects w

  20. 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...

  1. 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.

  2. 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.

  3. [Aspartate kinase from the cyanobacteriium Plectonema boryanum infected with the cyanophage LPP-3].

    Science.gov (United States)

    Perepelitsa, S I; Koltukova, N V; Mendzhul, M I

    1995-01-01

    The effect of development of cyanophage infection on the activity of aspartate kinase of Plectonema boryanum has been studied. It has been determined that activity of aspartate kinase increased during early period of reproduction of cyanophage. It coincided in time with the increase of the level of amino acids of aspartate family. Isoenzymes of aspartate kinase were isolated from the infected cells purified and studied. Expression of viral genome is accompanied with the appearance of four new isoenzymes determined by virus. The revealed aspartate kinases are not subject of regulation by amino acids (the end-products of biosynthesis) according to the principle of feed-back inhibition.

  4. 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

    -alpha;-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABA(B1......-2) and T1R2-3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity...

  5. Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase.

    Science.gov (United States)

    Ishibashi, Matsujiro; Ida, Keiko; Tatsuda, Shuhei; Arakawa, Tsutomu; Tokunaga, Masao

    2011-11-01

    We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

  6. The effect of lysophosphatidic acid and Rho-associated kinase patterning on adhesion of dental pulp cells.

    Science.gov (United States)

    Cheng, R; Shao, M-Y; Yang, H; Cheng, L; Wang, F-M; Zhou, X-D; Hu, T

    2011-01-01

    To investigate the effects of lysophosphatidic acid (LPA) and the Rho/Rho-associated kinase (ROCK) pathway on adhesion of dental pulp cells (DPCs). Human DPCs were cultured ex vivo. After treatment of LPA and Y-27632, a specific ROCK inhibitor, changes in focal contacts (FCs) were examined by immunofluorescent staining. Activation of FCs proteins was examined by measuring tyrosine 397 phosphorylation of focal adhesion kinase (FAK) and paxillin using immunoblotting. The data were analysed by Student's t-test. The immunofluorescent staining indicated LPA stimulation induced larger focal adhesion in the cell periphery, compared with the control. Inhibition of ROCK by Y-27632 decreased the formation of FCs markedly, even in the LPA-stimulated cells. LPA also increased the level of tyrosine phosphorylation of paxillin at 30min (P<0.05) and FAK at 5 and 30min (P<0.05). Furthermore, p-paxillin levels declined immediately after Y-27632 treatment and remained low at 5, 30, 60min. Y-27632 also suppressed the effects of LPA on p-paxillin and p-FAK at 5 and 30min (P<0.05). LPA activated Rho and then subsequently activated ROCK, suggesting that LPA influences the FCs of DPCs by modulating tyrosine phosphorylation of FAK and paxillin via the Rho/ROCK pathway. © 2010 International Endodontic Journal.

  7. Alternaric acid stimulates phosphorylation of His-tagged RiCDPK2, a calcium-dependent protein kinase in potato plants.

    Science.gov (United States)

    Hassan, A; Okuta, T; Kato, M; Hatsugai, N; Sano, Y; Ishimori, T; Okazaki, K; Doullah, M A; Shah, M M

    2012-08-16

    Calcium-dependent protein kinases (CDPK) are an essential component of plant defense mechanisms against pathogens. We investigated the effect of alternaric acid, a host-specific toxin produced by the plant fungal pathogen Alternaria solani (Pleosporaceae), on a putative plasma membrane and cytosolic kinase RiCDPK2 of potato (Solanum tuberosum) and on hypersensitive cell death of host potato cells. Alternaric acid, in the presence of Ca²⁺ and Mg²⁺, stimulated in vitro phosphorylation of His-tagged RiCDPK2, a Ca²⁺-dependent protein kinase found in potato plants. We concluded that Ca²⁺ and Mg²⁺ play an important role in the interaction between alternaric acid and RiCDPK2. Based on our observations, alternaric acid regulates RiCDPK2 kinase during the infection process in an interaction between host and A. solani, leading to the inhibition of hypersensitive cell death in the host. We suggest that alternaric acid is a primary determinant by which A. solani stimulates CDPK activity in the host, suppressing hypersensitive cell death.

  8. A new solvate of afatinib, a specific inhibitor of the ErbB family of tyrosine kinases

    Science.gov (United States)

    Zeller, Matthias; de Araujo, Gabriel Lima Barros; Parker, Trev; Singh Rai, Amrinder; Byrn, Stephen R.

    2017-01-01

    Afatinib (systematic name: N-{4-(3-chloro-4-fluoro­anilino)-7-[(tetra­hydro­furan-3-yl)­oxy]quinazolin-6-yl}-4-(di­methyl­amino)­but-2-enamide), is a specific in­hibitor of the ErbB family of tyrosine kinases. The free base form crystallizes from aceto­nitrile as a mixed water–aceto­nitrile solvent, C24H25ClFN5O3·0.25C2H3N·2H2O. It crystallizes with two independent mol­ecules (A and B) in the asymmetric unit of the chiral space group P4212, but exhibits close to perfect pseudo-inversion symmetry, emulating P4/ncc that relates the two mol­ecules to each other. Exact inversion symmetry is however broken by swapping of oxygen and CH2 moieties of the outer tetra­hydro­furanyl substituents of the two independent mol­ecules. This can, in turn, be traced back to C—H⋯N and C—H⋯O inter­actions of the aceto­nitrile solvent mol­ecules with the tetra­hydro­furan oxygen and CH2 units. In the crystal, neighboring mol­ecules are connected via N—H⋯O hydrogen bonds between the secondary amine and the amide keto O atom. Additional hydrogen bonds are formed through the water solvent mol­ecules, which are engaged in O—H⋯O and O—H⋯N hydrogen bonds connecting to the di­methyl­amino N atom, the amide keto O atom, and one of the quinazoline N atoms of a neighboring mol­ecule, leading to an intricate three-dimensional hydrogen-bonded superstructure. There are two types of channels stretching along the direction of the c axis; one along the fourfold rotational axis, occupied by aceto­nitrile solvent mol­ecules situated on that axis, and parallel channels which are not occupied by any solvent.

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

    Science.gov (United States)

    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.; Nalls, Mike A.; 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; Cookson, Mark R; 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, Ómar; 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; München, Helmholtz Zentrum; 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; Arepalli, Sampath; Cookson, Mark R; Dillman, Allissa; Ferrucci, Luigi; Gibbs, J Raphael; Hernandez, Dena G; Johnson, Robert; Longo, Dan L; Majounie, Elisa; Nalls, Michael A; O’Brien, Richard; Singleton, Andrew B; Traynor, Bryan J; 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 Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. PMID:24510904

  10. Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells.

    Science.gov (United States)

    Lu, Jing; Caplan, Michael S; Li, Dan; Jilling, Tamas

    2008-05-01

    We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.

  11. 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.

  12. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  13. 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.

  14. 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.

  15. 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...

  16. 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 wit

  17. 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

    Science.gov (United States)

    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.

  18. 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.

  19. Discovery of pyrido[3,4-g]quinazoline derivatives as CMGC family protein kinase inhibitors: Design, synthesis, inhibitory potency and X-ray co-crystal structure.

    Science.gov (United States)

    Esvan, Yannick J; Zeinyeh, Wael; Boibessot, Thibaut; Nauton, Lionel; Théry, Vincent; Knapp, Stefan; Chaikuad, Apirat; Loaëc, Nadège; Meijer, Laurent; Anizon, Fabrice; Giraud, Francis; Moreau, Pascale

    2016-08-08

    The design and synthesis of new pyrido[3,4-g]quinazoline derivatives is described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A). The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/DYRK1A activity was validated by nanomolar potencies (compounds 12 and 13). CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket.

  20. 脑缺血时NMDA受体通过Src激酶和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ调控ERKs激活%N-methyl-D-aspartate receptors mediate diphosphorylation of extracellular signal-regulated kinases through Src family tyrosine kinases and Ca2+/calmodulin-dependent protein kinase Ⅱ in rat hippocampus after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    吴辉文; 李洪福; 郭军

    2007-01-01

    目的 ERKs是钙依赖性激活蛋白,本研究旨在探讨钙依赖性蛋白激酶是否参与了脑缺血后ERK级联的调控.方法 采用四动脉结扎诱导大鼠前脑缺血,用免疫印迹的方法观察几个钙依赖性蛋白激酶含量及活性的变化.结果 致死性脑缺血以NMDA受体依赖的方式激活ERKs,并差异性上调Src和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的活性.Src激酶和CaMKⅡ的抑制剂PP2和KN62能显著的阻止缺血诱导的ERKs激活.然而,缺血诱导的Src过度激活也伴随着ERKs的活性抑制.结论 致死性脑缺血刺激NMDA受体通过Src激酶和CaMKⅡ介导ERKs活性上调,但是脑缺血诱导的Src过度激活可能也参与了ERKs信号通路的负性调控.%Objective: Extracellular signal-regulated kinases (ERKs) can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia.Methods Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot. Results Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate (NMDA)receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) activities. With the inhibition of Src family tyrosine kinases or CaMKⅡ by administration of PP2or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated. Conclusions Src family tyrosine kinases and CaMKⅡ might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.

  1. Mechanism of Retinoic Acid and Mitogen-activated Protein Kinases Regulating Hyperoxia Lung Injury

    Institute of Scientific and Technical Information of China (English)

    LI Wenbin; CHANG Liwen; RONG Zhihui; ZHANG Qianshen; WANG Hua; WANG Hong; LIU Chunmei; LIU Wei

    2006-01-01

    To investigate the protective effect of retinoicacid (RA) on hyperoxic lung injury and the role of RA as a modulator on mitogen-activated protein kinases (MAPKs), gastation 21 d SpragueDawley (SD) fetuses (term=22d) were delivered by hysterotomy. Within 12-24 h of birth,premature rat pups were randomly divided into 4 groups (n=12 each): air-exposed control group (group Ⅰ); hyperoxia-exposed group ( group Ⅱ), air-exposed plus RA group (group Ⅲ ), hyperoxia-exposed plus RA group (group Ⅳ). Group Ⅰ ,Ⅲ were kept in room air, and group Ⅱ , Ⅳwere placed in 85 % oxygen. The pups in groups Ⅲ and Ⅳ were intraperitoneally injected with RA (500 μg/kg every day). All lung tissues of premature rat pups were collected at the 4th day after birth. Terminal transferase d-UTP nick end labeling (TUNEL) staining was used for the detection of cell apoptosis. The expression of PCNA was immunohistochemically detected. Western blot analysis was employed for the determination of phosphorylated and total nonphosphorylated ERKs,JNKs or p38. Our results showed that lungs from the pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma (P<0.01),and decreased significantly after RA treatment (P<0.01). The index of PCNA-positive cells was significantly decreased (P<0.01), and was significantly increased by RA treatment (P<0.01).The air-space size was significantly enlarged, secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, but had no effect on hyperoxia-exposure pups. Western blotting showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were same as those in untreated lung tissues (P>0.05), whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased (P<0

  2. Tyrosine kinase inhibition reverses TDP-43 effects on synaptic protein expression, astrocytic function and amino acid dis-homeostasis.

    Science.gov (United States)

    Heyburn, Lanier; Hebron, Michaeline L; Smith, Jacqueline; Winston, Charisse; Bechara, John; Li, Zhaoxia; Lonskaya, Irina; Burns, Mark P; Harris, Brent T; Moussa, Charbel E-H

    2016-11-01

    The trans-activating response of DNA/RNA-binding protein (TDP)-43 pathology is associated with many neurodegenerative diseases via unknown mechanisms. Here, we use a transgenic mouse model over-expressing human wild-type neuronal TDP-43 to study the effects of TDP-43 pathology on glutamate metabolism and synaptic function. We found that neuronal TDP-43 over-expression affects synaptic protein expression, including Synapsin I, and alters surrounding astrocytic function. TDP-43 over-expression is associated with an increase in glutamate and γ-amino butyric acid and reduction of glutamine and aspartate levels, indicating impairment of presynaptic terminal. TDP-43 also decreases tricarboxylic acid cycle metabolism and induces oxidative stress via lactate accumulation. Neuronal TDP-43 does not alter microglia activity or significantly changes systemic and brain inflammatory markers compared to control. We previously demonstrated that brain-penetrant tyrosine kinase inhibitors (TKIs), nilotinib and bosutinib, reduce TDP-43-induced cell death in transgenic mice. Here, we show that TKIs reverse the effects of TDP-43 on synaptic proteins, increase astrocytic function and restore glutamate and neurotransmitter balance in TDP-43 mice. Nilotinib, but not bosutinib, reverses mitochondrial impairment and oxidative metabolism. Taken together, these data suggest that TKIs can attenuate TDP-43 toxicity and improve synaptic and astrocytic function, independent of microglial or other inflammatory effects. In conclusion, our data demonstrate novel mechanisms of the effects of neuronal TDP-43 over-expression on synaptic protein expression and alteration of astrocytic function. © 2016 International Society for Neurochemistry.

  3. 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

    not sustain their expression. Moreover, use of inhibitors implied that IL-3 was mainly exerting its effect on Bcl-2 at the level of transcription. The addition of LY294002 did not affect the expression of Bcl-2 and Bcl-XL, and thus, we conclude that expression of antiapoptotic Bcl-2 family member genes......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...... is not dependent on PI-3 kinase activity. Our results indicate that cytokines exert distinct survival effects and that FL and IL-3 are capable of sustaining progenitor survival by up-regulating the expression of Bcl-2 and related genes....

  4. Caffeic Acid Cyclohexylamide Rescues Lethal Inflammation in Septic Mice through Inhibition of IκB Kinase in Innate Immune Process

    Science.gov (United States)

    Choi, Jun Hyeon; Park, Sun Hong; Jung, Jae-Kyung; Cho, Won-Jea; Ahn, Byeongwoo; Yun, Cheong-Yong; Choi, Yong Pyo; Yeo, Jong Hun; Lee, Heesoon; Hong, Jin Tae; Han, Sang-Bae; Kim, Youngsoo

    2017-01-01

    Targeting myeloid differentiation protein 2 (MD-2) or Toll-like receptor 4 (TLR4) with small molecule inhibitor rescues the systemic inflammatory response syndrome (SIRS) in sepsis due to infection with Gram-negative bacteria but not other microbes. Herein, we provided IκB kinase β (IKKβ) in innate immune process as a molecular target of caffeic acid cyclohexylamide (CGA-JK3) in the treatment of polymicrobial TLR agonists-induced lethal inflammation. CGA-JK3 ameliorated E. coli lipopolysaccharide (LPS, MD-2/TLR4 agonist)-induced endotoxic shock, cecal ligation and puncture (CLP)-challenged septic shock or LPS plus D-galactosamine (GalN)-induced acute liver failure (ALF) in C57BL/6J mice. As a molecular basis, CGA-JK3 inhibited IKKβ-catalyzed kinase activity in a competitive mechanism with respect to ATP, displaced fluorescent ATP probe from the complex with IKKβ, and docked at the ATP-binding active site on the crystal structure of human IKKβ. Furthermore, CGA-JK3 inhibited IKKβ-catalyzed IκB phosphorylation, which is an axis leading to IκB degradation in the activating pathway of nuclear factor-κB (NF-κB), in macrophages stimulated with TLR (1/2, 2/6, 4, 5, 7, 9) agonists from Gram-positive/negative bacteria and viruses. CGA-JK3 consequently interrupted IKKβ-inducible NF-κB activation and NF-κB-regulated expression of TNF-α, IL-1α or HMGB-1 gene, thereby improving TLRs-associated redundant inflammatory responses in endotoxemia, polymicrobial sepsis and ALF. PMID:28145460

  5. 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.

  6. Global mapping of protein phosphorylation events identifies Ste20, Sch9 and the cell-cycle regulatory kinases Cdc28/Pho85 as mediators of fatty acid starvation responses in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pultz, Dennis; Bennetzen, Martin V; Rødkær, Steven Vestergaard

    2012-01-01

    Synthesis, degradation, and metabolism of fatty acids are strictly coordinated to meet the nutritional and energetic needs of cells and organisms. In the absence of exogenous fatty acids, proliferation and growth of the yeast Saccharomyces cerevisiae depends on endogenous synthesis of fatty acids...... identified as being regulated in a temporal manner in response to inhibition of fatty acid synthesis by cerulenin. By bioinformatic analysis of these phosphorylation events, we have identified the cell cycle kinases Cdc28 and Pho85, the PAK kinase Ste20 as well as the protein kinase Sch9 as central mediators...

  7. 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.

  8. 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...... the input domain of RpfC was active in BDSF signal perception when expressed in X. campestris. Mutation of BCAM0227 gave rise to reduced cytotoxicity to Chinese hamster ovary cells and reduced virulence to Wax moth larvae and in the agar-bead mouse model of pulmonary infection. The findings identify BCAM...

  9. Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes

    OpenAIRE

    Zhou, Jun; Wang, Qilong; Ding, Ye; Zou, Ming-hui

    2015-01-01

    We recently reported that genetic deletion of myeloperoxidase (MPO) alleviates obesity-related insulin resistance in mice in vivo. How MPO impairs insulin sensitivity in adipocytes is poorly characterized. As hypochlorous acid (HOCl) is a principal oxidant product generated by MPO, we evaluated the effects of HOCl on insulin signaling in adipocytes differentiated from 3T3-L1 cells. Exposure of 3T3-L1 adipocytes to exogenous HOCl (200 μmol/l) attenuated insulin-stimulated 2-deoxyglucose uptake...

  10. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes......, no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-subunit affecting: (i) stability, (ii) enzyme specificity and (iii) enzyme activity. The question where CK-2 and its subunits are located throughout the cell cycle has also been addressed, mainly because of the large discrepancies that still exist between results obtained by different investigators. Tissue...

  11. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Shinozaki, Y; Sato, Y; Koizumi, S; Ohno, Y; Nagao, T; Inoue, K

    2007-06-15

    Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.

  12. Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV.

    Science.gov (United States)

    Shepelev, M V; Korobko, I V

    2012-01-01

    Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.

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

    Science.gov (United States)

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  14. 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.

  15. 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.

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

    Science.gov (United States)

    Aouida, Mustapha; Rubio-Texeira, Marta; Rubio Texeira, Marta; Thevelein, Johan M; Poulin, Richard; Ramotar, Dindial

    2013-01-01

    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.

  17. 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.

    Science.gov (United States)

    Li, Ying; Wang, Jianwei; Gu, Tieguang; Yamahara, Johji; Li, Yuhao

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. 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.

  19. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  20. 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.

  1. Protection of INS-1 Cells from Free Fatty Acid-induced Apoptosis by Inhibiting the Glycogen Synthase Kinase-3

    Institute of Scientific and Technical Information of China (English)

    WU Wei; LUO Xiaoping

    2007-01-01

    To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 dia-betes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhib-ited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apop-tosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic β-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.

  2. Inhibition of Pyruvate Dehydrogenase Kinase 2 Protects Against Hepatic Steatosis Through Modulation of Tricarboxylic Acid Cycle Anaplerosis and Ketogenesis.

    Science.gov (United States)

    Go, Younghoon; Jeong, Ji Yun; Jeoung, Nam Ho; Jeon, Jae-Han; Park, Bo-Yoon; Kang, Hyeon-Ji; Ha, Chae-Myeong; Choi, Young-Keun; Lee, Sun Joo; Ham, Hye Jin; Kim, Byung-Gyu; Park, Keun-Gyu; Park, So Young; Lee, Chul-Ho; Choi, Cheol Soo; Park, Tae-Sik; Lee, W N Paul; Harris, Robert A; Lee, In-Kyu

    2016-10-01

    Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for β-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease.

  3. Antiplasmodial Activities of Homogentisic Acid Derivative Protein Kinase Inhibitors Isolated from a Vanuatu Marine Sponge Pseudoceratina sp.

    Directory of Open Access Journals (Sweden)

    Dominique Laurent

    2009-11-01

    Full Text Available As part of our search for new antimalarial drugs in South Pacific marine sponges, we have looked for inhibitors of Pfnek-1, a specific protein kinase of Plasmodium falciparum. On the basis of promising activity in a preliminary screening, the ethanolic crude extract of a new species of Pseudoceratina collected in Vanuatu was selected for further investigation. A bioassay-guided fractionation led to the isolation of a derivative of homogentisic acid [methyl (2,4-dibromo-3,6-dihydroxyphenylacetate, 4a] which inhibited Pfnek-1 with an IC50 around 1.8 μM. This product was moderately active in vitro against a FcB1 P. falciparum strain (IC50 = 12 μM. From the same sponge, we isolated three known compounds [11,19-dideoxyfistularin-3 (1, 11-deoxyfistularin-3 (2 and dibromo-verongiaquinol (3] which were inactive against Pfnek-1. Synthesis and biological evaluation of some derivatives of 4a are reported.

  4. Antiplasmodial activities of homogentisic acid derivative protein kinase inhibitors isolated from a Vanuatu marine sponge Pseudoceratina sp.

    Science.gov (United States)

    Lebouvier, Nicolas; Jullian, Valérie; Desvignes, Isabelle; Maurel, Séverine; Parenty, Arnaud; Dorin-Semblat, Dominique; Doerig, Christian; Sauvain, Michel; Laurent, Dominique

    2009-11-23

    As part of our search for new antimalarial drugs in South Pacific marine sponges, we have looked for inhibitors of Pfnek-1, a specific protein kinase of Plasmodium falciparum. On the basis of promising activity in a preliminary screening, the ethanolic crude extract of a new species of Pseudoceratina collected in Vanuatu was selected for further investigation. A bioassay-guided fractionation led to the isolation of a derivative of homogentisic acid [methyl (2,4-dibromo-3,6-dihydroxyphenyl)acetate, 4a] which inhibited Pfnek-1 with an IC(50) around 1.8 muM. This product was moderately active in vitro against a FcB1 P. falciparum strain (IC(50) = 12 muM). From the same sponge, we isolated three known compounds [11,19-dideoxyfistularin-3 (1), 11-deoxyfistularin-3 (2) and dibromo-verongiaquinol (3)] which were inactive against Pfnek-1. Synthesis and biological evaluation of some derivatives of 4a are reported.

  5. 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.

  6. AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats.

    Science.gov (United States)

    Assifi, Murwarid M; Suchankova, Gabriela; Constant, Scarlet; Prentki, Marc; Saha, Asish K; Ruderman, Neil B

    2005-11-01

    Acute increases in the concentration of malonyl-CoA play a pivotal role in mediating the decrease in fatty acid oxidation that occurs in many tissues during refeeding after a fast. In this study, we assess whether such increases in malonyl-CoA in liver could be mediated by malonyl-CoA decarboxylase (MCD), as well as acetyl-CoA carboxylase (ACC). In addition, we examine how changes in the activity of ACC, MCD, and other enzymes that govern fatty acid and glycerolipid synthesis relate temporally to alterations in the activities of the fuel-sensing enzyme AMP-activated protein kinase (AMPK). Rats starved for 48 h and refed a carbohydrate chow diet for 1, 3, 12, and 24 h were studied. Refeeding caused a 40% decrease in the activity of the alpha1-isoform of AMPK within 1 h, with additional decreases in AMPKalpha1 activity and a decrease in AMPKalpha2 occurring between 1 and 24 h. At 1 h, the decrease in AMPK activity was associated with an eightfold increase in the activity of the alpha1-isoform of ACC and a 30% decrease in the activity of MCD, two enzymes thought to be regulated by AMPK. Also, the concentration of malonyl-CoA was increased by 50%. Between 1 and 3 h of refeeding, additional increases in the activity of ACC and decreases in MCD were observed, as was a further twofold increase in malonyl-CoA. Increases in the activity (60%) and abundance (12-fold) of fatty acid synthase occurred predominantly between 3 and 24 h and increases in the activity of mitochondrial sn-glycerol-3-phosphate acyltransferase (GPAT) and acyl-CoA:diaclyglycerol acyltransferase (DGAT) at 12 and 24 h. The results strongly suggest that early changes in the activity of MCD, as well as ACC, contribute to the increase in hepatic malonyl-CoA in the starved-refed rat. They also suggest that the changes in these enzymes, and later occurring increases in enzymes regulating fatty acid and glycerolipid synthesis, could be coordinated by AMPK.

  7. 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.

  8. The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

    Institute of Scientific and Technical Information of China (English)

    Xuewen Liu; Cui Ma; Ruixian Xing; Weiwei Zhang; Buxian Tian; Xidong Li; Qiushi Li; Yanhui Zhang

    2013-01-01

    In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyl-D-aspartic acid-induced injury. Results showed that, compared with N-methyl-Daspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2+ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 μM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.

  9. CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

    Science.gov (United States)

    Bucurenci, N; Sakamoto, H; Briozzo, P; Palibroda, N; Serina, L; Sarfati, R S; Labesse, G; Briand, G; Danchin, A; Bărzu, O; Gilles, A M

    1996-02-02

    CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

  10. 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

  11. Effects on polo-like kinase 1 polo-box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position.

    Science.gov (United States)

    Qian, Wenjian; Park, Jung-Eun; Liu, Fa; Lee, Kyung S; Burke, Terrence R

    2013-07-15

    Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr β-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics. Published by Elsevier Ltd.

  12. Trypanosoma cruzi: Genome characterization of phosphatidylinositol kinase gene family (PIK and PIK-related) and identification of a novel PIK gene.

    Science.gov (United States)

    Oliveira, Priscila; Lima, Fabio Mitsuo; Cruz, Mario Costa; Ferreira, Renata Carmona; Sanchez-Flores, Alejandro; Cordero, Esteban Maurício; Cortez, Danielle Rodrigues; Ferreira, Éden Ramalho; Briones, Marcelo Ribeiro da Silva; Mortara, Renato Arruda; da Silveira, José Franco; Bahia, Diana

    2014-07-01

    Chagas disease is caused by the protozoan Trypanosoma cruzi which affects 10 million people worldwide. Very few kinases have been characterized in this parasite, including the phosphatidylinositol kinases (PIKs) that are at the heart of one of the major pathways of intracellular signal transduction. Recently, we have classified the PIK family in T. cruzi using five different models based on the presence of PIK conserved domains. In this study, we have mapped PIK genes to the chromosomes of two different T. cruzi lineages (G and CL Brener) and determined the cellular localization of two PIK members. The kinases have crucial roles in metabolism and are assumed to be conserved throughout evolution. For this reason, they should display a conserved localization within the same eukaryotic species. In spite of this, there is an extensive polymorphism regarding PIK localization at both genomic and cellular levels, among different T. cruzi isolates and between T. cruzi and Trypanosomabrucei, respectively. We showed in this study that the cellular localization of two PIK-related proteins (TOR1 and 2) in the T. cruzi lineage is distinct from that previously observed in T. brucei. In addition, we identified a new PIK gene with peculiar feature, that is, it codes for a FYVE domain at N-terminal position. FYVE-PIK genes are phylogenetically distant from the groups containing exclusively the FYVE or PIK domain. The FYVE-PIK architecture is only present in trypanosomatids and in virus such as Acanthamoeba mimivirus, suggesting a horizontal acquisition. Our Bayesian phylogenetic inference supports this hypothesis. The exact functions of this FYVE-PIK gene are unknown, but the presence of FYVE domain suggests a role in membranous compartments, such as endosome. Taken together, the data presented here strengthen the possibility that trypanosomatids are characterized by extensive genomic plasticity that may be considered in designing drugs and vaccines for prevention of Chagas disease.

  13. Src family kinase inhibitor PP2 efficiently inhibits cervical cancer cell proliferation through down-regulating phospho-Src-Y416 and phospho-EGFR-Y1173.

    Science.gov (United States)

    Kong, Lu; Deng, Zhihong; Shen, Haiying; Zhang, Yuxiang

    2011-02-01

    Tyrosine (Y) kinases inhibitors have been approved for targeted treatment of cancer. However, their clinical use is limited to some cancers and the mechanism of their action remains unclear. Previous study has indicated that PP2, a selective inhibitor of the Src family of non-receptor tyrosine kinases (nRTK), efficiently repressed cervical cancer growth in vitro and in vivo. In this regard, our aims are to explore the mechanism of PP2 on cervical cancer cell growth inhibition by investigating the suppressive divergence among PP1, PP2, and a negative control compound PP3. MTT results showed that three compounds had different inhibitory effects on proliferation of two cervical cancer cells, HeLa and SiHa, and PP2 was most efficient in a time- and dose-dependent manner. Moreover, we found 10 μM PP2 down-regulated pSrc-Y416 (P < 0.05), pEGFR-Y845 (P < 0.05), and -Y1173 (P < 0.05) expression levels, while 10 μM PP1 down-regulated pSrc-Y416 (P < 0.05) and pEGFR-Y845 (P < 0.05), but not pEGFR-Y1173; 10 μM PP3 down-regulated only pEGFR-Y1173 (P < 0.05). PP2 could modulate cell cycle arrest by up-regulating p21(Cip1) and p27(Kip1) in both HeLa and SiHa cells and down-regulating expression of cyclin A, and cyclin dependent kinase-2, -4 (Cdk-2, -4) in HeLa and of cyclin B and Cdk-2 in SiHa. Our results indicate that Src pathway and EGFR pathway play different roles in the proliferation of cervical cancer cells and PP2 efficiently reduces cervical cancer cell proliferation by reduction of both Src and EGFR activity.

  14. The Receptor-Like Cytoplasmic Kinase(OsRL CK) Gene Family in Rice:Organization,Phylogenetic Relationship,and Expressionduring Development and Stress

    Institute of Scientific and Technical Information of China (English)

    Shubha Vij; Jitender Giri; Prasant Kumar Dansana; Sanjay Kapoor; Akhilesh K.Tyagi

    2008-01-01

    Receptor-like cytoplasmic kinases(RLCKs)in plants belong to the super family of receptor-like kinases(RLKs).These proteins show homology to RLKs in kinase domain but Iack the transmembrane domain.Some of the functionally characterized RLCKs from plants have been shown to play roles jn development and stress responses.Previously,149 and 187 RLCK encoding genes were identified from Arabidopsis and rice,respectively.By using HMM-based domain structure and phylogenetic relationships,we have identified 379 OsRLCKs from rice.OsRLCKs are distributed on all 12 chromosomes of rice and some members are located on duplicated chromosomal segments.Several OsRLCKs probably also undergo alternative splicing,some having evidence only in the form of gene models.To understand their possible functions,expression patterns during Iandmark stages of vegetative and reproductive development as welI as abiotic and biotic stress using microarray and MPSS-based data were analyzed.Real-time PCR-based expression profiling for a selected few genes confirmed the outcome of microarray analysis.Differential expression patterns observed for majority of OsRLCKs during development and stress suggest their involvement in diverse functions in rice.Majority of the stress-responsive OsRLCKs were also found to be localized within mapped regions of abiotic stress QTLs.Outcome of this study would help in selecting organ/development stage specific OsRLCK genes/targets for functionaI validation studies.

  15. Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection.

    Science.gov (United States)

    Zhang, Jun; Ping, Peipei; Wang, Guang-Wu; Lu, Ming; Pantaleon, Dawn; Tang, Xian-Liang; Bolli, Roberto; Vondriska, Thomas M

    2004-11-01

    Previous studies have indicated that PKC-epsilon is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-epsilon exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-epsilon signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-epsilon signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.

  16. RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration.

    Science.gov (United States)

    Wallert, Mark A; Hammes, Daniel; Nguyen, Tony; Kiefer, Lea; Berthelsen, Nick; Kern, Andrew; Anderson-Tiege, Kristina; Shabb, John B; Muhonen, Wallace W; Grove, Bryon D; Provost, Joseph J

    2015-03-01

    The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

  17. 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

    2015-10-20

    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.

  18. Hereditary Xerocytosis due to Mutations in PIEZO1 Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families

    Science.gov (United States)

    Vercellati, Cristina; Marcello, Anna Paola; Zaninoni, Anna; van Wijk, Richard; Mirra, Nadia; Curcio, Cristina; Cortelezzi, Agostino; Zanella, Alberto; Barcellini, Wilma; Bianchi, Paola

    2017-01-01

    Hereditary xerocytosis (HX) is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve. PIEZO1 gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy. PMID:28367341

  19. Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways.

    Science.gov (United States)

    Semaan, Noha; Alsaleh, Ghada; Gottenberg, Jacques-Eric; Wachsmann, Dominique; Sibilia, Jean

    2008-03-01

    MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.

  20. Hereditary Xerocytosis due to Mutations in PIEZO1 Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families

    Directory of Open Access Journals (Sweden)

    Elisa Fermo

    2017-01-01

    Full Text Available Hereditary xerocytosis (HX is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve. PIEZO1 gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy.

  1. [Impact of family members and health care providers on the use of folic acid in pregnant women].

    Science.gov (United States)

    Zheng, Shan; Wang, Minzhen; Bai, Ya'na; Hu, Xiaobin; Zhang, Rongqiang; Du, Wenqi; Li, Liansheng; Li, Jingyu; Cheng, Ning

    2011-01-01

    To investigate the impact of family members and health care providers on the use of folic acid supplements in pregnant women, and to provide basic data for improving the effectiveness of folic acid intervention. A cross-sectional study was conducted in hospitals and households from June to September in 2009. Face-to-face anonymous questionnaires were distributed to 2094 women, who were pregnant at least three months or postpartum in one year, in two counties of Gansu Province. The awareness rate of folic acid was in 62.2% of 2094 pregnant women, and 25.4% of them have taken folic acid. Higher knowledge about folic acid of family members (OR = 0.268, 95% CI 0.208 - 0.346), agreed with taking folic acid by family members (OR = 0.103, 95% CI 0.031 -0.338), and urging pregnant women to take folic acid by family members (OR = 0.147, 95% CI 0.115 - 0.190) were significant predictors for having folic acid taken by pregnant women. Propagating knowledge related to folic acid (OR = 0.252, 95% CI 0.197 - 0.323) and directing pregnant women to use folic acid (OR = 0.168, 95% CI 0.096 - 0.296) by health care providers were also the important predictors for folic acid intake. Family members and health care providers play an important role in affecting the use of folic acid among pregnant women. In order to improve the effectiveness of intervention with folic acid, family members of pregnant women and health care providers should be included into the target population to receive an intensive propaganda campaign on folic acid education to improve the use of folic acid in pregnant women extensively.

  2. Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet's Disease

    Science.gov (United States)

    Pektanc, Gulsum; Akkurt, Zeynep M.; Bozkurt, Mehtap; Turkcu, Fatih M.; Kalkanli-Tas, Sevgi

    2016-01-01

    Behçet's disease (BD) is a multisystemic chronic inflammatory disease characterized by relapsing oral and genital ulcers, uveitis, and skin lesions. The pathogenesis of BD is still unknown. Aberrant production of some cytokines/chemokines plays an important role in the pathogenesis of various inflammatory diseases. Revealing a key signaling regulatory mechanism involved in proinflammatory cytokines/chemokines production is critical for understanding of the pathogenesis of BD. The aim of this study was to determine the role of Src family kinases (SFKs) in production of some LPS-induced proinflammatory cytokines/chemokines in peripheral blood mononuclear cells (PBMC) of active BD patients. Chemical inhibition of SFKs activity impaired LPS-induced TNF-α production in PBMC of active BD patients, suggesting that modulating SFKs activity may be a potential target for BD treatment. PMID:27445436

  3. 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.

  4. Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet’s Disease

    Directory of Open Access Journals (Sweden)

    Sevgi Irtegun

    2016-01-01

    Full Text Available Behçet’s disease (BD is a multisystemic chronic inflammatory disease characterized by relapsing oral and genital ulcers, uveitis, and skin lesions. The pathogenesis of BD is still unknown. Aberrant production of some cytokines/chemokines plays an important role in the pathogenesis of various inflammatory diseases. Revealing a key signaling regulatory mechanism involved in proinflammatory cytokines/chemokines production is critical for understanding of the pathogenesis of BD. The aim of this study was to determine the role of Src family kinases (SFKs in production of some LPS-induced proinflammatory cytokines/chemokines in peripheral blood mononuclear cells (PBMC of active BD patients. Chemical inhibition of SFKs activity impaired LPS-induced TNF-α production in PBMC of active BD patients, suggesting that modulating SFKs activity may be a potential target for BD treatment.

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

    Institute of Scientific and Technical Information of China (English)

    Ramón Peces; Joost PH Drenth; Rene HM te Morsche; Pedro González; Carlos Peces

    2005-01-01

    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.

  6. Phytic acid down-regulates IL-8 secretion from colonic epithelial cells by influencing mitogen-activated protein kinase signaling pathway.

    Science.gov (United States)

    Wawszczyk, Joanna; Orchel, Arkadiusz; Kapral, Małgorzata; Hollek, Andrzej; Weglarz, Ludmiła

    2012-01-01

    Phytic acid (IP6) is an essential component of high fiber diet physiologically present in human large gut. It has been recognized to possess various significant health benefits effects including chemopreventive and have antineoplastic activity against various types of cancer. Moreover, its role in immune response through modulation of the secretion of proinflammatory cytokines and chemokines has been postulated. One of the signal transduction pathways involved in a variety of inflammatory responses is p38 mitogen-activated protein kinase (MAPK) pathway. The aim of this study was to examine effect of IP6 on human p38alpha MAP kinase activity and the expression of gene encoding p38 MAP kinase in unstimulated and IL-1beta-stimulated Caco-2 cells. Furthermore, the role of signaling pathways involving p38 MAP kinase in IP6-induced down-regulation of IL-8 secretion by unstimulated and IL-1beta-stimulated cells in the presence of p38 MAP kinase activator (anisomycin) and inhibitor (SB 203580) was evaluated. IP6 inhibited activity of recombinant p38 MAPK activity in dose-dependent manner. Treatment of cells with IP6 for 3 h resulted in decreased p38 MAP kinase expression in both unstimulated and stimulated with IL-1beta cells. The similar level of p38alpha mRNA was found in untreated and treated with IP6 cells after 6 and 12 h. Incubation of Caco-2 cells with anisomycin resulted in upregulation of IL-8 secretion and their pretreatment with anisomycin prior to IP6 addition showed down-regulation of IL-8 secretion compared to cells treated with anisomycin alone. The findings of this study show that p38 MAPK could be one of the molecular targets for IP6 in the intestinal epithelial cells and that IP6 inhibitory effect on IL-8 secretion by Caco-2 cells could be mediated by its inhibition of p38 activity.

  7. The human fatty acid-binding protein family: Evolutionary divergences and functions

    Directory of Open Access Journals (Sweden)

    Smathers Rebecca L

    2011-03-01

    Full Text Available Abstract Fatty acid-binding proteins (FABPs are members of the intracellular lipid-binding protein (iLBP family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20 fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied.

  8. A novel Bruton's tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia.

    Science.gov (United States)

    Chear, Chai Teng; Gill, Harvindar Kaur; Ramly, Nazatul Haslina; Dhaliwal, Jasbir Singh; Bujang, Noraini; Ripen, Adiratna Mat; Mohamad, Saharuddin Bin

    2013-12-01

    X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1Gagammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

  9. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

    Science.gov (United States)

    Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2014-09-01

    Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement.

  10. Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells.

    Science.gov (United States)

    Timiri Shanmugam, Prakash Srinivasan; Nair, Renjith Parameshwaran; De Benedetti, Arrigo; Caldito, Gloria; Abreo, Fleurette; Sunavala-Dossabhoy, Gulshan

    2016-04-01

    Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.

  11. Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Aicher, T D; Anderson, R C; Gao, J; Shetty, S S; Coppola, G M; Stanton, J L; Knorr, D C; Sperbeck, D M; Brand, L J; Vinluan, C C; Kaplan, E L; Dragland, C J; Tomaselli, H C; Islam, A; Lozito, R J; Liu, X; Maniara, W M; Fillers, W S; DelGrande, D; Walter, R E; Mann, W R

    2000-01-27

    N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

  12. N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

    Science.gov (United States)

    Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

    2017-06-24

    N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.

  13. Interaction of dietary fatty acids with tumour necrosis factor family cytokines during colon inflammation and cancer.

    Science.gov (United States)

    Hofmanová, Jiřina; Straková, Nicol; Vaculová, Alena Hyršlová; Tylichová, Zuzana; Safaříková, Barbora; Skender, Belma; Kozubík, Alois

    2014-01-01

    Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs) and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF- α, TRAIL, and FasL) have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA) or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NF κ B activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined.

  14. Interaction of Dietary Fatty Acids with Tumour Necrosis Factor Family Cytokines during Colon Inflammation and Cancer

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    Jiřina Hofmanová

    2014-01-01

    Full Text Available Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF-α, TRAIL, and FasL have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NFκB activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined.

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

    Science.gov (United States)

    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 minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca2+ channel, CCH1, and a functional vacuolar membrane H+-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid. PMID:15060153

  16. Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress.

    Science.gov (United States)

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

    2004-04-01

    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 minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca(2+) channel, CCH1, and a functional vacuolar membrane H(+)-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

  17. Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

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    Guo Honghui

    2012-01-01

    Full Text Available Abstract Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC and carnitine palmitoyl transferase 1 (CPT-1 pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g, a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease.

  18. Identification of an erythrocyte pyruvate kinase variant in a family from Latium with non-spherocytic congenital haemolytic anaemia.

    Science.gov (United States)

    Papa, G; De Laurenzi, A; Isacchi, G C; Bonifazi, G; Parziale, L; Salvati, A M

    1979-01-01

    Erythrocyte PK deficiency was detected in a family from Latium in Italy. This PK variant is characterized by normal or increased activity immediately after blood collection, instability to storage, to heat and to urea. Only in the propositus the mutant enzyme exhibited an increased Michaelis constant for PEP, slightly increased inhibition by ATP and an altered optimum pH value. The kinetic anomaly was only partially corrected by activation with F-1, 6-DP and by addition of 2-ME. From these results it can be concluded that in the family observed two distinct erythrocyte PK alterations were demonstrable: instability in the propositus and his father; low affinity for PEP and altered optimum pH value only in the propositus.

  19. Mutation analysis of the gene encoding Bruton`s tyrosine kinase in a family with a sporadic case of X-linked agammaglobulinemia reveals three female carriers

    Energy Technology Data Exchange (ETDEWEB)

    Hagemann, T.L.; Kwan, Sau-Ping [Rush Medical School, Chicago, IL (United States); Assa`ad, A.H. [Children`s Hospital Medical Center, Cincinnati, OH (United States)

    1995-11-06

    Bruton`s tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA). We and others have cloned the gene for Btk and recently reported the genomic organization. Nineteen exons were positioned within the 37 kb gene. With the sequence data derived from our genomic map, we have designed a PCR based assay to directly identify mutations of the Btk gene in germline DNA of patients with XLA. In this report, the assay was used to analyze a family with a sporadic case of XLA to determine if other female relatives carry the disease. A four base-pair deletion was found in the DNA of the affected boy and was further traced through three generations. With the direct identification of the mutations responsible for XLA, we can now diagnose conclusively the disease and identify the immunologically normal female carriers. This same technique can easily be applied to prenatal diagnosis in families where the mutation can be identified. 34 refs., 3 figs.

  20. FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor.

    Science.gov (United States)

    Azuma, Koichi; Kawahara, Akihiko; Sonoda, Kahori; Nakashima, Kazutaka; Tashiro, Kousuke; Watari, Kosuke; Izumi, Hiroto; Kage, Masayoshi; Kuwano, Michihiko; Ono, Mayumi; Hoshino, Tomoaki

    2014-08-15

    Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [1] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [2] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [3] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [4] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [5] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.

  1. The evolutionary history and diverse physiological roles of the grapevine calcium-dependent protein kinase gene family.

    Science.gov (United States)

    Chen, Fei; Fasoli, Marianna; Tornielli, Giovanni Battista; Dal Santo, Silvia; Pezzotti, Mario; Zhang, Liangsheng; Cai, Bin; Cheng, Zong-Ming

    2013-01-01

    Calcium-dependent protein kinases (CDPKs) are molecular switches that bind Ca(2+), ATP, and protein substrates, acting as sensor relays and responders that convert Ca(2+) signals, created by developmental processes and environmental stresses, into phosphorylation events. The precise functions of the CDPKs in grapevine (Vitis vinifera) are largely unknown. We therefore investigated the phylogenetic relationships and expression profiles of the 17 CDPK genes identified in the 12x grapevine genome sequence, resolving them into four subfamilies based on phylogenetic tree topology and gene structures. The origins of the CDPKs during grapevine evolution were characterized, involving 13 expansion events. Transcriptomic analysis using 54 tissues and developmental stages revealed three types of CDPK gene expression profiles: constitutive (housekeeping CDPKs), partitioned functions, and prevalent in pollen/stamen. We identified two duplicated CDPK genes that had evolved from housekeeping to pollen-prevalent functions and whose origin correlated with that of seed plants, suggesting neofunctionalization with an important role in pollen development and also potential value in the breeding of seedless varieties. We also found that CDPKs were involved in three abiotic stress signaling pathways and could therefore be used to investigate the crosstalk between stress responses.

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

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    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.

  4. 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

  5. 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

  6. 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...

  7. 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

  8. Mth10b, a unique member of the Sac10b family, does not bind nucleic acid.

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    Yan-Feng Liu

    Full Text Available The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.

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

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    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

  10. 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.

  11. Genome-wide analysis and evolutionary study of sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family members in Arabidopsis and Oryza.

    Science.gov (United States)

    Saha, Jayita; Chatterjee, Chitrita; Sengupta, Atreyee; Gupta, Kamala; Gupta, Bhaskar

    2014-04-01

    The over-expression of plant specific SnRK2 gene family members by hyperosmotic stress and some by abscisic acid is well established. In this report, we have analyzed the evolution of SnRK2 gene family in different plant lineages including green algae, moss, lycophyte, dicot and monocot. Our results provide some evidences to indicate that the natural selection pressure had considerable influence on cis-regulatory promoter region and coding region of SnRK2 members in Arabidopsis and Oryza independently through time. Observed degree of sequence/motif conservation amongst SnRK2 homolog in all the analyzed plant lineages strongly supported their inclusion as members of this family. The chromosomal distributions of duplicated SnRK2 members have also been analyzed in Arabidopsis and Oryza. Massively Parallel Signature Sequencing (MPSS) database derived expression data and the presence of abiotic stress related promoter elements within the 1 kb upstream promoter region of these SnRK2 family members further strengthen the observations of previous workers. Additionally, the phylogenetic relationships of SnRK2 have been studied in all plant lineages along with their respective exon-intron structural patterns. Our results indicate that the ancestral SnRK2 gene of land plants gradually evolved by duplication and diversification and modified itself through exon-intron loss events to survive under environmental stress conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

    Science.gov (United States)

    Marcotte, Douglas; Rushe, Mia; M Arduini, Robert; Lukacs, Christine; Atkins, Kateri; Sun, Xin; Little, Kevin; Cullivan, Michael; Paramasivam, Murugan; Patterson, Thomas A; Hesson, Thomas; D McKee, Timothy; May-Dracka, Tricia L; Xin, Zhili; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda R; Lyssikatos, Joseph P; Silvian, Laura F

    2017-02-01

    Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

  13. 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.

  14. Salvianolic Acid B Protects Normal Human Dermal Fibroblasts Against Ultraviolet B Irradiation-Induced Photoaging Through Mitogen-Activated Protein Kinase and Activator Protein-1 Pathways.

    Science.gov (United States)

    Sun, Zhengwang; Park, Sang-Yong; Hwang, Eunson; Zhang, Mengyang; Jin, Fengxie; Zhang, Baochun; Yi, Tae Hoo

    2015-01-01

    Exposure to ultraviolet (UV) light causes increased matrix metalloproteinase (MMP) activity and decreased collagen synthesis, leading to skin photoaging. Salvianolic acid B (SAB), a polyphenol, was extracted and purified from salvia miltiorrhiza. We assessed effects of SAB on UVB-induced photoaging and investigated its molecular mechanism of action in UVB-irradiated normal human dermal fibroblasts. Our results show that SAB significantly inhibited the UVB-induced expression of metalloproteinases-1 (MMP-1) and interleukin-6 (IL-6) while promoting the production of type I procollagen and transforming growth factor β1 (TGF-β1). Moreover, treatment with SAB in the range of 1-100 μg/mL significantly inhibited UVB-induced extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 phosphorylation, which resulted in decreasing UVB-induced phosphorylation of c-Fos and c-Jun. These results indicate that SAB downregulates UV-induced MMP-1 expression by inhibiting Mitogen-activated protein kinase (MAPK) signaling pathways and activator protein-1 (AP-1) activation. Our results suggest a potential use for SAB in skin photoprotection.

  15. Induction of Protection against Paraquat-induced Oxidative Damage by Abscisic Acid in Maize Leaves is Mediated through Mitogen-activated Protein Kinase

    Institute of Scientific and Technical Information of China (English)

    Hai-Dong Ding; Xiao-Hua Zhang; Shu-Cheng Xu; Li-Li Sun; Ming-Yi Jiang; A-Ying Zhang; Yin-Gen Jin

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascade has been shown to be important components In stress signal trans-duction pathway. In the present study, protection of maize seedlings (Zea mays L.) against paraquat-generated oxidative toxicity by abscisic acid (ABA), its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation (in terms of carbonyl groups) under paraquat (PQ) stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in-gel kinase assay with immunoprecipitation. These results reveal that ABA-induced protection against PQ-generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.

  16. 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...

  17. Cdc2-like kinase 2 suppresses hepatic fatty acid oxidation and ketogenesis through disruption of the PGC-1α and MED1 complex.

    Science.gov (United States)

    Tabata, Mitsuhisa; Rodgers, Joseph T; Hall, Jessica A; Lee, Yoonjin; Jedrychowski, Mark P; Gygi, Steven P; Puigserver, Pere

    2014-05-01

    Hepatic ketogenesis plays an important role in catabolism of fatty acids during fasting along with dietary lipid overload, but the mechanisms regulating this process remain poorly understood. Here, we show that Cdc2-like kinase 2 (Clk2) suppresses fatty acid oxidation and ketone body production during diet-induced obesity. In lean mice, hepatic Clk2 protein is very low during fasting and strongly increased during feeding; however, in diet-induced obese mice, Clk2 protein remains elevated through both fed and fasted states. Liver-specific Clk2 knockout mice fed a high-fat diet exhibit increased fasting levels of blood ketone bodies, reduced respiratory exchange ratio, and increased gene expression of fatty acid oxidation and ketogenic pathways. This effect of Clk2 is cell-autonomous, because manipulation of Clk2 in hepatocytes controls genes and rates of fatty acid utilization. Clk2 phosphorylation of peroxisome proliferator-activated receptor γ coactivator (PGC-1α) disrupts its interaction with Mediator subunit 1, which leads to a suppression of PGC-1α activation of peroxisome proliferator-activated receptor α target genes in fatty acid oxidation and ketogenesis. These data demonstrate the importance of Clk2 in the regulation of fatty acid metabolism in vivo and suggest that inhibition of hepatic Clk2 could provide new therapies in the treatment of fatty liver disease.

  18. Activation of the aryl hydrocarbon receptor by the calcium/calmodulin-dependent protein kinase kinase inhibitor 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid (STO-609).

    Science.gov (United States)

    Monteiro, Patricia; Gilot, David; Langouet, Sophie; Fardel, Olivier

    2008-12-01

    This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

  19. MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases.

    Science.gov (United States)

    Botta, A; Malena, A; Tibaldi, E; Rocchi, L; Loro, E; Pena, E; Cenci, L; Ambrosi, E; Bellocchi, M C; Pagano, M A; Novelli, G; Rossi, G; Monaco, H L; Gianazza, E; Pantic, B; Romeo, V; Marin, O; Brunati, A M; Vergani, L

    2013-08-15

    Myotonic dystrophy type-1 (DM1) is the most prevalent form of muscular dystrophy in adults. This disorder is an RNA-dominant disease, caused by expansion of a CTG repeat in the DMPK gene that leads to a misregulation in the alternative splicing of pre-mRNAs. The longer muscleblind-like-1 (MBNL1) transcripts containing exon 5 and the respective protein isoforms (MBNL142-43) were found to be overexpressed in DM1 muscle and localized exclusively in the nuclei. In vitro assays showed that MBNL142-43 bind the Src-homology 3 domain of Src family kinases (SFKs) via their proline-rich motifs, enhancing the SFK activity. Notably, this association was also confirmed in DM1 muscle and myotubes. The recovery, mediated by an siRNA target to Ex5-MBNL142-43, succeeded in reducing the nuclear localization of both Lyn and MBNL142-43 proteins and in decreasing the level of tyrosine phosphorylated proteins. Our results suggest an additional molecular mechanism in the DM1 pathogenesis, based on an altered phosphotyrosine signalling pathway.

  20. 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.

  1. Phylogeny of Tec family kinases identification of a premetazoan origin of Btk, Bmx, Itk, Tec, Txk, and the Btk regulator SH3BP5.

    Science.gov (United States)

    Ortutay, Csaba; Nore, Beston F; Vihinen, Mauno; Smith, C I Edvard

    2008-01-01

    It is generally considered mammals and birds have five Tec family kinases (TFKs): Btk, Bmx (also known as Etk), Itk, Tec, and Txk (also known as Rlk). Here, we discuss the domains and their functions and regulation in TFKs. Over the last few years, a large number of genomes from various phyla have been sequenced making it possible to study evolutionary relationships at the molecular and sequence level. Using bioinformatics tools, we for the first time demonstrate that a TFK ancestor exists in the unicellular choanoflagellate Monosiga brevicollis, which is the closest known relative to metazoans with a sequenced genome. The analysis of the genomes for sponges, insects, hagfish, and frogs suggests that these species encode a single TFK. The insect form has a divergent and unique N-terminal region. Duplications generating the five members took place prior to the emergence of vertebrates. Fishes have two or three forms and the platypus, Ornithorhynchus anatinus, has four (lacks Txk). Thus, not all mammals have all five TFKs. The single identified TFK in frogs is an ortholog of Tec. Bmx seems to be unique to mammals and birds. SH3BP5 is a negative regulator of Btk. It is conserved in choanoflagellates and interestingly exists also in nematodes, which do not express TFKs, suggesting a broader function in addition to Btk regulation. The related SH3BP5-like protein is not found in Nematodes.

  2. Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

    Science.gov (United States)

    Burnett, James C.; Nuss, Jonathan E.; Wanner, Laura M.; Peyser, Brian D.; Du, Hao T.; Gomba, Glenn Y.; Kota, Krishna P.; Panchal, Rekha G.; Gussio, Rick; Kane, Christopher D.; Tessarollo, Lino

    2015-01-01

    Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins’ proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin’s enzymatic components, to antagonize multiple BoNT serotypes in motor neurons. PMID:25782580

  3. Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

    Directory of Open Access Journals (Sweden)

    Hyun-Soo Kim

    2014-03-01

    Full Text Available Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02, casein kinase II (CKII, and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

  4. Protein tyrosine kinase, JNK, and ERK involvement in p seudolaric acid B-induced apoptosis of human breast cancer MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Jing-hua YU; Hong-jun WANG; Xiang-ru LI; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2008-01-01

    Aim:To investigate the apoptotic mechanism ofpseudolaric acid B (PAB) in hu-man breast cancer MCF-7 cells. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide analysis and morphological changes were applied to detect apoptosis. The percentage of apoptotic and necrotic cells were calculated by the lactate dehydrogenase activity-based cytotoxicity assay, and the protein expression was examined by Western blot analysis. Results: PAB and/or the mitogen-activated protein kinases, including p38, c-Jun-N-terrninal kinase (JNK) and extracellular signal-regulated kinase (ERK), did not participate in necrosis. P38 had no obvious function on apoptosis after 4 μmol/L PAB treatment for 36 h, but PAB induced JNK phosphorylation and inhibited ERK phosphorylation in the apoptotic process. In this study the inhibitor of protein tyrosine kinase (PTK) genistein inverted the inhibitory effect of PAB, instead promoting the survival of MCF-7 cells. Like genistein, another PTK inhibitor AG1024 had a similar ef-fect on PAB-treated MCF-7 cells, indicating that PAB activated PTK to induce apoptosis. Together with PAB, genistein increased the expression of p-ERK, and decreased the expressions of JNK and p-JNK in PAB-treated MCF-7 cells at 36 h. And it is considered that the p-ERK and p-JNK were active patterns of ERK and JNK, respectively. Conclusion: PTK were upstream of ERK and JNK, and PTK induced apoptosis through activating JNK and inactivating ERK in PAB-treated MCF-7 cells.

  5. Chrysophanic acid reduces testosterone-induced benign prostatic hyperplasia in rats by suppressing 5α-reductase and extracellular signal-regulated kinase.

    Science.gov (United States)

    Youn, Dong-Hyun; Park, Jinbong; Kim, Hye-Lin; Jung, Yunu; Kang, JongWook; Jeong, Mi-Young; Sethi, Gautam; Seok Ahn, Kwang; Um, Jae-Young

    2017-02-07

    Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further in vitro studies with RWPE-1 cells confirmed TP-induced proliferation and elevated AR, PSA and p-ERK were all reduced by CA treatment. Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH.

  6. Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Long Zi-Jie

    2011-05-01

    Full Text Available Abstract Background Aurora kinase ensures accurate chromosome segregation during cell cycle, maintaining genetic integrity in cell division. VX-680, a small-molecule Aurora kinase inhibitor, interferes with mitotic entry and formation of bipolar spindles. Here, we evaluated VX-680 as a potential agent for treatment of all-trans retinoid acid (ATRA-resistant acute promyelocytic leukemia (APL in vitro. Methods CD11b expression was utilized to assess cell differentiation by flow cytometry. Immunofluorescence staining was conducted to analyze formation of cell monopolar spindle. Cell proliferation was evaluated by MTT assay. Sub-G1 population and Annexin V/PI staining were used to measure cell apoptosis. Hoechst 33342 staining was applied for identifying morphological changes in nucleus of apoptotic cell. Aurora-A (Aur-A activation and the signaling pathways involved in apoptosis were detected by Western blot. JC-1 probe was employed to measure mitochondrial depolarization. Results VX-680 inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288, accompanied by producing monopolar mitotic spindles in APL cell line NB4-R2 that was resistant to ATRA. In addition, we found that VX-680 inhibited cell proliferation as assessed by MTT assay. Flow cytometry showed that VX-680 led to apoptotic cell death in both dose- and time-dependent manners by either Sub-G1 or Annexin V/PI analysis. Hoechst 33342 staining represented typical apoptotic cells with nuclear fragmentation in VX-680 treated cells. Importantly, VX-680 inhibition of Aurora kinase suppressed Akt-1 activation and induced mitochondrial depolarization, which eventually resulted in apoptosis by activation of caspase pathway, as indicated by increasing proteolytic cleavage of procaspase-3 and poly ADP ribose polymerase (PARP in NB4-R2 cells. Conclusions Our study suggested potential clinical use of mitotic Aurora kinase inhibitor in targeting ATRA-resistant leukemic cells.

  7. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

  8. 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.

  9. The role of arachidonic acid/cyclooxygenase cascade, phosphodiesterase IV and Rho-kinase in H2S-induced relaxation in the mouse corpus cavernosum.

    Science.gov (United States)

    Aydinoglu, Fatma; Ogulener, Nuran

    2017-08-01

    Penile corpus cavernosum is an extremely vascularized tissue and cavernosal smooth muscle tone is regulated by the balance between contractile and relaxant factor. We investigated the possible role of arachidonic acid/cyclooxygenase cascade, phosphodiesterase IV (PDEIV) and Rho-kinase in exogenous hydrogen sulfide (H2S)-induced relaxation in mouse corpus cavernosum. The relaxant response to H2S (NaHS as exogenous H2S; 1-1000μM) were obtained in isolated mouse corpus cavernosum tissues which pre-contracted by phenylephrine (5μM). The effects of 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA; 10μM), a selective phospholipase A2 (PLA2) inhibitor, indomethacin (1μM), a non-selective cyclooxygenase (COX) inhibitor, baicalein (10μM), a lipoxygenase (LOX) inhibitor, and proadifen (10μM), cytochrome P450 inhibitor, on the relaxant responses to H2S were investigated. Furthermore, the effects of theophylline (500μM) and rolipram (1μM), a non-selective and selective PDEIV inhibitor, and fasudil (3μM), a specific Rho-kinase inhibitor, were studied on H2S-induced relaxation. H2S-induced relaxations were significantly reduced by OBAA, indomethacin and proadifen but not baicalein. Furthermore, theophylline, rolipram and fasudil reduced H2S-induced relaxations. These results suggest that PLA2, COX, cytochrome P450, PDEIV and Rho-kinase pathway may involve in H2S-induced relaxation in mouse corpus cavernosum tissues. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  10. Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

    Science.gov (United States)

    Sugden, M C; Lall, H S; Harris, R A; Holness, M J

    2000-11-01

    The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

  11. A retinoic acid receptor beta agonist (CD2019) overcomes inhibition of axonal outgrowth via phosphoinositide 3-kinase signalling in the injured adult spinal cord.

    Science.gov (United States)

    Agudo, Marta; Yip, Ping; Davies, Meirion; Bradbury, Elizabeth; Doherty, Patrick; McMahon, Stephen; Maden, Malcolm; Corcoran, Jonathan P T

    2010-01-01

    After spinal cord injury in the adult mammal, axons do not normally regrow and this commonly leads to paralysis. Retinoic acid (RA) can stimulate neurite outgrowth in vitro of both the embryonic central and peripheral nervous system, via activation of the retinoic acid receptor (RAR) beta2. We show here that regions of the adult CNS, including the cerebellum and cerebral cortex, express RARbeta2. We show that when cerebellar neurons are grown in the presence of myelin-associated glycoprotein (MAG) which inhibits neurite outgrowth, RARbeta can be activated in a dose dependent manner by a RARbeta agonist (CD2019) and neurite outgrowth can occur via phosphoinositide 3-kinase (PI3K) signalling. In a model of spinal cord injury CD2019 also acts through PI3K signalling to induce axonal outgrowth of descending corticospinal fibres and promote functional recovery. Our data suggest that RARbeta agonists may be of therapeutic potential for human spinal cord injuries.

  12. New Treatment of Medullary and Papillary Human Thyroid Cancer: Biological Effects of Hyaluronic Acid Hydrogel Loaded With Quercetin Alone or in Combination to an Inhibitor of Aurora Kinase.

    Science.gov (United States)

    Quagliariello, Vincenzo; Armenia, Emilia; Aurilio, Caterina; Rosso, Francesco; Clemente, Ottavia; de Sena, Gabriele; Barbarisi, Manlio; Barbarisi, Alfonso

    2016-08-01

    The aim of this paper is based on the use of a hyaluronic acid hydrogel of Quercetin tested alone and in combination to an inhibitor of Aurora Kinase type A and B (SNS-314) on human medullary and papillary thyroid cancer cells. Biological investigations were focused on the cellular uptake of the hydrogel, cell viability, antioxidant, and cytokines secretion studies. Quercetin delivered from hydrogel show a time and CD44 dependent interaction with both cell lines with significant anti-inflammatory effects. Combination of Quercetin and SNS-314 leads to a synergistic cytotoxic effect on medullary TT and papillary BCPAP cell lines with a significant reduction of the IC50 value. These results, highlights the importance of synergistic effect of the hyaluronic acid hydrogel of Quercetin with SNS-314 in the regulation of human thyroid cancer cell proliferation and emphasize the anti-tumor activity of these molecules. J. Cell. Physiol. 231: 1784-1795, 2016. © 2015 Wiley Periodicals, Inc.

  13. 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.

  14. A critical role of Src family kinase in SDF-1/CXCR4-mediated bone-marrow progenitor cell recruitment to the ischemic heart.

    Science.gov (United States)

    Cheng, Min; Huang, Kai; Zhou, Junlan; Yan, Dewen; Tang, Yao-Liang; Zhao, Ting C; Miller, Richard J; Kishore, Raj; Losordo, Douglas W; Qin, Gangjian

    2015-04-01

    The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP(+) MNCs and eGFP(+)c-kit(+) PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP(+) and eGFP(+)c-kit(+) cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment.

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

    Science.gov (United States)

    Sauviac, Laurent; Bruand, Claude

    2014-07-01

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

  16. Phosphorylation of acidic ribosomal proteins by ribosome-associated protein kinases of ``Saccharomyces cerevisiae`` and ``Schizosaccharomyces pombe``

    Energy Technology Data Exchange (ETDEWEB)

    Jakubowicz, T.; Cytrynska, M.; Kowalczyk, W.; Gasior, E. [Uniwersytet Marii Curie-Sklodowskiej, Lublin (Poland)

    1993-12-31

    Two proteins of 13 kDa and 38 kDa, the components of 60S ribosomal subunits, were identified as phosphorylation substrates for protein tightly associated with ``S. cerevisiae`` and ``Schizosaccharomyces pombe`` ribosomes. An enzyme with properties of multifunctional casein kinase II was detected in ribosome preparations from both yeast species. In S. cerevisiae another protein kinase with high substrate specificity toward those proteins was also identified. By using isoelectric focusing, the protein band of 13 kDa from ``S. cerevisiae`` and ``S. pombe`` was resolved respectively into three and four major forms of different charge. The same protein forms were phosphorylated in the in vivo {sup 32}P-labelling experiments. (author). 33 refs, 6 figs.

  17. Metabolic pathways and fermentative production of L-aspartate family amino acids.

    Science.gov (United States)

    Park, Jin Hwan; Lee, Sang Yup

    2010-06-01

    The L-aspartate family amino acids (AFAAs), L-threonine, L-lysine, L-methionine and L-isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L-threonine, L-lysine and L-methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.

  18. Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption

    Institute of Scientific and Technical Information of China (English)

    Marco Montagnani; Anna Abrahamsson; Cecilia G(a)lman; G(o)sta Eggertsen; Hanns-Ulrich Marschall; Elisa Ravaioli; Curt Einarsson; Paul A Dawson

    2006-01-01

    The etiology of most cases of idiopathic bile acid malabsorption (TBAM) is unknown. Tn this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR)and peroxisome proliferator activated receptor alpha (PPARα). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using 75Se-homocholic acid taurine(SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4). The ASBT,FXR, and PPARα genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC,and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARα genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.

  19. Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids.

    Science.gov (United States)

    Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; McKeehan, Wallace L

    2005-05-06

    The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

  20. Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases.

    Science.gov (United States)

    Siniossoglou, Symeon

    2013-03-01

    Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Regulation of the expression and activity of glucose and lactic acid metabolism-related genes by protein kinase C in skeletal muscle cells.

    Science.gov (United States)

    Otake, Sho; Kobayashi, Masaki; Narumi, Katsuya; Sasaki, Shotaro; Kikutani, Yurika; Furugen, Ayako; Watanabe, Meguho; Takahashi, Natsuko; Ogura, Jiro; Yamaguchi, Hiroaki; Iseki, Ken

    2013-01-01

    Protein kinase C (PKC) modulators are very attractive therapeutic targets in cancer. Since most cancer cells display increased glycolysis, elucidations of the effects of PKC activation on glycolysis is necessary for the development of effective medicine. In the present study, to clarify the role of PKC in the regulation of glycolysis, we examined the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on the expression and activity of glucose and lactic acid metabolism-related genes in human rhabdomyosarcoma cells (RD cells). In parallel to increases in glucose uptake and mRNA levels of glucose transporters (GLUTs) induced by PMA treatment for 6 h, the hexokinase (HK) mRNA level and activity were also significantly increased in RD cells. On the other hand, a significant increase in lactate dehydrogenase (LDH) mRNA level and activity was seen when the cells were incubated with PMA for 24 h, but not for 6 or 12 h, and was associated with lactic acid production. These effects by PMA treatment were markedly suppressed by Bisindolylmaleimide (BIM), a PKC inhibitor. Furthermore, chetomin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, completely abrogated the increment of LDH mRNA level and activity as well as monocarboxylate transporter (MCT) 4, a lactic acid efflux transporter. In conclusion, we found that HK and LDH activity induced by PKC activation was associated with the glucose uptake and lactic acid level and that LDH and MCT4 are modulated by a common factor, HIF-1.

  2. Inhibition of protein kinase B by Palmitate in the insulin signaling of HepG2 cells and the preventive effect of Arachidonic acid on insulin resistance

    Institute of Scientific and Technical Information of China (English)

    XIA Yanzhi; WAN Xuedong; DUAN Qiuhong; HE Shansu; WANG Ximing

    2007-01-01

    Elevated plasma levels of free fatty acids(FFAs)may contribute to insulin resistance (IR)that is characteristic of type 2 diabetes mellitus.In this study,we investigated the effects of two fatty acids,palmitate(PA)and arachidonic acid (AA)on glycogenesis under insulin signaling in HepG2cells,a transformed hepatic carcinoma cell line.In the presence of 200 μmol of palmitate,insulin(10-7 mol/L)stimulation of glycogenesis was inhibited,as evidenced by increased glucose in the medium and decreased intracellular glycogen.Wortmannin(WM),a specific inhibitor of PI3K,dramatically decreased the amount of intracellular glycogen in cells without PA incubation.However,glycogen in PA treated cells was not significantly changed by WM,indicating that PA may also act on PI3K.Interestingly,AA restored the effects of WM inhibition on glycogenesis in PA cells.Western blot analysis demonstrated that PA in the absence of WM increased phosphorylated glycogen synthase(inactive form of GS)and decreased phosphorylated protein kinase B(active form of PKB),causing a reduction of intracellular glycogen.AA,however,reversed the effects of PA on GS and PKB.Furthermore,inhibition of protein kinase C(PKC)by a specific inhibitor chelerythrine chloride (CC)abolished the inhibitory efrect of PA on glycogen synthesis by decreasing phosphorylated GS and increasing phosphorylated PKB.However,the effect of CC in the presence of PA disappeared when AA was also present.Our results suggest that there is a disruption of the insulin signaling pathway between PKB and GS when the cells were exposed to PA,contributing to IR.PA may also interrupt the PKC signaling pathway.In contrast,AA could rescue glycogenesis impaired by PA.

  3. Nutritional modulation of guinea pig skin hyperproliferation by essential fatty acid deficiency is associated with selective down regulation of protein kinase C-beta.

    Science.gov (United States)

    Cho, Y; Ziboh, V A

    1995-11-01

    In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-beta) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-alpha and beta-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-beta expression and activity and the reversal of hyperproliferation. In contrast, although, the expression and activity of PKC-alpha was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-alpha expression was not down regulated after refeeding the safflower oil diet to the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Common Variants in Serum/Glucocorticoid Regulated Kinase 1 (SGK1) and Blood Pressure Responses to Dietary Sodium or Potassium Interventions: A family-Based Association Study.

    Science.gov (United States)

    Chu, Chao; Wang, Yang; Wang, Man; Mu, Jian-Jun; Liu, Fu-Qiang; Wang, Lan; Ren, Ke-Yu; Wang, Dan; Yuan, Zu-Yi

    2015-01-01

    Serum/Glucocorticoid Regulated Kinase 1 (SGK1) plays a significant role in regulating renal Na(+) reabsorption, K(+) secretion, and blood pressure (BP). This study aimed to assess the association of common genetic variants in the SGK1 gene with BP responses to controlled dietary sodium or potassium interventions. A total of 334 subjects from 124 families were recruited from the rural areas of northern China. After a three-day baseline observation, they were sequentially maintained a seven-day low-sodium diet (3g/day of NaCl or 51.3 mmol/day of sodium), a seven-day high-sodium diet (18 g/day of NaCl or 307.8 mmol/day of sodium) and a seven-day high-sodium plus potassium supplementation intervention (4.5 g/day of KCl or 60 mmol/day of potassium). Six single-nucleotide polymorphisms (SNPs) in the SGK1 gene were selected. After adjustment for multiple testing, SNP rs9376026 was significantly associated with diastolic BP (DBP) and mean arterial pressure (MAP) responses to low-sodium intervention (P = 0.018 and 0.022, respectively). However, the associations between selected SNPs in the SGK1 gene and BP responses to high-sodium or high-sodium plus potassium-supplementation intervention did not reach statistical significance. In addition, SNP rs9389154 and two other SNPs (rs1763509 and rs9376026) were associated respectively with systolic BP (SBP) and DBP at baseline (P = 0.040, 0.032, and 0.031, respectively). SNP rs3813344 was significantly associated with SBP, DBP, and MAP (P = 0.049, 0.015 and 0.018, respectively). Our study indicates that the genetic polymorphism in the SGK1 gene is significantly associated with BP responses to dietary sodium intervention. © 2015 S. Karger AG, Basel.

  5. Common variation in with no-lysine kinase 1 (WNK1) and blood pressure responses to dietary sodium or potassium interventions- family-based association study.

    Science.gov (United States)

    Liu, Fuqiang; Zheng, Shuhui; Mu, Jianjun; Chu, Chao; Wang, Lan; Wang, Yang; Xiao, Hongyu; Wang, Dan; Cao, Yu; Ren, Keyu; Liu, Enqi; Yuan, Zuyi

    2013-01-01

    Common variations in the gene with no-lysine kinase 1 (WNK1) are associated with hypertension, but because of gene-environment interaction, it is difficult to fully identify the genetic contribution of WNK1 gene polymorphism to blood pressure (BP) variability. The aim of this study was to identify the effect of common WNK1 variants on the shift of BP during strict dietary interventions of salt or potassium intake. A total of 342 subjects from 126 families were selected and sequentially maintained on normal diet for 3 days at baseline, a low-salt diet for 7 days (3g/day, NaCl), then a high-salt diet for 7 days (18 g/day), and high-salt diet with potassium supplementation for another 7 days (4.5 g/day, KCl). Five single nucleotide polymorphisms (SNPs) were selected from the WNK1 gene. rs880054 and rs12828016 were associated with diastolic BP (DBP) response during the low-or high-sodium intervention, and rs2301880 was significantly associated with systolic BP, DBP and mean arterial pressure responses to the high-sodium intervention (all P<0.05). Unfortunately, no associations for WNK1 SNPs and the constructed haplotype blocks of WNK1 with BP responses to high-salt-and-potassium supplement intervention reached nominal statistical significance. The WNK1 gene might be mechanistically involved in the variation in BP response to dietary sodium and potassium intake among individuals, and might contribute to the variation of this complex phenotype.

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

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    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.

  7. 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.

  8. Src family protein tyrosine kinase regulates the basolateral K channel in the distal convoluted tubule (DCT) by phosphorylation of KCNJ10 protein.

    Science.gov (United States)

    Zhang, Chengbiao; Wang, Lijun; Thomas, Sherin; Wang, Kemeng; Lin, Dao-Hong; Rinehart, Jesse; Wang, Wen-Hui

    2013-09-01

    The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba(2+)-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from -65 to -43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr(8) and Tyr(9). The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr(9) did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr(9) on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

  9. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    Science.gov (United States)

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  10. Physiological responses of needles of Pinus massoniana elite families to phosphorus stress in acid soil

    Institute of Scientific and Technical Information of China (English)

    HE You-lan; LIU Ai-qin; Mulualem Tigabu; WUPeng-fei; MA Xiang-qing; WANG Chen; Per Christer Oden

    2013-01-01

    Pinus massoniana Lamb.is a major timber species widely planted in the South China,where the soil is acidic and deficient in phosphorus (P) due to fixation by aluminum and iron.Understanding the physiological responses to rhizospheric insoluble P is essential for enhancing plantation productivity.Thus,a sand culture experiment was conducted with four levels of P treatment (0,5,20 g insoluble P and 10 g soluble P),and 11 P.massoniana elite families.Physiological responses were measured after two months of stress.Compared to the normal soluble P treatment,the insoluble P treatment significantly reduced the proline content and the APase activity in the needles,while it significantly increased the catalase activity by 1.3-fold and malondialdehyde content by 1.2-fold.Soluble protein content was unaffected by the treatments,but chlorophyll content was significantly lower in P-deprived treatment compared with soluble and insoluble P treatments.These physiological responses also exhibited highly significant variation among families (p < 0.01).The findings suggest that increased catalase activities in the presence of insoluble P might be involved in the activation of an anti-oxidation defense mechanism that scavenges the reactive oxygen species elicited by the stress.And this response has a strong genetic control that can be exploited to identify desirable genotypes.

  11. Profile-QSAR: a novel meta-QSAR method that combines activities across the kinase family to accurately predict affinity, selectivity, and cellular activity.

    Science.gov (United States)

    Martin, Eric; Mukherjee, Prasenjit; Sullivan, David; Jansen, Johanna

    2011-08-22

    Profile-QSAR is a novel 2D predictive model building method for kinases. This "meta-QSAR" method models the activity of each compound against a new kinase target as a linear combination of its predicted activities against a large panel of 92 previously studied kinases comprised from 115 assays. Profile-QSAR starts with a sparse incomplete kinase by compound (KxC) activity matrix, used to generate Bayesian QSAR models for the 92 "basis-set" kinases. These Bayesian QSARs generate a complete "synthetic" KxC activity matrix of predictions. These synthetic activities are used as "chemical descriptors" to train partial-least squares (PLS) models, from modest amounts of medium-throughput screening data, for predicting activity against new kinases. The Profile-QSAR predictions for the 92 kinases (115 assays) gave a median external R²(ext) = 0.59 on 25% held-out test sets. The method has proven accurate enough to predict pairwise kinase selectivities with a median correlation of R²(ext) = 0.61 for 958 kinase pairs with at least 600 common compounds. It has been further expanded by adding a "C(k)XC" cellular activity matrix to the KxC matrix to predict cellular activity for 42 kinase driven cellular assays with median R²(ext) = 0.58 for 24 target modulation assays and R²(ext) = 0.41 for 18 cell proliferation assays. The 2D Profile-QSAR, along with the 3D Surrogate AutoShim, are the foundations of an internally developed iterative medium-throughput screening (IMTS) methodology for virtual screening (VS) of compound archives as an alternative to experimental high-throughput screening (HTS). The method has been applied to 20 actual prospective kinase projects. Biological results have so far been obtained in eight of them. Q² values ranged from 0.3 to 0.7. Hit-rates at 10 uM for experimentally tested compounds varied from 25% to 80%, except in K5, which was a special case aimed specifically at finding "type II" binders, where none of the compounds were predicted to be

  12. Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

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    C.M. Queiroz

    2008-05-01

    Full Text Available Intrahippocampal administration of kainic acid (KA induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4 which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4 and K252a (10 pmol, N = 4, respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

  13. 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.

  14. 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.

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    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

  15. The small SLC43 family: facilitator system l amino acid transporters and the orphan EEG1.

    Science.gov (United States)

    Bodoy, Susanna; Fotiadis, Dimitrios; Stoeger, Claudia; Kanai, Yoshikatsu; Palacín, Manuel

    2013-01-01

    The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein.

  16. 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 (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.

  17. Gallic acid induces the apoptosis of human osteosarcoma cells in vitro and in vivo via the regulation of mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Liang, Cheng-zhen; Zhang, Xin; Li, Hao; Tao, Yi-qing; Tao, Li-jiang; Yang, Zi-ru; Zhou, Xiao-peng; Shi, Zhong-li; Tao, Hui-min

    2012-12-01

    To examine the antitumor effects of gallic acid (GA) on osteosarcoma, two human osteosarcoma cell lines U-2OS and MNNG/HOS were treated by GA and subjected to cell proliferation and apoptosis assays. In addition, MNNG/HOS xenograft tumors were established in nude BALB/c mice to evaluate the anticancer capacity of GA in vivo. The results showed that GA inhibited the proliferation and induced the apoptosis of osteosarcoma cells, accompanied by the upregulation of p-38 activation and the downregulation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK1/2) activation. Additionally, p38 MAPK inhibitor abrogated GA-induced growth inhibition of osteosarcoma cells, whereas JNK or ERK1/2 inhibitors sensitized osteosarcoma cells to GA-induced growth inhibition. In vivo studies further showed that GA administration decreased xenograft tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of PCNA and CD31 expression and upregulation of apoptosis in MNNG/HOS tumor tissues following GA treatment. This study demonstrates the antitumor efficacy of GA for osteosarcoma that is mediated by the modulation of cell proliferation, apoptosis, and angiogenesis. Our findings suggest that GA could be a potent agent for osteosarcoma intervention.

  18. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

    Science.gov (United States)

    Chen, Y.; Hughes-Fulford, M.

    2000-01-01

    Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

  19. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

    Science.gov (United States)

    Chen, Y.; Hughes-Fulford, M.

    2000-01-01

    Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

  20. Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation*

    Science.gov (United States)

    Norambuena, Andrés; Metz, Claudia; Vicuña, Lucas; Silva, Antonia; Pardo, Evelyn; Oyanadel, Claudia; Massardo, Loreto; González, Alfonso; Soza, Andrea

    2009-01-01

    Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity. PMID:19276072

  1. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis.

    Science.gov (United States)

    Jiang, Shanshan; Zhang, Dan; Wang, Li; Pan, Jiaowen; Liu, Yang; Kong, Xiangpei; Zhou, Yan; Li, Dequan

    2013-10-01

    Calcium-dependent protein kinases (CDPKs) play essential roles in calcium-mediated signal transductions in plant response to abiotic stress. Several members have been identified to be regulators for plants response to abscisic acid (ABA) signaling. Here, we isolated a subgroup I CDPK gene, ZmCPK4, from maize. Quantitative real time PCR (qRT-PCR) analysis revealed that the ZmCPK4 transcripts were induced by various stresses and signal molecules. Transient and stable expression of the ZmCPK4-GFP fusion proteins revealed ZmCPK4 localized to the membrane. Moreover, overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth and stomatal movement. The transgenic plants also enhanced drought stress tolerance. Taken together, the results suggest that ZmCPK4 might be involved in ABA-mediated regulation of stomatal closure in response to drought stress.

  2. Synergistic effect of a combination of nanoparticulate Fe3O4 and gambogic acid on phosphatidylinositol 3-kinase/Akt/Bad pathway of LOVO cells

    Directory of Open Access Journals (Sweden)

    Hu S

    2012-07-01

    Full Text Available Lianghua Fang,1,3 Baoan Chen,2 Shenlin Liu,3 Ruiping Wang,3 Shouyou Hu,3 Guohua Xia,2 Yongli Tian,3 Xiaohui Cai21No 1 Clinical Medical College of Nanjing University of Chinese Medicine, 2Department of Hematology, Zhongda Hospital, Medical School, Southeast University, 3Department of Oncology, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing, People's Republic of ChinaBackground: The present study evaluated whether magnetic nanoparticles containing Fe3O4 could enhance the activity of gambogic acid in human colon cancer cells, and explored the potential mechanisms involved.Methods: Cytotoxicity was evaluated by MTT assay. The percentage of cells undergoing apoptosis was analyzed by flow cytometry, and cell morphology was observed under both an optical microscope and a fluorescence microscope. Reverse transcriptase polymerase chain reaction and Western blot assay were performed to determine the transcription of genes and expression of proteins, respectively.Results: Gambogic acid could inhibit proliferation of LOVO cells in a dose-dependent and time-dependent manner and induce apoptosis, which was dramatically enhanced by magnetic nanoparticles containing Fe3O4. The typical morphological features of apoptosis in LOVO cells were observed after treatment comprising gambogic acid with and without magnetic nanoparticles containing Fe3O4. Transcription of cytochrome c, caspase 9, and caspase 3 genes was higher in the group treated with magnetic nanoparticles containing Fe3O4 and gambogic acid than in the groups that received gambogic acid or magnetic nanoparticles containing Fe3O4, but transcription of phosphatidylinositol 3-kinase, Akt, and Bad genes decreased. Notably, expression of cytochrome c, caspase 9, and caspase 3 proteins in the group treated with gambogic acid and magnetic nanoparticles containing Fe3O4 was higher than in the groups receiving magnetic nanoparticles containing Fe3O4 or gambogic acid, while expression of p-PI3

  3. Valproic acid and butyrate induce apoptosis in human cancer cells through inhibition of gene expression of Akt/protein kinase B

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    Li Qiao

    2006-12-01

    Full Text Available Abstract Background In eukaryotic cells, the genomic DNA is packed with histones to form the nucleosome and chromatin structure. Reversible acetylation of the histone tails plays an important role in the control of specific gene expression. Mounting evidence has established that histone deacetylase inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in variety of cancer cells, making them a promising class of anticancer drugs. However, the molecular mechanisms of the anti-cancer effects of these inhibitors have yet to be understood. Results Here, we report that a key determinant for the susceptibility of cancer cells to histone deacetylase inhibitors is their ability to maintain cellular Akt activity in response to the treatment. Also known as protein kinase B, Akt is an essential pro-survival factor in cell proliferation and is often deregulated during tumorigenesis. We show that histone deacetylase inhibitors, such as valproic acid and butyrate, impede Akt1 and Akt2 expression, which leads to Akt deactivation and apoptotic cell death. In addition, valproic acid and butyrate induce apoptosis through the caspase-dependent pathway. The activity of caspase-9 is robustly activated upon valproic acid or butyrate treatment. Constitutively active Akt is able to block the caspase activation and rescues cells from butyrate-induced apoptotic cell death. Conclusion Our study demonstrates that although the primary target of histone deacetylase inhibitors is transcription, it is the capacity of cells to maintain cellular survival networks that determines their fate of survival.

  4. Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

    Science.gov (United States)

    Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

    2015-11-19

    Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5'-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5'-TGTAAGCCCTAACA-3') upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

  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

    2016-12-07

    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 analyzed 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 (AC ) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and RuBP-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 (OPDA), while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signaling mediated by mitogen activated protein kinase 4 (MPK4). The analysis also revealed a role for cytokinins interacting with MPK4 in CO2 -mediated stomatal regulation. Hence oral-secretions, while eliciting jasmonic acid-mediated defense responses, also elicits OPDA-mediated changes in stomatal conductance and AC , an observation illustrating the complexity and economy of the signaling that regulates defense and carbon assimilation pathways in response to herbivore attack.

  6. A novel family of (1-aminoalkyl(trifluoromethyl- and -(difluoromethylphosphinic acids – analogues of α-amino acids

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    Natalia V. Pavlenko

    2014-03-01

    Full Text Available A series of novel (1-aminoalkyl(trifluoromethyl- and -(difluoromethylphosphinic acids – analogues of proteinogenic and nonproteinogenic α-amino acids were prepared. The synthetic methodology was based on nucleophilic addition of (trifluoromethylphosphinic acid or (difluoromethylphosphinic acid or its ethyl ester to substrates with C=N or activated C=C double bonds. Analogues of glycine, phenylglycine, alanine, valine, proline, aminomalonic and aspartic acids were thus prepared. Three-component one-pot reactions of (trifluoromethylphosphinic acid and dibenzylamine with aldehydes were also tested to prepare the title compounds.

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

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    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.

  8. Nuciferine downregulates Per-Arnt-Sim kinase expression during its alleviation of lipogenesis and inflammation on oleic acid-induced hepatic steatosis in HepG2 cells

    Science.gov (United States)

    Zhang, Dan-Dan; Zhang, Ji-Gang; Wu, Xin; Liu, Ying; Gu, Sheng-Ying; Zhu, Guan-Hua; Wang, Yu-Zhu; Liu, Gao-Lin; Li, Xiao-Yu

    2015-01-01

    Non-alcoholic fatty liver disease (NAFLD) is a prevalent liver disease associated with lipotoxicity, lipid peroxidation, oxidative stress, and inflammation. Nuciferine, an active ingredient extracted from the natural lotus leaf, has been reported to be effective for the prevention and treatment of NAFLD. Per-Arnt-Sim kinase (PASK) is a nutrient responsive protein kinase that regulates lipid and glucose metabolism, mitochondrial respiration, and gene expression. The aim of the present study was to investigate the protective effect of nuciferine against NAFLD and its inhibitory effect on PASK, exploring the possible underlying mechanism of nuciferine-mediated inhibition on NAFLD. Relevant biochemical parameters (lipid accumulation, extent of oxidative stress and release of inflammation cytokines) in oleic acid (OA)-induced HepG2 cells that mimicked steatosis in vitro were measured and compared with the control. It was found that nuciferine and silenced-PASK (siRNA PASK) both inhibited triglyceride (TG) accumulation and was effective in decreasing fatty acid (FFAs). The content of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) were increased respectively by nuciferine and siRNA PASK without increase in glutathione (GSH). Malondialdehyde (MDA) was decreased respectively by nuciferine and siRNA PASK. In addition, nuciferine decreased TNF-a, IL-6 and IL-8 as well as the siRNA PASK group. IL-10 was increased by nuciferine and siRNA PASK respectively. Further investigation revealed that nuciferine and siRNA PASK could respectively regulate the expression of target genes involved in lipogenesis and inflammation, suggesting that nuciferine may be a potential therapeutic treatment for NAFLD. Furthermore, the modulated effect of nuciferine on (OA)-induced HepG2 cells lipogenesis and inflammation, which was accompanied with PASK inhibition, was also consistent with siRNA PASK, implying that PASK might play a role in nuciferine-mediated regulation on NAFLD

  9. [Aspartate kinase complex of Anabaena variabilis during the early period of development of cyanophage A-1].

    Science.gov (United States)

    Koltukova, N V; Kadyrova, G Kh; Lysenko, T G; Mendzhul, M I

    1994-01-01

    Aspartate kinase activity in cells of A. variabilis has been studied in the dynamics of development of virus infection. An early period of reproduction of cyanophage A-1 has been determined to be conjugated with the increase of biosynthesis of amino acids from aspartate family. Five isoenzymes of aspartate kinase were isolated and purified from A. variabilis cells during early development period of cyanophage A-1. Physicochemical properties and influence of amino acids of aspartate family on the activity of homogeneous isoenzymes have been studied. Retroinhibition effect was not observed in infected cyanobacteria cells, which probably enables one to increase 2-7 times the concentration of amino acids in a cell. Such an increase of the amino acids pool is apparently necessary for realization of viral genome strategy.

  10. W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress Responses

    Institute of Scientific and Technical Information of China (English)

    Zhao-Shi Xu; Li Liu; Zhi-Yong Ni; Pei Liu; Ming Chen; Lian-Cheng Li; Yao-Feng Chen; You-Zhi Ma

    2009-01-01

    Protein kinases play crucial roles In response to external environment stress signals. A putative protein kinase, W55a, belonging to SNF1-related protein kinase 2 (SnRK2) subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5' cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction(RT-PCR). It contains a 1029-bp open reading frame (ORF) encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonata, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexprassing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.

  11. Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

    Science.gov (United States)

    Ghosh, S; Strum, J C; Sciorra, V A; Daniel, L; Bell, R M

    1996-04-01

    Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

  12. Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

    NARCIS (Netherlands)

    Sanchez-Torres, P.; Visser, J.; Benen, J.A.E.

    2003-01-01

    Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. O

  13. Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

    NARCIS (Netherlands)

    Sanchez-Torres, P.; Visser, J.; Benen, J.A.E.

    2003-01-01

    Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH.

  14. International Retrospective Chart Review of Treatment Patterns in Severe Familial Mediterranean Fever, Tumor Necrosis Factor Receptor-Associated Periodic Syndrome, and Mevalonate Kinase Deficiency/Hyperimmunoglobulinemia D Syndrome.

    Science.gov (United States)

    Ozen, Seza; Kuemmerle-Deschner, Jasmin B; Cimaz, Rolando; Livneh, Avi; Quartier, Pierre; Kone-Paut, Isabelle; Zeft, Andrew; Spalding, Steve; Gul, Ahmet; Hentgen, Veronique; Savic, Sinisa; Foeldvari, Ivan; Frenkel, Joost; Cantarini, Luca; Patel, Dony; Weiss, Jeffrey; Marinsek, Nina; Degun, Ravi; Lomax, Kathleen G; Lachmann, Helen J

    2017-04-01

    Periodic fever syndrome (PFS) conditions are characterized by recurrent attacks of fever and localized inflammation. This study examined the diagnostic pathway and treatments at tertiary centers for familial Mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic syndrome (TRAPS), and mevalonate kinase deficiency (MKD)/hyperimmunoglobulinemia D syndrome (HIDS). PFS specialists at medical centers in the US, the European Union, and the eastern Mediterranean participated in a retrospective chart review, providing de-identified data in an electronic case report form. Patients were treated between 2008 and 2012, with at least 1 year of followup; all had clinical and/or genetically proven disease and were on/eligible for biologic treatment. A total of 134 patients were analyzed: FMF (n = 49), TRAPS (n = 47), and MKD/HIDS (n = 38). Fever was commonly reported as severe across all indications. Other frequently reported severe symptoms were serositis for FMF patients and elevated acute-phase reactants and gastrointestinal upset for TRAPS and MKD/HIDS. A long delay from disease onset to diagnosis was seen within TRAPS and MKD/HIDS (5.8 and 7.1 years, respectively) compared to a 1.8-year delay in FMF patients. An equal proportion of TRAPS patients first received anti-interleukin-1 (anti-IL-1) and anti-tumor necrosis factor (anti-TNF) biologic agents, whereas IL-1 blockade was the main choice for FMF patients resistant to colchicine and MKD/HIDS patients. For TRAPS patients, treatment with anakinra versus anti-TNF treatments as first biologic agent resulted in significantly higher clinical and biochemical responses (P = 0.03 and P < 0.01, respectively). No significant differences in responses were observed between biologic agents among other cohorts. Referral patterns and diagnostic delays highlight the need for greater awareness and improved diagnostics for PFS. This real-world treatment assessment supports the need for further

  15. 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.

  16. New functions of the chloroplast Preprotein and Amino acid Transporter (PRAT) family members in protein import.

    Science.gov (United States)

    Rossig, Claudia; Reinbothe, Christiane; Gray, John; Valdes, Oscar; von Wettstein, Diter; Reinbothe, Steffen

    2014-01-01

    Plant cells contain distinct compartments such as the nucleus, the endomembrane system comprising the endoplasmic reticulum and Golgi apparatus, peroxisomes, vacuoles, as well as mitochondria and chloroplasts. All of these compartments are surrounded by 1 or 2 limiting membranes and need to import proteins from the cytosol. Previous work led to the conclusion that mitochondria and chloroplasts use structurally different protein import machineries in their outer and inner membranes for the uptake of cytosolic precursor proteins. Our most recent data show that there is some unexpected overlap. Three members of the family of preprotein and amino acid transporters, PRAT, were identified in chloroplasts that mediate the uptake of transit sequence-less proteins into the inner plastid envelope membrane. By analogy, mitochondria contain with TIM22 a related PRAT protein that is involved in the import of transit sequence-less proteins into the inner mitochondrial membrane. Both mitochondria and chloroplasts thus make use of similar import mechanisms to deliver some of their proteins to their final place. Because single homologs of HP20- and HP30-like proteins are present in algae such as Chlamydomonas, Ostreococcus, and Volvox, which diverged from land plants approximately 1 billion years ago, it is likely that the discovered PRAT-mediated mechanism of protein translocation evolved concomitantly with the secondary endosymbiotic event that gave rise to green plants.

  17. Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

    Science.gov (United States)

    Zeng, Huawei; Botnen, James H; Briske-Anderson, Mary

    2010-01-01

    The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75-300 micromol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

  18. Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy

    Science.gov (United States)

    Hoo, Ruby L. C.; Shu, Lingling; Cheng, Kenneth K. Y.; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

    2017-01-01

    Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases. PMID:28094778

  19. 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.

  20. 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

  1. Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation

    Science.gov (United States)

    Ruzafa-Martínez, María; Ramos-Morcillo, Antonio Jesús

    2017-01-01

    During wound healing, skin function is restored by the action of several cell types that undergo differentiation, migration, proliferation and/or apoptosis. These dynamics are tightly regulated by the evolution of the extra cellular matrix (ECM) contents along the process. Pharmacologically active flavonoids have shown to exhibit useful physiological properties interesting in pathological states. Among them, oleanolic acid (OA), a pentacyclic triterpene, shows promising properties over wound healing, as increased cell migration in vitro and improved wound resolution in vivo. In this paper, we pursued to disclose the molecular mechanisms underlying those effects, by using an in vitro scratch assay in two epithelial cell lines of different linage: non-malignant mink lung epithelial cells, Mv1Lu; and human breast cancer cells, MDA-MB-231. In every case, we observed that OA clearly enhanced cell migration for in vitro scratch closure. This correlated with the stimulation of molecular pathways related to mitogen-activated protein (MAP) kinases, as ERK1,2 and Jun N-terminal kinase (JNK) 1,2 activation and c-Jun phosphorylation. Moreover, MDA-MB-231 cells treated with OA displayed an altered gene expression profile affecting transcription factor genes (c-JUN) as well as proteins involved in migration and ECM dynamics (PAI1), in line with the development of an epithelial to mesenchymal transition (EMT) status. Strikingly, upon OA treatment, we observed changes in the epidermal growth factor receptor (EGFR) subcellular localization, while interfering with its signalling completely prevented migration effects. This data provides a physiological framework supporting the notion that lipophilic plant extracts used in traditional medicine, might modulate wound healing processes in vivo through its OA contents. The molecular implications of these observations are discussed. PMID:28231262

  2. 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.

  3. 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.

  4. The maize (Zea mays ssp. mays var. B73) genome encodes 33 members of the purple acid phosphatase family.

    Science.gov (United States)

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J H

    2015-01-01

    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.

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

    Science.gov (United States)

    Choy, C. Anela; Davison, Peter C.; Drazen, Jeffrey C.; Flynn, Adrian; Gier, Elizabeth J.; Hoffman, Joel C.; McClain-Counts, Jennifer P.; Miller, Todd W.; Popp, Brian N.; Ross, Steve W.; Sutton, Tracey T.

    2012-01-01

    The δ15N 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 δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N 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.

  6. 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.

  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...... acid uptake by >80% in both wild-type and PKC-¿-knockout cardiomyocytes. In PKC-¿ knockout cardiomyocytes, PKC-¿ 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-¿ activity in PKC-¿-knockout cardiomyocytes is sufficient...

  8. 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.

  9. Phylogenetic analysis, structural evolution and functional divergence of the 12-oxo-phytodienoate acid reductase gene family in plants

    Directory of Open Access Journals (Sweden)

    Wang Hongbin

    2009-05-01

    Full Text Available Abstract Background The 12-oxo-phytodienoic acid reductases (OPRs are enzymes that catalyze the reduction of double-bonds in α, β-unsaturated aldehydes or ketones and are part of the octadecanoid pathway that converts linolenic acid to jasmonic acid. In plants, OPRs belong to the old yellow enzyme family and form multigene families. Although discoveries about this family in Arabidopsis and other species have been reported in some studies, the evolution and function of multiple OPRs in plants are not clearly understood. Results A comparative genomic analysis was performed to investigate the phylogenetic relationship, structural evolution and functional divergence among OPR paralogues in plants. In total, 74 OPR genes were identified from 11 species representing the 6 major green plant lineages: green algae, mosses, lycophytes, gymnosperms, monocots and dicots. Phylogenetic analysis showed that seven well-conserved subfamilies exist in plants. All OPR genes from green algae were clustered into a single subfamily, while those from land plants fell into six other subfamilies, suggesting that the events leading to the expansion of the OPR family occurred in land plants. Further analysis revealed that lineage-specific expansion, especially by tandem duplication, contributed to the current OPR subfamilies in land plants after divergence from aquatic plants. Interestingly, exon/intron structure analysis showed that the gene structures of OPR paralogues exhibits diversity in intron number and length, while the intron positions and phase were highly conserved across different lineage species. These observations together with the phylogenetic tree revealed that successive single intron loss, as well as indels within introns, occurred during the process of structural evolution of OPR paralogues. Functional divergence analysis revealed that altered functional constraints have occurred at specific amino acid positions after diversification of the paralogues

  10. A clinicobiochemical study of tryptophan and other plasma and urinary amino acids in the family with Hartnup disease.

    Science.gov (United States)

    Milovanović, Dragoslav D

    2003-01-01

    Two cases of Hartnup disease were diagnosed in a five member family. A changeable polymorph and severe clinical features of a 16 year old girl was described. Total plasma amino acids value was significantly decreased in the girl compared to the sum of plasma amino acids value in the brother, mother, father and to the summed maximal values of normal range. Intermediate aminoaciduria was also found with atypical amino acids pattern. Total plasma amino acids concentration was significantly reduced (27.20%) in the mother, while no significant decrease in the son (1.83%) and father (7.51%) were found compared to the summed maximal values of normal range. In the clinicaly healthy father, 38 years of age, a gross aminoaciduria with atypical pattern of amino acids was also found. Urinary amino acids concentration in the son and his mother were rather normal, although low concentration of eight amino acids was found in the mother's urine. Cerebrospinal fluid 5-hydroxyindoleacetic acid level was reduced in the girl.

  11. 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.

  12. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    Science.gov (United States)

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH.

  13. Involvement of MINK, a Ste20 Family Kinase, in Ras Oncogene-Induced Growth Arrest in Human Ovarian Surface Epithelial Cells

    NARCIS (Netherlands)

    Nicke, B.; Bastien, J.; Khanna, S.J.; Warne, P.H.; Cowling, V.; Cook, S.J.; Peters, G.; Delpuech, O.; Schulze, A.; Berns, K.; Mullenders, J.; Beijersbergen, R.L.; Bernards, R.A.; Ganesan, T.S.; Downward, J.; Hancock, D.C.

    2005-01-01

    The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kino

  14. A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct

    NARCIS (Netherlands)

    W.M. Baarends (Willy); M.J. van Helmond (Marjolein); M. Verhoef-Post (Miriam); P.J.C.M. van der Schoot; J.W. Hoogerbrugge (Jos); J.P. de Winter (Johan); J.Th.J. Uilenbroek (Jan); B. Karels (Bas)

    1994-01-01

    textabstractThe activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, which also includes the recently cloned TGF-beta type I receptor. We have isolated and characterized a cDNA clon

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  20. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  1. Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays

    DEFF Research Database (Denmark)

    Amanchy, Ramars; Zhong, Jun; Molina, Henrik;

    2008-01-01

    c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human...... embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c......-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues...

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

    Institute of Scientific and Technical Information of China (English)

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

    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.

  3. 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

  4. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes.

    Science.gov (United States)

    Thaller, M C; Lombardi, G; Berlutti, F; Schippa, S; Rossolini, G M

    1995-01-01

    The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

  5. Diacylglycerol Kinase Inhibition and Vascular Function.

    Science.gov (United States)

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.

  6. Genome-Wide Identification of Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D and D-E-L Motifs in EF-Hand Domain.

    Science.gov (United States)

    Mohanta, Tapan K; Mohanta, Nibedita; Mohanta, Yugal K; Bae, Hanhong

    2015-01-01

    Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca(2+) concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca(2+) ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins.

  7. Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Yu X. H.; Shanklin J.; Rawat, R.

    2011-05-01

    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. 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. 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 plant systems, it represents a strong candidate gene

  8. 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

  9. Activation of the AMP-activated protein kinase by eicosapentaenoic acid (EPA, 20:5 n-3 improves endothelial function in vivo.

    Directory of Open Access Journals (Sweden)

    Yong Wu

    Full Text Available The aim of the present study was to test the hypothesis that the cardiovascular-protective effects of eicosapentaenoic acid (EPA may be due, in part, to its ability to stimulate the AMP-activated protein kinase (AMPK-induced endothelial nitric oxide synthase (eNOS activation. The role of AMPK in EPA-induced eNOS phosphorylation was investigated in bovine aortic endothelial cells (BAEC, in mice deficient of either AMPKα1 or AMPKα2, in eNOS knockout (KO mice, or in Apo-E/AMPKα1 dual KO mice. EPA-treatment of BAEC increased both AMPK-Thr172 phosphorylation and AMPK activity, which was accompanied by increased eNOS phosphorylation, NO release, and upregulation of mitochondrial uncoupling protein-2 (UCP-2. Pharmacologic or genetic inhibition of AMPK abolished EPA-enhanced NO release and eNOS phosphorylation in HUVEC. This effect of EPA was absent in the aortas isolated from either eNOS KO mice or AMPKα1 KO mice fed a high-fat, high-cholesterol (HFHC diet. EPA via upregulation of UCP-2 activates AMPKα1 resulting in increased eNOS phosphorylation and consequent improvement of endothelial function in vivo.

  10. Caffeic Acid Phenethyl Ester Inhibits Oral Cancer Cell Metastasis by Regulating Matrix Metalloproteinase-2 and the Mitogen-Activated Protein Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Chih-Yu Peng

    2012-01-01

    Full Text Available Caffeic acid phenethyl ester (CAPE, an active component extracted from honeybee hives, exhibits anti-inflammatory and anticancer activities. However, the molecular mechanism by which CAPE affects oral cancer cell metastasis has yet to be elucidated. In this study, we investigated the potential mechanisms underlying the effects of CAPE on the invasive ability of SCC-9 oral cancer cells. Results showed that CAPE attenuated SCC-9 cell migration and invasion at noncytotoxic concentrations (0 μM to 40 μM. Western blot and gelatin zymography analysis findings further indicated that CAPE downregulated matrix metalloproteinase-2 (MMP-2 protein expression and inhibited its enzymatic activity. CAPE exerted its inhibitory effects on MMP-2 expression and activity by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2 and potently decreased migration by reducing focal adhesion kinase (FAK phosphorylation and the activation of its downstream signaling molecules p38/MAPK and JNK. These data indicate that CAPE could potentially be used as a chemoagent to prevent oral cancer metastasis.

  11. Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Peskin, Alexander V; Winterbourn, Christine C

    2006-01-01

    Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.

  12. TRB3 is involved in free fatty acid-induced INS-1-derived cell apoptosis via the protein kinase C δ pathway.

    Science.gov (United States)

    Qin, Jun; Fang, Ni; Lou, Jinning; Zhang, Wenjian; Xu, Shiqing; Liu, Honglin; Fang, Qing; Wang, Zai; Liu, Jiang; Men, Xiuli; Peng, Liang; Chen, Li

    2014-01-01

    Chronic exposure to free fatty acids (FFAs) may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.

  13. TRB3 is involved in free fatty acid-induced INS-1-derived cell apoptosis via the protein kinase C δ pathway.

    Directory of Open Access Journals (Sweden)

    Jun Qin

    Full Text Available Chronic exposure to free fatty acids (FFAs may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3 is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.

  14. Protection from procedural myocardial injury by omega-3 polyunsaturated fatty acids (PUFAs): is related with lower levels of creatine kinase-MB (CK-MB) and troponin I?

    Science.gov (United States)

    Foroughinia, Farzaneh; Salamzadeh, Jamshid; Namazi, Mohammad H

    2013-10-01

    This study sought to investigate the effect of omega-3 polyunsaturated fatty acids (PUFAs) on cardiac biomarkers, CK-MB, and troponin I in patients undergoing PCI. Restenosis remains as a major long-term complication following percutaneous coronary intervention (PCI). It appears that there is strong relationship between post-PCI creatine kinase-MB (CK-MB) and troponin I elevation and cardiovascular events after PCI. In this randomized clinical trial, a total of 90 patients planned to undergo PCI were randomly assigned into two groups: Group A-receiving omega-3 PUFAs (3 g, 12 h before PCI) plus standard treatment (n = 43) and Group B-control group, receiving only standard therapy (n = 47). Standard treatment included aspirin 325 mg and clopidogrel 600 mg loading dose. The plasma CK-MB level was measured before the procedure (baseline), at 8 and 24 h after PCI. The plasma troponin I was measured at baseline and 24 h after PCI. In comparison with control, omega-3 PUFAs could significantly reduce the level of CK-MB in 8 (P = 0.001) and 24 h (P = 0.012) after its prescription in the omega-3 PUFAs group. Omega-3 PUFAs could not significantly decrease troponin I. Our results revealed that omega-3 PUFAs can be considered as a safe adjunctive medication to the standard regimen before PCI for the aim of decreasing cardiovascular event after PCI. © 2012 John Wiley & Sons Ltd.

  15. A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco.

    Science.gov (United States)

    Gomi, Kenji; Ogawa, Daisuke; Katou, Shinpei; Kamada, Hiroshi; Nakajima, Nobuyoshi; Saji, Hikaru; Soyano, Takashi; Sasabe, Michiko; Machida, Yasunori; Mitsuhara, Ichiro; Ohashi, Yuko; Seo, Shigemi

    2005-12-01

    The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.

  16. 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......-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor...... subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases....

  17. A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

    Institute of Scientific and Technical Information of China (English)

    韩秋敏; 姜华武; 齐晓朋; 于洁; 吴平

    2004-01-01

    AtCRE1 is known to be a cytokinin receptor in Arabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRL1a, OsCRL1b, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain. The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.

  18. A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

    Institute of Scientific and Technical Information of China (English)

    韩秋敏; 姜华武; 齐晓朋; 丁洁; 吴平

    2004-01-01

    AtCRE1 is known to be a cytokinin receptor inArabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRLla, OsCRLlb, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.

  19. 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

  20. Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*

    OpenAIRE

    2011-01-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology model...

  1. 蛋白激酶蛋白家族的功能研究进展%Advances in the function of protein kinase D family

    Institute of Scientific and Technical Information of China (English)

    刘思旸; 邹志鹏; 郦俊

    2011-01-01

    蛋白激酶D(protein kinase D,PKD)属丝氨酸/苏氨酸(serine/threonine,Ser/Thr)蛋白质家族,主要包括3个成员:PKD1/PKCμ、PKD2、PKD3/PKCν.其结构相对保守,均具有相似的调节结构域和激酶结构域.近年来的研究表明PKD家族蛋白参与细胞生长、增殖、迁移、分化、凋亡,高尔基体的组织和重建,免疫应答,内分泌调节、增强心肌收缩力等多种生理功能.%Protein kinase D ( PKD) is a kind of Ser/Thr protein kinases including three members : PKD1/PKCμ, PKD2 , PKD3/PKC. All of them have conserved structures and similar regular domains. Recently many investigations have found that PKDs contribute to many cellucar biological functions such as cell survival, migration, differentiation, apoptosis, organization and regeneration of Golgi ' s apparatus, immune responses, regulation of endocrine secretion and myocardial contraccility.

  2. 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.

  3. 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.

  4. 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

  5. Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.

    Science.gov (United States)

    Hur, Wooyoung; Velentza, Anastasia; Kim, Sungjoon; Flatauer, Laura; Jiang, Xinnong; Valente, David; Mason, Daniel E; Suzuki, Melissa; Larson, Brad; Zhang, Jianming; Zagorska, Anna; Didonato, Michael; Nagle, Advait; Warmuth, Markus; Balk, Steven P; Peters, Eric C; Gray, Nathanael S

    2008-11-15

    Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

  6. 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.

  7. Inhibition of aldo-keto reductase family 1 member B10 by unsaturated fatty acids.

    Science.gov (United States)

    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; Soda, Midori; El-Kabbani, Ossama; Yashiro, Koji

    2016-11-01

    A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC50 value of 4.2 μM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC50 values of 1.1 μM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the Ki values of 0.24-1.1 μM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The Src family tyrosine kinases src and yes have differential effects on inflammation-induced apoptosis in human pulmonary microvascular endothelial cells.

    Science.gov (United States)

    Nelin, Leif D; White, Hilary A; Jin, Yi; Trittmann, Jennifer K; Chen, Bernadette; Liu, Yusen

    2016-05-01

    Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1β, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomix-induced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis.

  9. 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.

  10. 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.

  11. Periexercise coingestion of branched-chain amino acids and carbohydrate in men does not preferentially augment resistance exercise-induced increases in phosphatidylinositol 3 kinase/protein kinase B-mammalian target of rapamycin pathway markers indicative of muscle protein synthesis.

    Science.gov (United States)

    Ferreira, Maria Pontes; Li, Rui; Cooke, Matthew; Kreider, Richard B; Willoughby, Darryn S

    2014-03-01

    The effects of a single bout of resistance exercise (RE) in conjunction with periexercise branched-chain amino acid (BCAA) and carbohydrate (CHO) ingestion on skeletal muscle signaling markers indicative of muscle protein synthesis were determined. It was hypothesized that CHO + BCAA would elicit a more profound effect on these signaling markers compared with CHO. Twenty-seven males were randomly assigned to CHO, CHO + BCAA, or placebo (PLC) groups. Four sets of leg presses and leg extensions were performed at 80% 1 repetition maximum. Supplements were ingested 30 minutes and immediately before and after RE. Venous blood and muscle biopsy samples were obtained immediately before supplement ingestion and 0.5, 2, and 6 hours after RE. Serum insulin and glucose and phosphorylated levels of muscle insulin receptor substrate 1 (IRS-1), protein kinase B, mammalian target of rapamycin, phosphorylated 70S6 kinase, and 4E binding protein 1 were assessed. Data were analyzed by 2-way repeated-measures analysis of variance. Significant group × time interactions were observed for glucose and insulin (P protein kinase B (P = .031), mammalian target of rapamycin (P = .003), and phosphorylated 70S6 kinase (P = .001). Carbohydrate and CHO + BCAA supplementation significantly increased IRS-1 compared with PLC (P = .002). However, periexercise coingestion of CHO and BCAA did not augment RE-induced increases in skeletal muscle signaling markers indicative of muscle protein synthesis when compared with CHO.

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

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas;

    2009-01-01

    The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numer...

  13. Exercise pre‑conditioning alleviates brain damage via excitatory amino acid transporter 2 and extracellular signal‑regulated kinase 1/2 following ischemic stroke in rats.

    Science.gov (United States)

    Wang, Xiao; Zhang, Min; Feng, Rui; Li, Wen-Bin; Ren, Shi-Qing; Zhang, Feng

    2015-02-01

    Previous studies have reported that physical exercise may exert a neuroprotective effect in humans as well as animals. However, the detailed mechanisms underlying the neuroprotective effect of exercise has remained to be elucidated. The aim of the present study was to explore the possible signaling pathways involved in the protective effect of pre‑ischemic treadmill training for ischemic stroke in rats. A total of 36 male Sprague‑Dawley rats were divided at random into three groups as follows (n=12 for each): Sham surgery group; middle cerebral artery occlusion (MCAO) group; and exercise with MCAO group. Following treadmill training for three weeks, the middle cerebral artery was occluded for 90 min in order to induce ischemic stroke, followed by reperfusion. Following 24 h post‑reperfusion, six rats from each group were assessed for neurological deficits and then sacrificed to calculate the infarct volume. The remaining rats (n=6 for each group) were sacrificed and the expression levels of excitatory amino acid transporter 2 (EAAT‑2) and extracellular signal‑regulated kinase 1/2 (ERK1/2) were detected using western blot analysis. The results of the present study demonstrated that rats that underwent pre‑ischemic exercise intervention had a significantly decreased brain infarct volume and neurological deficits; in addition, the pre‑ischemic exercise group showed decreased overexpression of phosphorylated ERK1/2 and increased expression of EAAT‑2 following ischemic stroke. In conclusion, treadmill training exercise prior to ischemic stroke alleviated brain damage in rats via regulation of EAAT‑2 and ERK1/2.

  14. Retinoic acid enhances expression of neural specific genes in Sca-1+ cells of mouse fetal liver through activating protein kinase C

    Institute of Scientific and Technical Information of China (English)

    Gexiu Liu; Yuan Zhang; Dongmei He

    2006-01-01

    BACKGROUND: Interstitial stem cell is characterized by multiple differentiations,and retinoic acid (RA) can induce differentiation of stromal cells into nerve tissue cells in fetal liver of mice, so, its signal transduction pathway should be discussed to trigger differentiation.OBJECTIVE: To study the effect of RA on expression of neural specific gene and its signal transduction in fetal liver of mice.DESIGN: Paired controlled study on the basis of cell.SETTING: Institute of Hematology, Medical College of Jinan University.MATERIALS: The experiment was completed in the Institute of Hematology, Medical College of Jinan University from April to December 2005. C57BL/6 mice, of clean grade, aged 8-10 weeks, weighting 20-35 g,10 females and 4 males, were selected in this study.METHODS: Sca-1+ cells in fetal liver were prepared with MACS kit and cultured with DMEM + 10% fetal bovine serum (FBS). On the fourth day, it was added with or without protein kinase C (PKC) inhibitor chelerythrine chloride (3 μmol/L) and 5×10-7 mol/L RA for 24 hours, and then incubated in serum-free medium for 5 days. Expressions of genes were assayed by Western blotting and semi-quantitative RT-PCR.MAIN OUTCOME MEASURES: Expression of neural specific gene NF-L, NF-H, BF-1 and TH.RESULTS: Expression of neural specific gene NF-L, NF-H, BF-1 and TH was significantly increased after treatment with RA and they were increased 5.06, 5.15, 4.63 and 3.33 times, respectively. However, chelerythrine chloride could inhibit expression of neural specific gene NF-L, NF-H, BF-1 and TH induced by RA.CONCLUSION: RA can promote the expression of neural specific genes in Sca-1+ cells of fetal liver, and its pathway may be related to PKC.

  15. Glycyrrhizic acid prevents ultraviolet-B-induced photodamage: a role for mitogen-activated protein kinases, nuclear factor kappa B and mitochondrial apoptotic pathway.

    Science.gov (United States)

    Afnan, Quadri; Kaiser, Peerzada J; Rafiq, Rather A; Nazir, Lone A; Bhushan, Shashi; Bhardwaj, Subhash C; Sandhir, Rajat; Tasduq, Sheikh A

    2016-06-01

    Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV-B-induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV-B-induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV-B-mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV-B-mediated increase in intracellular reactive oxygen species (ROS) and down-regulated the release of pro-inflammatory cytokines interleukin (IL)-1α, -1β and -6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2). GA inhibited UV-B-mediated activation of p38 and JNK MAP kinases, COX-2 expression and nuclear translocation of NF-κB. Furthermore, GA inhibited UV-B-mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA-treated HaCaT cells also exhibited elevated antiapoptotic Bcl-2 protein, concomitant with reduced caspase-3 cleavage and decreased PARP-1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV-B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX-2, NF-κB and ICAM-1. Based on the above findings, we conclude that GA protects against UV-B-mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF-κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress.

    Science.gov (United States)

    Yoshida, Takuya; Fujita, Yasunari; Maruyama, Kyonoshin; Mogami, Junro; Todaka, Daisuke; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2015-01-01

    Under osmotic stress conditions such as drought and high salinity, the plant hormone abscisic acid (ABA) plays important roles in stress-responsive gene expression mainly through three bZIP transcription factors, AREB1/ABF2, AREB2/ABF4 and ABF3, which are activated by SNF1-related kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). However, since the three AREB/ABFs are crucial, but not exclusive, for the SnRK2-mediated gene expression, transcriptional pathways governed by SRK2D/E/I are not fully understood. Here, we show that a bZIP transcription factor, ABF1, is a functional homolog of AREB1, AREB2 and ABF3 in ABA-dependent gene expression in Arabidopsis. Despite lower expression levels of ABF1 than those of the three AREB/ABFs, the areb1 areb2 abf3 abf1 mutant plants displayed increased sensitivity to drought and decreased sensitivity to ABA in primary root growth compared with the areb1 areb2 abf3 mutant. Genome-wide transcriptome analyses revealed that expression of downstream genes of SRK2D/E/I, which include many genes functioning in osmotic stress responses and tolerance such as transcription factors and LEA proteins, was mostly impaired in the quadruple mutant. Thus, these results indicate that the four AREB/ABFs are the predominant transcription factors downstream of SRK2D/E/I in ABA signalling in response to osmotic stress during vegetative growth.

  17. Mitogen-activated protein kinase 4 is a salicylic acid-independent regulator of growth but not of photosynthesis in Arabidopsis.

    Science.gov (United States)

    Gawroński, Piotr; Witoń, Damian; Vashutina, Kateryna; Bederska, Magdalena; Betliński, Błażej; Rusaczonek, Anna; Karpiński, Stanisław

    2014-07-01

    Mitogen-activated protein kinase (MAPK) pathways regulate signal transduction from different cellular compartments and from the extracellular environment to the nucleus in all eukaryotes. One of the best-characterized MAPKs in Arabidopsis thaliana is MPK4, which was shown to be a negative regulator of systemic-acquired resistance. The mpk4 mutant accumulates salicylic acid (SA), possesses constitutive expression of pathogenesis-related (PR) genes, and has an extremely dwarf phenotype. We show that suppression of SA and phylloquinone synthesis in chloroplasts by knocking down the ICS1 gene (by crossing it with the ics1 mutant) in the mpk4 mutant background did not revert mpk4-impaired growth. However, it did cause changes in the photosynthetic apparatus and severely impaired the quantum yield of photosystem II. Transmission microscopy analysis revealed that the chloroplasts' structure was strongly altered in the mpk4 and mpk4/ics1 double mutant. Analysis of reactive oxygen species (ROS)-scavenging enzymes expression showed that suppression of SA and phylloquinone synthesis in the chloroplasts of the mpk4 mutant caused imbalances in ROS homeostasis which were more pronounced in mpk4/ics1 than in mpk4. Taken together, the presented results strongly suggest that MPK4 is an ROS/hormonal rheostat hub that negatively, in an SA-dependent manner, regulates immune defenses, but at the same time positively regulates photosynthesis, ROS metabolism, and growth. Therefore, we concluded that MPK4 is a complex regulator of chloroplastic retrograde signaling for photosynthesis, growth, and immune defenses in Arabidopsis.

  18. Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

    Science.gov (United States)

    Tao, Bingdong; Liu, Lidan; Wang, Ni; Tong, Dongyi; Wang, Wei; Zhang, Jin

    2015-12-01

    Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

  19. 酸沉降胁迫对不同家系马尾松幼苗耐酸性的影响%Assessment of Acid-Tolerant Parameters of Different Pinus massoniana Families under Acid Deposition Stress

    Institute of Scientific and Technical Information of China (English)

    王水良; 王平; 许建华

    2013-01-01

    The seedlings of 16 elite Masson pine (Pinus massoniana) families,with stable high-yield,including family 35,38,76,78,79,80,88,89,90,114,115,116,117,119,16,147,and 151,were used to explore the effects of precipitation acidity on their acid tolerance.The physiological parameters (biomass,relative plasma membrane permeability,chlorophyll) of the families cultivated in root-boxes outdoor were measured under treatments by spraying simulated acid rain at different pH values (2.5,3.5,4.5 and 5.6).The results showed that the biomass and chlorophyll content decreased with the increase of rain acidity,but the ratios of chlorophyll a/b increased with the increase of rain acidity.In addition,with the rain acidity increased,the relative plasma membrane permeability of the pine seedlings increased,suggesting that the injury of cell membrane was enhanced.These parameters varied with different P.massoniana families.Meanwhile,when rain pH decreased from 5.6 to 4.5,these parameters were slightly decline,but a significant decline was observed from pH 3.5 to 2.5,indicating that these pine families had relatively high productivity under mild acid environment at pH 4.5 to 5.6,and suggesting the tree species,Masson pine,would be suitable for being planted in mild acid soils.

  20. Detection of a novel mutation in the SRC homology domain 2 (SH2) of Bruton`s tyrosine kinase and direct female carrier evaluation in a family with x-linked agammaglobulinemia

    Energy Technology Data Exchange (ETDEWEB)

    Schuster, V.; Seidenspinner, S.; Wolfgang Kreth, H. [Univ. of Wuerzburg (Germany)

    1996-05-03

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease with a block in differentiation from pre-B to B cells resulting in a selective defect in the humoral immune response. Affected males have very low concentrations of serum immunoglobulins leading predominantly to recurrent bacterial infections beginning at age 6 to 18 months. The gene responsible for XLA was identified recently to encode a cytoplasmatic tyrosine kinase (Bruton`s tyrosine kinase, BTK). We have analyzed the BTK gene in a large family in which two brothers presented with the severe phenotype of XLA. Genomic DNA of affected boys and from healthy relatives was amplified by PCR with primers specific for the putative promoter region and for all 19 exons, including flanking intron boundaries, and subsequently screened for mutations using single strand conformation polymorphism (SSCP) analysis. Altered single strand band patterns were found using primers specific for exon 10, 15, and 18. Direct cycle-sequencing of these BTK segments detected two known polymorphisms in intron 14 and in exon 18. Sequencing of exon 10 from two boys with XLA demonstrated a novel point mutation in the SH2 domain of BTK. Direct identification of healthy female carriers in three generations was performed by amplification mutagenesis using PCR with a modified first primer. This method can easily be applied also to prenatal diagnosis. 25 refs., 3 figs.

  1. The role of the acidity of N-heteroaryl sulfonamides as inhibitors of bcl-2 family protein-protein interactions.

    Science.gov (United States)

    Touré, B Barry; Miller-Moslin, Karen; Yusuff, Naeem; Perez, Lawrence; Doré, Michael; Joud, Carol; Michael, Walter; DiPietro, Lucian; van der Plas, Simon; McEwan, Michael; Lenoir, Francois; Hoe, Madelene; Karki, Rajesh; Springer, Clayton; Sullivan, John; Levine, Kymberly; Fiorilla, Catherine; Xie, Xiaoling; Kulathila, Raviraj; Herlihy, Kara; Porter, Dale; Visser, Michael

    2013-02-14

    Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263.

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

    OpenAIRE

    Mustapha Aouida; Marta Rubio-Texeira; Thevelein, Johan M.; Richard Poulin; Dindial Ramotar

    2013-01-01

    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 e...

  3. Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

  4. Tomato ABSCISIC ACID STRESS RIPENING (ASR) Gene Family Revisited: e107117

    National Research Council Canada - National Science Library

    Ido Golan; Pia Guadalupe Dominguez; Zvia Konrad; Doron Shkolnik-Inbar; Fernando Carrari; Dudy Bar-Zvi

    2014-01-01

      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...

  5. Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry

    Science.gov (United States)

    Greaves, Jennifer; Munro, Kevin R.; Davidson, Stuart C.; Riviere, Matthieu; Wojno, Justyna; Smith, Terry K.; Tomkinson, Nicholas C. O.; Chamberlain, Luke H.

    2017-01-01

    S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity. PMID:28167757

  6. Density functional theory fragment descriptors to quantify the reactivity of a molecular family: application to amino acids.

    Science.gov (United States)

    Senet, P; Aparicio, F

    2007-04-14

    By using the exact density functional theory, one demonstrates that the value of the local electronic softness of a molecular fragment is directly related to the polarization charge (Coulomb hole) induced by a test electron removed (or added) from (at) the fragment. Our finding generalizes to a chemical group a formal relation between these molecular descriptors recently obtained for an atom in a molecule using an approximate atomistic model [P. Senet and M. Yang, J. Chem. Sci. 117, 411 (2005)]. In addition, a practical ab initio computational scheme of the Coulomb hole and related local descriptors of reactivity of a molecular family having in common a similar fragment is presented. As a blind test, the method is applied to the lateral chains of the 20 isolated amino acids. One demonstrates that the local softness of the lateral chain is a quantitative measure of the similarity of the amino acids. It predicts the separation of amino acids in different biochemical groups (aliphatic, basic, acidic, sulfur contained, and aromatic). The present approach may find applications in quantitative structure activity relationship methodology.

  7. MST kinases in development and disease

    OpenAIRE

    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 (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 cance...

  8. 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.

  9. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.

    Science.gov (United States)

    Ezumi, Y; Shindoh, K; Tsuji, M; Takayama, H

    1998-07-20

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

  10. The mechanism of protein kinase C regulation

    Institute of Scientific and Technical Information of China (English)

    Julhash U. KAZI

    2011-01-01

    Protein kinase C (PKC) is a family ofserine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis.Nine PKC genes have been identified in the human genome,which encode 10 proteins.Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations.Activation of PKC has been implicated in the regulation of cell growth and differentiation.This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.

  11. The EZ:Faast family of amino acid analysis kits: application of the GC-FID kit for rapid determination of plasma tryptophan and other amino acids.

    Science.gov (United States)

    Badawy, Abdulla A-B

    2012-01-01

    Plasma tryptophan (Trp) and other amino acids (AA) can be determined rapidly by gas (GC) or liquid (LC) chromatography using the Phenomenex EZ:Faast(™) family of kits. Three kits are available: (1) GC-FID or -NPD, (2) GC-MS, (3) LC-MS. The two GC kits can determine 32 AA, whereas the LC-MS can determine five additional AA. All three kits, however, share the same experimental procedure up to and including the preparation of derivatised AA. The method is based on solid-phase extraction (SPE), thus saving time on prior removal of plasma or other proteins and interfering substances, and can be applied to other body fluids and experimental media and to supernatants of extracts of solid material. Briefly, SPE is performed using a proprietary cation-exchange mechanism. The acid solution of the internal standard ensures that the free amino acids are in an anionic form suitable for cationic binding. The alkaline nature of the elution medium ensures that the AA cations are released prior to derivatisation. The latter involves production of chloroformate derivatives of both the amino and carboxylic acid groups. With experience, six plasma samples can be so processed within 12 min. The shortest analytical run is FID/NPD kit. Despite its many steps, the procedure becomes second nature and an enjoyable task. I have now used the GC-FID kit with manual injection to process >1,600 plasma and other samples. Limit of detection of AA is 1 μM or less. The procedure has been validated and optimised for Trp and its main five brain uptake competitors.

  12. Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus.

    Science.gov (United States)

    Hanlon, W A; Inouye, M; Inouye, S

    1997-02-01

    The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identify with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

  13. 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).

  14. Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    Directory of Open Access Journals (Sweden)

    Hsieh Hsi-Lung

    2010-11-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA is a component of Gram-positive bacterial cell walls, which has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, matrix metalloproteinases (MMPs, MMP-9 especially, have been observed in patients with brain inflammatory diseases and may contribute to brain disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in brain astrocytes remain unclear. Objective The goal of this study was to examine whether LTA-induced cell migration is mediated by calcium/calmodulin (CaM/CaM kinase II (CaMKII-dependent transactivation of the PDGFR pathway in rat brain astrocytes (RBA-1 cells. Methods Expression and activity of MMP-9 induced by LTA was evaluated by zymographic, western blotting, and RT-PCR analyses. MMP-9 regulatory signaling pathways were investigated by treatment with pharmacological inhibitors or using dominant negative mutants or short hairpin RNA (shRNA transfection, and chromatin immunoprecipitation (ChIP-PCR and promoter activity reporter assays. Finally, we determined the cell functional changes by cell migration assay. Results The data show that c-Jun/AP-1 mediates LTA-induced MMP-9 expression in RBA-1 cells. Next, we demonstrated that LTA induces MMP-9 expression via a calcium/CaM/CaMKII-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and JNK1/2 and then activated c-Jun/AP-1 signaling. Activated-c-Jun bound to the AP-1-binding site of the MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, up-regulation of MMP-9 by LTA enhanced cell migration of astrocytes. Conclusions These results demonstrate that in RBA-1 cells, activation of c-Jun/AP-1 by a CaMKII-dependent PI3K/Akt-JNK activation mediated through transactivation of PDGFR is essential for up-regulation of MMP-9 and cell migration induced by LTA. Understanding the regulatory mechanisms

  15. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    OpenAIRE

    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 echino...

  16. 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.

  17. An intervention study to increase knowledge and use of folic acid among relatives in neural tube defect-affected families in Washington, D.C.

    Science.gov (United States)

    Byrne, Julianne; Carolan, Susan; Arcement, Rashad; Kozlowski, Matthew; Taller, Inna; Ried, Stephanie; Keating, Robert

    2005-06-01

    Little is known about the level of knowledge and use of folic acid among near relatives in U.S. families of a child with spina bifida. We hypothesized that relatives would be more knowledgeable than the general population and more likely to take folic acid. Further, we hypothesized that relatives would be more motivated by an intervention to increase their use of folic acid. We conducted an intervention study among females in families attending a hospital spina bifida clinic in Washington, DC. The 231 subjects consisted of the affected individuals, mothers, sisters, and aunts. The average age was 34 years. At baseline, most (87.4%) reported that they had heard of folic acid; 37.6% were currently taking multivitamins with folic acid and 6.9% were taking folic acid tablets. The intervention significantly increased both knowledge (to 99%) and intake of folic acid from 41.9 to 48.5%. Folic acid intake increased significantly among African-American women and women with less education, women who were older, married, with children, and nonsmokers. This intervention was successful in increasing folic acid intake among female relatives in spina bifida-affected families. By the end of the study, almost all women had heard of folic acid and folic acid use had increased by 16%. Among these women at higher than expected risk for having an affected child, this rate of intake, while more than the general population, still falls short of optimum. Fortification of food with folic acid may be the only way to ensure increased folic acid intake. (c) 2005 Wiley-Liss, Inc.

  18. Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

    Science.gov (United States)

    Ezumi, Yasuharu; Shindoh, Keisuke; Tsuji, Masaaki; Takayama, Hiroshi

    1998-01-01

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex. PMID:9670039

  19. The src-family kinase inhibitor PP2 suppresses the in vitro invasive phenotype of bladder carcinoma cells via modulation of Akt.

    Science.gov (United States)

    Chiang, George J; Billmeyer, Brian R; Canes, David; Stoffel, John; Moinzadeh, Alireza; Austin, Christina A; Kosakowski, Monika; Rieger-Christ, Kimberly M; Libertino, John A; Summerhayes, Ian C

    2005-08-01

    To evaluate PP2 as a modulator of the cadherin/catenin complex in late-stage bladder carcinoma cells, and to assess its potential invasion-suppressor activity in this model. A panel of five human bladder carcinoma cells, characterizing late-stage disease, was used to determine the concentration for 50% inhibition of PP2 in cell-proliferation assays. Modulation of cadherin/catenin expression by PP2 was determined in Western blot analysis, with an assessment of the activation status of mitogen-activated protein kinase and Akt signalling pathways. Altered invasive capacity linked to these variables was determined in standard in vitro invasion assays. PP2 elicited concentration-dependent growth inhibition in all bladder cell lines within the panel, with growth suppression recorded at 10-35 micromol/L PP2. Distinct morphological changes were recorded in cell lines exposed to PP2, accompanied by up-regulation of plakoglobin expression in a subset of lines. Exposure of cells to PP2 resulted in inactivation of Akt in all cells and a concomitant reduction in in vitro invasive capacity. These results show that PP2 inhibits bladder carcinoma cell growth and can modulate plakoglobin expression in a subset of cell lines. In addition, PP2 can suppress the in vitro invasive capacity of bladder carcinoma cells by modulating the activation status of Akt.

  20. Comparative study of rosmarinic acid content in some plants of Labiatae family.

    Science.gov (United States)

    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. 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 officinalis, Thymus daenensis, Thymus citriodorous, Thymus pubescens, Thymus vulgaris, Zataria multiflora, Mentha piperita, Mentha pulegium, Mentha longifolia, Mentha spicata, Mentha aquatica, Mentha crispa, Perovskia artemisoides, Zhumeria majdae, Satureja hortensis, Satureja khuzistanica, Satureja bachtiarica, Satureja atropatana, Satureja mutica and Satureja macrantha were determined by using high-performance liquid chromatographic method. The results showed that RA content in different species of Labiatae was 0.0-58.5 mg g(-1) of dried plants. The highest amount of RA was found in Mentha species especially M. spicata. M. spicata can be considered as a new source of rosmarinic acid .

  1. Synthesis and spectroscopic studies of iron (III) complex with a quinolone family member (pipemidic acid)

    Science.gov (United States)

    Skrzypek, D.; Szymanska, B.; Kovala-Demertzi, Dimitra; Wiecek, Joanna; Talik, E.; Demertzis, Mavroudis A.

    2006-12-01

    The interaction of iron (III) with pipemidic acid, Hpipem, afforded the complex [Fe (pipem) (HO)2 (H2O)]2. The new complex has been characterised by elemental analyses, infra-red, EPR and XPS spectroscopies. The monoanion, pipem, exhibits O, O ligation through the carbonyl and carboxylato oxygen atoms. Six coordinate dimer distorted octahedral configuration has been proposed for [Fe (pipem) (HO)2 (H2O)]2.

  2. Amino acids and peptides activate at least five members of the human bitter taste receptor family.

    Science.gov (United States)

    Kohl, Susann; Behrens, Maik; Dunkel, Andreas; Hofmann, Thomas; Meyerhof, Wolfgang

    2013-01-09

    Amino acids and peptides represent important flavor molecules eliciting various taste sensations. Here, we present a comprehensive assessment of the interaction of various peptides and all proteinogenic amino acids with the 25 human TAS2Rs expressed in cell lines. L-Phenylalanine and L-tryptophan activate TAS2R1 and TAS2R4, respectively, whereas TAS2R4 and TAS2R39 responded to D-tryptophan. Structure-function analysis uncovered the basis for the lack of stereoselectivity of TAS2R4. The same three TAS2Rs or subsets thereof were also sensitive to various dipeptides containing L-tryptophan, L-phenylalanine, or L-leucine and to Trp-Trp-Trp, whereas Leu-Leu-Leu specifically activated TAS2R4. Trp-Trp-Trp also activated TAS2R46 and TAS2R14. Two key bitter peptides from Gouda cheese, namely, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser and Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, both activated TAS2R1 and TAS2R39. Thus, the data demonstrate that the bitterness of amino acids and peptides is not mediated by specifically tuned TAS2Rs but rather is brought about by an unexpectedly complex pattern of sensitive TAS2Rs.

  3. Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase.

    Science.gov (United States)

    Hughes, Paul E; Oertli, Beat; Hansen, Malene; Chou, Fan-Li; Willumsen, Berthe M; Ginsberg, Mark H

    2002-07-01

    The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.

  4. 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.

  5. 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.

  6. The Raine syndrome protein FAM20C is a Golgi kinase that phosphorylates bio-mineralization proteins.

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    Hiroyuki O Ishikawa

    Full Text Available Raine syndrome is caused by mutations in FAM20C, which had been reported to encode a secreted component of bone and teeth. We found that FAM20C encodes a Golgi-localized protein kinase, distantly related to the Golgi-localized kinase Four-jointed. Drosophila also encode a Golgi-localized protein kinase closely related to FAM20C. We show that FAM20C can phosphorylate secreted phosphoproteins, including both Casein and members of the SIBLING protein family, which modulate biomineralization, and we find that FAM20C phosphorylates a biologically active peptide at amino acids essential for inhibition of biomineralization. We also identify autophosphorylation of FAM20C, and characterize parameters of FAM20C's kinase activity, including its Km, pH and cation dependence, and substrate specificity. The biochemical properties of FAM20C match those of an enzymatic activity known as Golgi casein kinase. Introduction of point mutations identified in Raine syndrome patients into recombinant FAM20C impairs its normal localization and kinase activity. Our results identify FAM20C as a kinase for secreted phosphoproteins and establish a biochemical basis for Raine syndrome.

  7. 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...

  8. 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.

  9. 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

  10. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses.

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A; Kay, Steve A; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R; Schroeder, Julian I

    2015-09-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.

  11. Indoleamine 2,3-dioxygenase depletes tryptophan, activates general control non-derepressible 2 kinase and down-regulates key enzymes involved in fatty acid synthesis in primary human CD4+ T cells.

    Science.gov (United States)

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2015-10-01

    Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and exerts immunosuppressive effects on CD4(+) T cells. One mechanism is through the inhibition of aerobic glycolysis. Another prerequisite for T-cell proliferation and differentiation into effector cells is increased fatty acid (FA) synthesis. The effect of IDO on enzymes involved in FA synthesis was evaluated in primary human cells both in mixed lymphocyte reactions in the presence or not of the IDO inhibitor 1-dl-methyl-tryptophan, and in stimulated CD4(+) T cells in the presence or not of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP). IDO or TRP inhibited cell proliferation. By assessing the level of GCN2 kinase or mammalian target of rapamycin complex 1 substrates along with a kynurenine free system we showed that IDO exerts its effect mainly through activation of GCN2 kinase. IDO or TRP down-regulated ATP-citrate lyase and acetyl coenzyme A carboxylase 1, key enzymes involved in FA synthesis. Also, IDO or TRP altered the expression of enzymes that control the availability of carbon atoms for FA synthesis, such as lactate dehydrogenase-A, pyruvate dehydrogenase, glutaminase 1 and glutaminase 2, in a way that inhibits FA synthesis. In conclusion, IDO through GCN2 kinase activation inhibits CD4(+) T-cell proliferation and down-regulates key enzymes that directly or indirectly promote FA synthesis, a prerequisite for CD4(+) T-cell proliferation and differentiation into effector cell lineages. © 2015 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    1998-09-04

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

  13. 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