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...

The Р60-S6K1 isoform of ribosomal protein S6 kinase 1 is a product of alternative mRNA translation

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I. V. Zaiets

2018-07-01

Full Text Available Ribosomal protein S6 kinase 1 (S6K1 is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1 participating primarily in the regulation of cell growth and metabolism. Deregulation of mTOR/S6K1 signaling can promote numerous human pathologies, including cancer, neurodegeneration, cardiovascular disease, and metabolic disorders. As existing data suggest, the S6K1 gene encodes several protein isoforms, including p85-S6K1, p70-S6K1, and p60-S6K1. The two of these isoforms, p85-S6K1 and p70-S6K1, were extensively studied to date. The origin and functional significance of the p60-S6K1 isoform remains a mystery, however, it was suggested that the isoform could be a product of alternative S6K1 mRNA translation. Herein we report the generation of HEK-293 cells exclusively expressing p60-S6K1 as a result of CRISPR/Cas9-mediated inactivation of p85/p70-S6K1 translation. Moreover, the generated modified cells displayed the elevated level of p60-S6K1 expression compared to that in wild-type HEK-293 cells. Our data confirm an assumption that p60-S6K1 is alternatively translated, most probably, from the common for both p70- and p85-S6K1 mRNA transcript and reveal a link between p60-S6K1 expression and such cellular processes as cell proliferation and motility. In addition, our findings indicate that the p60-S6K1 isoform of S6K1 may undergo a mode of regulation distinct from p70- and p85-S6K1 due to the absence of mTOR-regulated p60-S6K1 phosphorylation at T389 that is important for S6K1 activation.

Role of Bruton's tyrosine kinase (BTK) in growth and metastasis of INA6 myeloma cells

International Nuclear Information System (INIS)

Bam, R; Venkateshaiah, S U; Khan, S; Ling, W; Randal, S S; Li, X; Zhang, Q; Rhee, F van; Barlogie, B; Epstein, J; Yaccoby, S

2014-01-01

Bruton's tyrosine kinase (BTK) and the chemokine receptor CXCR4 are linked in various hematologic malignancies. The aim of the study was to understand the role of BTK in myeloma cell growth and metastasis using the stably BTK knockdown luciferase-expressing INA6 myeloma line. BTK knockdown had reduced adhesion to stroma and migration of myeloma cells toward stromal cell-derived factor-1. BTK knockdown had no effect on short-term in vitro growth of myeloma cells, although clonogenicity was inhibited and myeloma cell growth was promoted in coculture with osteoclasts. In severe combined immunodeficient-rab mice with contralaterally implanted pieces of bones, BTK knockdown in myeloma cells promoted their proliferation and growth in the primary bone but suppressed metastasis to the contralateral bone. BTK knockdown myeloma cells had altered the expression of genes associated with adhesion and proliferation and increased mammalian target of rapamycin signaling. In 176 paired clinical samples, BTK and CXCR4 expression was lower in myeloma cells purified from a focal lesion than from a random site. BTK expression in random-site samples was correlated with proportions of myeloma cells expressing cell surface CXCR4. Our findings highlight intratumoral heterogeneity of myeloma cells in the bone marrow microenvironment and suggest that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of myeloma cells

Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

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Savinska L. O.

2015-08-01

Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana

International Nuclear Information System (INIS)

Baños-Sanz, Jose Ignacio; Villate, Maider; Sanz-Aparicio, Julia; Brearley, Charles Alistair; González, Beatriz

2009-01-01

Inositol 1,3,4,5,6-pentakisphosphate kinase from A. thaliana has been expressed in E. coli, purified and crystallized and diffraction data have been collected to 2.3 Å resolution. Two heavy-atom crystal derivatives are under study. Inositol 1,3,4,5,6-pentakisphosphate kinase (IP 5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP 6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) and ATP. IP 6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP 6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP 5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP 5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP 6 . The best crystals obtained for X-ray crystallographic analysis belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 58.124, b = 113.591, c = 142.478 Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source

Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle

DEFF Research Database (Denmark)

Deshmukh, A; Salehzadeh, F; Metayer-Coustard, S

2009-01-01

Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1...... to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle....

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

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Hernández-Padilla, Laura; Vázquez-Rivera, Dolores; Sánchez-Briones, Luis A; Díaz-Pérez, Alma L; Moreno-Rodríguez, José; Moreno-Eutimio, Mario A; Meza-Carmen, Victor; Cruz, Homero Reyes-De la; Campos-García, Jesús

2017-06-20

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD 50 of 60-250 µM) and inducing apoptosis with EC 50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-10⁵ fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

Science.gov (United States)

Son, Ora; Kim, Sunghan; Hur, Yoon-Sun; Cheon, Choong-Ill

2016-03-25

TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction. Copyright © 2016 Elsevier Inc. All rights reserved.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

Science.gov (United States)

Li, H.; Dauwalder, M.; Roux, S. J.

1991-01-01

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

International Nuclear Information System (INIS)

Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

1985-01-01

A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and]2number 2 PO 4 /mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 34 PO 4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro

Regulated eukaryotic DNA replication origin firing with purified proteins.

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Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

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Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

2016-07-01

Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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Li, H.; Roux, S. J.

1992-01-01

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

[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

Science.gov (United States)

Wang, Lili; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Zhao, Song; Ma, Dongchu

2016-10-01

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum.

Science.gov (United States)

Gray, K A; Grossman, S H; Summers, D D

1986-01-01

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.

Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

DEFF Research Database (Denmark)

Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

2007-01-01

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

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

DEFF Research Database (Denmark)

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

1994-01-01

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

Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana BRI1-associated kinase 1 (BAK1) cytoplasmic domain

International Nuclear Information System (INIS)

Gao, Jian; Ma, Yuanyuan; Sun, Yuna; Zhao, Huadong; Hong, Dapeng; Yan, Liming; Lou, Zhiyong

2012-01-01

The cytoplasmic domain of BRI1-associated kinase 1 from A. thaliana has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution. BRI1-associated kinase 1 (BAK1) is a member of the plant receptor-like kinase (RLK) superfamily. BAK1 has been shown to initiate brassinosteroid (BR) signalling and innate immune responses in plants by forming receptor complexes with both brassinosteroid-insensitive 1 (BRI1) and flagellin-sensing 2 (FLS2). To gain a better understanding of the structural details and the mechanism of action of the BAK1 kinase domain, recombinant BAK1 cytoplasmic domain has been expressed, purified and crystallized at 291 K using PEG 3350 as a precipitant. A 2.6 Å resolution data set was collected from a single flash-cooled crystal at 100 K. This crystal belonged to space group C2, with unit-cell parameters a = 70.3, b = 75.6, c = 71.9 Å, β = 93.1°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient was 2.6 Å 3 Da −1

PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

Energy Technology Data Exchange (ETDEWEB)

Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

2015-10-23

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

Molecular Characterization and Expression Analysis of S6K1 in Cashmere Goats (

Directory of Open Access Journals (Sweden)

Wu Manlin

2013-08-01

Full Text Available p70 ribosomal S6 kinase (p70S6K can integrate nutrient and growth factor signals to promote cell growth and survival. We report our molecular characterization of the complementary DNA (cDNA that encodes the goat p70S6K gene 40S ribosomal S6 kinase 1 (S6K1 (GenBank accession GU144017 and its 3′ noncoding sequence in Inner Mongolia Cashmere goats (Capra hircus. Goat S6K1 cDNA was 2,272 bp and include an open reading frame (ORF of 1,578 bp, corresponding to a polypeptide of 525 amino acids, and a 694-residue 3′ noncoding sequence with a polyadenylation signal at nucleotides 2,218 to 2,223. The relative abundance of S6K1 mRNA was measured by real-time PCR in 6 tissues, and p70S6K expression was examined by immunohistochemistry in heart and testis. The phosphorylation of p70S6K is regulated by mitogen-activated protein kinase (MAPK signaling in fetal fibroblasts.

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

International Nuclear Information System (INIS)

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

1985-01-01

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

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

Science.gov (United States)

Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2017-07-05

Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

Science.gov (United States)

Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

2014-06-25

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v=src oncogene

Czech Academy of Sciences Publication Activity Database

Vojtěchová, Martina; Šloncová, Eva; Kučerová, Dana; Jiřička, Jaroslav; Sovová, Vlasta; Tuháčková, Zdena

2003-01-01

Roč. 543, 1-3 (2003), s. 81-86 ISSN 0014-5793 R&D Projects: GA ČR GV312/96/K205; GA ČR GA301/00/0269; GA MZd NC5428 Institutional research plan: CEZ:AV0Z5052915 Keywords : phosphoinositide 3-kinase signalling pathway * mRNA translation * 70 kDa ribosomal protein S6 kinase Subject RIV: CE - Biochemistry Impact factor: 3.609, year: 2003

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

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

2005-01-01

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

Radioimmunoassay of inactive creatine kinase B protein in human plasma

Energy Technology Data Exchange (ETDEWEB)

Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

1981-08-27

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

Radioimmunoassay of inactive creatine kinase B protein in human plasma

International Nuclear Information System (INIS)

Burnam, M.H.; Shell, W.E.

1981-01-01

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

OpenAIRE

Rider, M H; Kuntz, D A; Hue, L

1988-01-01

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofru...

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

International Nuclear Information System (INIS)

Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

1988-01-01

Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca 2+ -dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca 2+ -independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10 -6 M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10 -4 M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 μM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [γ- 32 P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention.

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Miaozong Wu

Full Text Available BACKGROUND: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. PRINCIPAL FINDINGS: Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308 was higher in soleus muscles of very aged rats (33-months. Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR phosphorylation, along with decreased levels of insulin receptor beta (IR-beta, phosphoinositide 3-kinase (PI3K, phosphatase and tensin homolog deleted on chromosome 10 (PTEN and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1 (Ser241. In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS. Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month animals with acetaminophen (30 mg/kg body weight/day for 6-months. CONCLUSIONS: These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.

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

KAUST Repository

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

2014-01-01

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

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

KAUST Repository

Zourelidou, Melina

2014-06-19

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

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

Science.gov (United States)

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

2003-08-01

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

The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

Science.gov (United States)

Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

2016-01-01

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

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

DEFF Research Database (Denmark)

Schneider, H R; Issinger, O G

1988-01-01

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

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

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Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; Liu, Zhen; Qiu, Wen-Li; Whitham, Steven A.; Qian, Wei-Jun

2017-09-29

It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

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Yuan Jian

2010-06-01

Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

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Sonja Rakette

Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

DEFF Research Database (Denmark)

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

1993-01-01

Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C...

The Sensor Kinase GacS Negatively Regulates Flagellar Formation and Motility in a Biocontrol Bacterium, Pseudomonas chlororaphis O6

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Ji Soo Kim

2014-06-01

Full Text Available The GacS/GacA two component system regulates various traits related to the biocontrol potential of plant-associated pseudomonads. The role of the sensor kinase, GacS, differs between strains in regulation of motility. In this study, we determined how a gacS mutation changed cell morphology and motility in Pseudomonas chlororaphis O6. The gacS mutant cells were elongated in stationary-phase compared to the wild type and the complemented gacS mutant, but cells did not differ in length in logarithmic phase. The gacS mutant had a two-fold increase in the number of flagella compared with the wild type strain; flagella number was restored to that of the wild type in the complemented gacS mutant. The more highly flagellated gacS mutant cells had greater swimming motilities than that of the wild type strain. Enhanced flagella formation in the gacS mutant correlated with increased expression of three genes, fleQ, fliQ and flhF, involved in flagellar formation. Expression of these genes in the complemented gacS mutant was similar to that of the wild type. These findings show that this root-colonizing pseudomonad adjusts flagella formation and cell morphology in stationary-phase using GacS as a major regulator.

Calcium-dependent but calmodulin-independent protein kinase from soybean

International Nuclear Information System (INIS)

Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

1987-01-01

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

Science.gov (United States)

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

The TRPM6 Kinase Domain Determines the Mg·ATP Sensitivity of TRPM7/M6 Heteromeric Ion Channels*

Science.gov (United States)

Zhang, Zheng; Yu, Haijie; Huang, Junhao; Faouzi, Malika; Schmitz, Carsten; Penner, Reinhold; Fleig, Andrea

2014-01-01

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg2+) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg2+ and therefore unlikely to be active at physiological levels of [Mg2+]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex. PMID:24385424

The TRPM6 kinase domain determines the Mg·ATP sensitivity of TRPM7/M6 heteromeric ion channels.

Science.gov (United States)

Zhang, Zheng; Yu, Haijie; Huang, Junhao; Faouzi, Malika; Schmitz, Carsten; Penner, Reinhold; Fleig, Andrea

2014-02-21

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg(2+)) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg(2+) and therefore unlikely to be active at physiological levels of [Mg(2+)]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.

Cloning and expression of human deoxycytidine kinase cDNA

International Nuclear Information System (INIS)

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

1991-01-01

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

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

Science.gov (United States)

Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

Directory of Open Access Journals (Sweden)

Chang-Ling Li

Full Text Available Megakaryocytes (MKs are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1 at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in

Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction

DEFF Research Database (Denmark)

Frödin, M; Gammeltoft, S

1999-01-01

), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK......-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c...

Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase

International Nuclear Information System (INIS)

Qian, Kevin C.; Studts, Joey; Wang, Lian; Barringer, Kevin; Kronkaitis, Anthony; Peng, Charline; Baptiste, Alistair; LaFrance, Roger; Mische, Sheenah; Farmer, Bennett

2004-01-01

Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P6 5 , with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK.

Directory of Open Access Journals (Sweden)

Fabrice Lucien

Full Text Available The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na(+/H(+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na(+/H(+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK, which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

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

Science.gov (United States)

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

2008-01-01

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

Radioimmunoassay of creatine kinase BB isoenzyme

International Nuclear Information System (INIS)

Geng Jianguo

1988-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1981-06-18

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

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase

International Nuclear Information System (INIS)

Balogun, Emmanuel Oluwadare; Inaoka, Daniel Ken; Kido, Yasutoshi; Shiba, Tomoo; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Michels, Paul A. M.; Harada, Shigeharu; Kita, Kiyoshi

2010-01-01

Glycerol kinase from human African trypanosomes has been purified and crystallized for X-ray structure analysis. In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His 6 tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 Å resolution indicated that the crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 Å. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V M ) of 2.5 Å 3 Da −1 , corresponding to 50% solvent content

Pentacene field-effect transistors by in situ and real time electrical characterization: Comparison between purified and non-purified thin films

International Nuclear Information System (INIS)

Liu, Shun-Wei; Wen, Je-Min; Lee, Chih-Chien; Su, Wei-Cheng; Wang, Wei-Lun; Chen, Ho-Chien; Lin, Chun-Feng

2013-01-01

We present an electrical characterization of the organic field-effect transistor with purified and non-purified pentacene by using in situ and real time measurements. The field-effect phenomenon was observed at the thickness of 1.5 nm (approximately one monolayer of pentacene) for purified pentacene, as compared to 3.0 nm for the non-purified counterpart. Moreover, the hole mobility is improved from 0.13 to 0.23 cm 2 /V s after the sublimation process to purify the pentacene. With atomic force microscopic measurements, the purified pentacene thin film exhibits a larger grain size and film coverage, resulting in better crystallinity of the thin film structure due to the absence of the impurities. This is further confirmed by X-ray diffraction patterns, which show higher intensities for the purified pentacene. - Highlights: • We present in-situ characterization for pentacene field-effect transistors. • The hole mobility is improved after the sublimation process to purify the pentacene. • Purified pentacene thin film exhibits a larger grain size and film coverage. • Hole mobility of pentacene is improved from 0.13 to 0.23 cm 2 /V s. • The discontinuity of grain boundary may cause the shift of threshold voltage

Pentacene field-effect transistors by in situ and real time electrical characterization: Comparison between purified and non-purified thin films

Energy Technology Data Exchange (ETDEWEB)

Liu, Shun-Wei, E-mail: swliu@mail.mcut.edu.tw [Department of Electronic Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan, ROC (China); Wen, Je-Min; Lee, Chih-Chien; Su, Wei-Cheng; Wang, Wei-Lun; Chen, Ho-Chien [Department of Electronic Engineering, National Taiwan University of Science and Technology, Taipei, 10607 Taiwan, ROC (China); Lin, Chun-Feng [Department of Electronic Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan, ROC (China)

2013-05-01

We present an electrical characterization of the organic field-effect transistor with purified and non-purified pentacene by using in situ and real time measurements. The field-effect phenomenon was observed at the thickness of 1.5 nm (approximately one monolayer of pentacene) for purified pentacene, as compared to 3.0 nm for the non-purified counterpart. Moreover, the hole mobility is improved from 0.13 to 0.23 cm{sup 2}/V s after the sublimation process to purify the pentacene. With atomic force microscopic measurements, the purified pentacene thin film exhibits a larger grain size and film coverage, resulting in better crystallinity of the thin film structure due to the absence of the impurities. This is further confirmed by X-ray diffraction patterns, which show higher intensities for the purified pentacene. - Highlights: • We present in-situ characterization for pentacene field-effect transistors. • The hole mobility is improved after the sublimation process to purify the pentacene. • Purified pentacene thin film exhibits a larger grain size and film coverage. • Hole mobility of pentacene is improved from 0.13 to 0.23 cm{sup 2}/V s. • The discontinuity of grain boundary may cause the shift of threshold voltage.

Chronic Alcohol Consumption Alters Mammalian Target of Rapamycin (mTOR), Reduces Ribosomal p70S6 Kinase and p4E-BP1 Levels in Mouse Cerebral Cortex

OpenAIRE

Li, Qun; Ren, Jun

2007-01-01

Reduced insulin sensitivity following chronic alcohol consumption may contribute to alcohol-induced brain damage although the underlying mechanism(s) has not been elucidated. This study was designed to examine the effect of chronic alcohol intake on insulin signaling in mouse cerebral cortex. FVB mice were fed with a 4% alcohol diet for 16 weeks. Insulin receptor substrates (IRS-1, IRS-2) and post-receptor signaling molecules Akt, mammalian target of rapamycin (mTOR), ribosomal p70s6 kinase (...

Radioimmunoassay for herpes simplex virus (HSV) thymidine kinase

International Nuclear Information System (INIS)

McGuirt, P.V.; Keller, P.M.; Elion, G.B.

1982-01-01

A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified 3 H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150-460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation

Expression and subcellular localization of p70S6 kinase under heart failure

Directory of Open Access Journals (Sweden)

Usenko V. S.

2010-11-01

Full Text Available The PI3K/PDK/Akt/mTOR/p70S6K signaling pathway is primary associated with the activation of insulin receptors and is important for cardiomyocytes survival. p70S6K is a key regulator of the speed and efficiency of protein biosynthesis within the cell. Recently the pro-apoptotic protein BAD has been identified as a new target for р70S6K1. BAD is inactivated in normal cardiomyocytes by р70S6K1 phosphorylation which prevents the cardiomyocytes apoptosis. Aim. To study possible changes in р70S6K1 expression and/or cellular localization at heart failure progression – in DCM- affected human myocardia and murine hearts with experimental DCM-like pathology. Methods. Western-blot analysis and immunohystochemistry. Results. The substantial decrease in р70S6K1 level was observed at the final stage of pathology progression and in the dynamics of DCM pathogenesis as well. For the first time relocalization of the protein to the connective tissue was shown according to the Western-blot results. Conclusions. The data obtained allow us to understand a possible role of р70S6K1 in the regulation of stress-induced apoptotic signaling in cardiomyocytes.

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

Science.gov (United States)

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

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

International Nuclear Information System (INIS)

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

1981-01-01

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

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao, E-mail: li@sri.org [Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Alabama 35205 (United States)

2005-06-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3{sub 1}21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao

2005-01-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3 1 21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme

HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

Directory of Open Access Journals (Sweden)

Kazuhiko Fujita

Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

Association between PI3K/Akt/mTOR/p70S6K signaling pathway and hepatic fibrosis

Directory of Open Access Journals (Sweden)

WU Changhui

2015-11-01

Full Text Available Phosphoinositide 3-kinase (PI3K/protein kinase-B (AkT/mammalian target of rapamycin (mTOR/70-kDa ribosomal protein S6 kinase (p70S6K, PI3K/Akt/mTOR/p70S6K, is an important signaling pathway in the life activities of cells, and it plays an important role in promoting the growth, proliferation, invasion, and anti-apoptosis of cells and promoting angiogenesis. It was clarified that the PI3K/Akt/mTOR/p70S6K signaling pathway is involved in regulating the activities of hepatic stellate cell(HSC, thus influencing the development and progression of hepatic fibrosis. Analysis demonstrated that blocking any target of the PI3K/Akt/mTOR/p70S6K signaling pathway can inhibit the activation and proliferation of HSC, promote the apoptosis of HSC, inhibit the extracellular matrix secretion from HSC, and delay the progression of hepatic fibrosis. Blocking the pathway is expected to be a treatment strategy for hepatic fibrosis.

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

Science.gov (United States)

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

2013-01-01

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

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

Energy Technology Data Exchange (ETDEWEB)

Takahashi, Hitoshi; Akazawa, Daisuke [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Kato, Takanobu; Date, Tomoko [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Shirakura, Masayuki [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Nakamura, Noriko; Mochizuki, Hidenori [Toray Industries, Inc., Kanagawa (Japan); Tanaka-Kaneko, Keiko; Sata, Tetsutaro [Department of Pathology, National Institute of Infectious Diseases, Tokyo (Japan); Tanaka, Yasuhito [Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medicine, Nagoya (Japan); Mizokami, Masashi [Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba (Japan); Suzuki, Tetsuro [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Wakita, Takaji, E-mail: wakita@nih.go.jp [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan)

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

International Nuclear Information System (INIS)

Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

2010-01-01

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

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

DEFF Research Database (Denmark)

Götz, C; Koenig, M G; Issinger, O G

1995-01-01

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

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

International Nuclear Information System (INIS)

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

1989-01-01

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

A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.

Science.gov (United States)

Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

2012-03-01

This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

Radioimmunoassay of creatine kinase BB isoenzyme

Energy Technology Data Exchange (ETDEWEB)

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

1988-11-01

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

Lack of Csk-mediated negative regulation in a unicellular SRC kinase.

Science.gov (United States)

Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, Iñaki; Miller, W Todd

2012-10-16

Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

Science.gov (United States)

Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

Side-effects of protein kinase inhibitors on ion channels

Indian Academy of Sciences (India)

2013-11-06

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

The Protein Kinase RSK Family - Roles in Prostate Cancer

National Research Council Canada - National Science Library

Lannigan, Deborah

2006-01-01

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

Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

International Nuclear Information System (INIS)

Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

Improved radioimmunoassay for creatine kinase isoenzymes in plasma

International Nuclear Information System (INIS)

Ritter, C.S.; Mumm, S.R.; Roberts, R.

1981-01-01

We describe convenient and relatively rapid procedures for purifying creatine kinase isoenzymes MM, BB, and MB, and their use in an improved radioimmunoassay for creatine kinase isoenzymes in plasma. The modifications include use of: (a) BB with a specific activity of 400 kU/G, which can be labeled with a specific radioactivity of 20 Ci/g; (b) albumin-free purified MB as inhibitor; (c) antiserum to MB creatine kinase; and (d) a second-antibody technique that necessitates only a 15-min incubation. The radioimmunoassay for MB has a sensitivity of 0.2 μg/L (80 mU/L) and a CV of <5%. Plasma MB average 22 (SD 12) μg/L in 200 normal subjects; 24 (SD 12) μg/L in 200 patients with chest pain without infarction; and 23 (SD 7) μg/L in 43 patients with renal disease, whether measured before or after dialysis. Peak values for plasma MB averaged 191 (SD 86) μg/L in 325 patients with documented myocardial infarction; BB was negligible. Extensive clinical experience indicates the radioimmunoassay to be suitably rapid, highly sensitive, and reliable as a diagnostic assay for MB on plasma

Identification of the protein kinase C phosphorylation site in neuromodulin

International Nuclear Information System (INIS)

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

1990-01-01

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

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Science.gov (United States)

Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

2017-06-15

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Levoglucosan kinase involved in citric acid fermentation by Aspergillus niger CBX-209 using levoglucosan as sole carbon and energy source

Energy Technology Data Exchange (ETDEWEB)

Zhuang, X.L.; Zhang, H.X.; Tang, J.J. [Chinese Academy of Sciences, Beijing (China). Research Center for Eco-Environmental Sciences

2001-07-01

Conditions were optimized for the production of citric acid by a mutant A. niger CBX-209 using levoglucosan derived from pyrolysis of wastepaper as a sole carbon and energy source in a simple medium. The optimum concentration of levoglucosan and wheat bran in the medium was 8% and 3%, respectively, at an optimum initial pH between 5.5 and 6.0 at 35{sup o}C. During fermentation, direct formation of glucose 6-phosphate from levogluocosan in the presence of ATP and Mg{sup 2+} as observed by HPLC in the reaction with both cell extracts and partially purified enzyme, suggested that the enzyme acting on levoglucosan isa kinase. Time-course changes in the levels of this special levoglucosan kinase in A. niger CBX-209 grown on levoglucosan and glucose revealed that levoglucosan kinase was an inductive enzyme. (Author)

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

Directory of Open Access Journals (Sweden)

Daniel Thomas

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

Myosin light chain kinase phosphorylation in tracheal smooth muscle

International Nuclear Information System (INIS)

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

1990-01-01

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

Activation of purified calcium channels by stoichiometric protein phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

1989-09-01

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

Activation of purified calcium channels by stoichiometric protein phosphorylation

International Nuclear Information System (INIS)

Nunoki, K.; Florio, V.; Catterall, W.A.

1989-01-01

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45 Ca 2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45 Ca 2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd 2+ , Ni 2+ , and Mg 2+ . The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

Action of mercurials on activity of partially purified soluble protein kinase C from mice brain

International Nuclear Information System (INIS)

Inoue, Y.; Saijoh, K.; Sumino, K.

1988-01-01

The enzymatic activity of soluble protein kinase C from mice brain was inhibited by mercuric chloride (II) (HgCl 2 ) and organic mercurials, i.e. methyl mercury, phenyl mercury and p-chloromercuribenzoic acid (PCMB). The IC50 was 0.08 μM for HgCl 2 and about 1 μM for organic mercurials. Sulfhydryl blocking reagents such as 5.5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) were less potent but nevertheless inhibited the enzymic activity of protein kinase C. The Hill coefficients of HgCl 2 , DTNB and NEM were close to unity whereas the values for organic mercurials were 1.3 to 1.5. The inhibition was of a non-competitive type with respect to Hl histone. 3 H-PDBu binding activity was also inhibited by all of the reagents in a non-competitive manner. Mercurials apparently bind to sulfhydryl groups of protein kinase C to inhibit the enzymatic activity. (author)

Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Mieskes, G; Issinger, O G

1993-01-01

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylat......The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation...... and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All...... kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR...

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

Science.gov (United States)

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

2018-05-03

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

Purification and characterization of a thylakoid protein kinase

International Nuclear Information System (INIS)

Coughlan, S.J.; Hind, G.

1986-01-01

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

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

DEFF Research Database (Denmark)

Madec, Edwige; Stensballe, Allan; Kjellström, Sven

2003-01-01

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing...... the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high...... mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant...

Ibrutinib: a first in class covalent inhibitor of Bruton’s tyrosine kinase

Science.gov (United States)

Davids, Matthew S; Brown, Jennifer R

2015-01-01

Ibrutinib (formerly PCI-32765) is a potent, covalent inhibitor of Bruton’s tyrosine kinase, a kinase downstream of the B-cell receptor that is critical for B-cell survival and proliferation. In preclinical studies, ibrutinib bound to Bruton’s tyrosine kinase with high affinity, leading to inhibition of B-cell receptor signaling, decreased B-cell activation and induction of apoptosis. In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL. Ongoing studies are evaluating ibrutinib in other types of non-Hodgkin’s lymphoma, such as diffuse large B-cell lymphoma and Waldenström’s macrogobulinemia, in larger Phase III studies in CLL and MCL, and in combination studies with monoclonal antibodies and chemotherapy. Future studies will combine ibrutinib with other promising novel agents currently in development in hematologic malignancies. PMID:24941982

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase

International Nuclear Information System (INIS)

Gunn, Natalie J.; Gorman, Michael A.; Dobson, Renwick C. J.; Parker, Michael W.; Mulhern, Terrence D.

2011-01-01

The Src-homology 2 (SH2) domain of Csk-family protein tyrosine kinases acts as a conformational switch to regulate their catalytic activity, which in turn promotes the inhibition of their proto-oncogenic targets, the Src-family kinases. Here, the expression, purification, small-angle X-ray scattering and preliminary diffraction analysis of the SH2 domain of the Csk-homologous kinase is reported. The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a = 25.8, b = 34.6, c = 63.2 Å, β = 99.4°

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

Science.gov (United States)

Barbosa, Inês C R; Shikata, Hiromasa; Zourelidou, Melina; Heilmann, Mareike; Heilmann, Ingo; Schwechheimer, Claus

2016-12-15

Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases. © 2016. Published by The Company of Biologists Ltd.

Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases

DEFF Research Database (Denmark)

Issinger, O G; Benne, R; Hershey, J W

1976-01-01

Purified protein synthesis initiation factors IF-E2 and IF-E3 from rabbit reticulocytes were phosphorylated in vitro with protein kinases isolated from the same source. The highest levels of phosphorylation resulted from incubation of the factors with a cyclic nucleotide-independent protein kinase...

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Light aging of reactive fuels purified by various methods

Energy Technology Data Exchange (ETDEWEB)

Khodzhaeva, M G; Burtyshev, N Ya; Molodozhenyuk, T B; Ryabovda, N D

1976-01-01

A study of the effect of uv-radiation on aging of Fergana fuel TS-1 has been extended to the uv-effect on alkali-purified fuels (e.g., Krasnovodsk, Omsk, and Orsk TS-1), on hydro-purified (Syzran T-8, Syzran T-7, and Novokuybyshev T-7) and on adsorption-purified Fergana TS-1. The PRK-4 lamp was employed. Aging criteria were formation of insoluble gums, soluble gums separable on silicagel, acidity, and optical density. Fuels purified in the same manner aged practically identically; after 6 months storage the greatest gum formation was seen in the fuels Orsk TS-1 and Syzran T-8. 3 references, 1 figure, 1 table.

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase.

Science.gov (United States)

Gunn, Natalie J; Gorman, Michael A; Dobson, Renwick C J; Parker, Michael W; Mulhern, Terrence D

2011-03-01

The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a=25.8, b=34.6, c=63.2 Å, β=99.4°.

Purifying Nanomaterials

Science.gov (United States)

Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

2014-01-01

A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

RPS6KA2, a putative tumour suppressor gene at 6q27 in sporadic epithelial ovarian cancer

DEFF Research Database (Denmark)

Bignone, P A; Lee, K Y; Liu, Y

2007-01-01

We had previously defined by allele loss studies a minimal region at 6q27 (between D6S264 and D6S297) to contain a putative tumour suppressor gene. The p90 ribosomal S6 kinase-3 gene (p90 Rsk-3, RPS6KA2) maps in this interval. It is a serine-threonine kinase that signals downstream of the mitogen...

Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

International Nuclear Information System (INIS)

Das, Mahua R.; Bag, Arup K.; Saha, Shekhar; Ghosh, Alok; Dey, Sumit K.; Das, Provas; Mandal, Chitra; Ray, Subhankar; Chakrabarti, Saikat; Ray, Manju; Jana, Siddhartha S.

2016-01-01

For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. PKM2

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

Science.gov (United States)

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

Science.gov (United States)

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

Science.gov (United States)

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

mTORC1 Targets the Translational Repressor 4E-BP2, but Not S6 Kinase 1/2, to Regulate Neural Stem Cell Self-Renewal In Vivo

Directory of Open Access Journals (Sweden)

Nathaniel W. Hartman

2013-10-01

Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 integrates signals important for cell growth, and its dysregulation in neural stem cells (NSCs is implicated in several neurological disorders associated with abnormal neurogenesis and brain size. However, the function of mTORC1 on NSC self-renewal and the downstream regulatory mechanisms are ill defined. Here, we found that genetically decreasing mTORC1 activity in neonatal NSCs prevented their differentiation, resulting in reduced lineage expansion and aborted neuron production. Constitutive activation of the translational repressor 4E-BP1, which blocked cap-dependent translation, had similar effects and prevented hyperactive mTORC1 induction of NSC differentiation and promoted self-renewal. Although 4E-BP2 knockdown promoted NSC differentiation, p70 S6 kinase 1 and 2 (S6K1/S6K2 knockdown did not affect NSC differentiation but reduced NSC soma size and prevented hyperactive mTORC1-induced increase in soma size. These data demonstrate a crucial role of mTORC1 and 4E-BP for switching on and off cap-dependent translation in NSC differentiation.

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

DEFF Research Database (Denmark)

Grankowski, N; Gasior, E; Issinger, O G

1993-01-01

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

Hsp60 and p70S6K form a complex in human cardiomyocytes

Directory of Open Access Journals (Sweden)

Kroupskaya I. V.

2011-02-01

Full Text Available Molecular chaperon Hsp60 and protein kinase p70S6K play an important functional role in the regulation of cardiomyocytes vital function or apoptosis. Aim. To study a possibility of in vivo complex formation between Hsp60 and p70S6K in cardiomyocytes. Methods. Co-immunoprecipitation, Western-blot analysis. Results. We have identified in vivo interaction between molecular chaperone Hsp60 and two isoforms of proteinkinase p70S6K in human myocardium, normal and affected by cardiomyopathy. Conclusions. The results obtained suggest a possible participation of molecular chaperon Hsp60 in regulation of p70S6K activity in stressinduced apoptotic signaling pathway in cardiomyocytes.

Purification of the alpha and beta subunits of phosphorylase kinase for structural studies

International Nuclear Information System (INIS)

Sotiroudis, T.G.; Heilmeyer, L.M.G. Jr.; Crabb, J.W.

1987-01-01

Structural analysis of the alpha (Mr, 132,000) and beta (Mr, 113,000) subunits of phosphorylase kinase may provide clues to their yet unknown functions however purification remains problematic. Preparative RP-HPLC procedures yield inconveniently large, dilute solutions and concentration steps are required prior to subunit modification and fragmentation. Concentration of the β subunit usually results in significant losses due to insolubility. Using preparative SDS-polyacrylamide gel electrophoresis, they have purified the α, 7 , and β subunits from rabbit muscle phosphorylase kinase in a soluble and concentrated form suitable for structural studies. Phosphorylase kinase labelled with fluorescein isothiocyanate in the α and α' subunits and fully 14 C-S-carboxymethylated was fractionated on a 5% acrylamide Laemmli slab gel. The subunit bands were visualized by fluorescence and by SDS precipitation then excised and electroeluted in the presence of SDS using an ELUTRAP device. From 4.5 mg of enzyme applied to a 4.5 mm thick gel about 70% of the α subunit and about 90% of the β subunit were typically recovered in less than 1 ml with overnight elution

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

Science.gov (United States)

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

2017-09-15

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

Influence of supplementation with branched-chain amino acids in combination with resistance exercise on p70S6 kinase phosphorylation in resting and exercising human skeletal muscle.

Science.gov (United States)

Apró, W; Blomstrand, E

2010-11-01

Skeletal muscle growth is thought to be regulated by the mammalian target of rapamycin (mTOR) pathway, which can be activated by resistance exercise and branched-chain amino acids (BCAA). The major aim of the present study was to distinguish between the influence of resistance exercise and BCAA on key enzymes considered to be involved in the regulation of protein synthesis, including p70(S6) kinase (p70(S6k)). Nine healthy subjects (four men and five women) performed unilateral resistance exercise on two occasions separated by 1 month. Subjects were randomly supplied either a mixture of BCAA or flavoured water. Muscle biopsies were taken from both resting and exercising muscle before, after and 1 h after exercise. Phosphorylation of Akt was unaltered by either resistance exercise and/or BCAA supplementation whereas mTOR phosphorylation was enhanced (Pexercising and resting muscle following exercise in the absence (70-90%) and presence of BCAA supplementation (80-130%). Phosphorylation of p70(S6k) was unaffected by resistance exercise alone; however, BCAA intake increased (Pexercise. In resting muscle, a 5- and 16-fold increase in p70(S6k) was observed immediately after and 1 h after exercise, respectively, as compared to 11- and 30-fold increases in the exercising muscle. Phosphorylation of eukaryotic elongation factor 2 was attenuated 1 h after exercise (Pexercising muscle (30-50%) under both conditions. The present findings indicate that resistance exercise and BCAA exert both separate and combined effects on the p70(S6k) phosphorylation in an Akt-independent manner. © 2010 The Authors. Journal compilation © 2010 Scandinavian Physiological Society.

Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines

Directory of Open Access Journals (Sweden)

Kotani Hidehito

2009-07-01

Full Text Available Abstract Background The Hedgehog (HH pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3β, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC. Results To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3β, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3β (Ser9 was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation. Conclusion We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3β followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

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

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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.

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

Science.gov (United States)

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

2015-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

An HTRF® Assay for the Protein Kinase ATM.

Science.gov (United States)

Adams, Phillip; Clark, Jonathan; Hawdon, Simon; Hill, Jennifer; Plater, Andrew

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase that plays a key role in the regulation of DNA damage pathways and checkpoint arrest. In recent years, there has been growing interest in ATM as a therapeutic target due to its association with cancer cell survival following genotoxic stress such as radio- and chemotherapy. Large-scale targeted drug screening campaigns have been hampered, however, by technical issues associated with the production of sufficient quantities of purified ATM and the availability of a suitable high-throughput assay. Using a purified, functionally active recombinant ATM and one of its physiological substrates, p53, we have developed an in vitro FRET-based activity assay that is suitable for high-throughput drug screening.

Insulin resistance in uremia: Insulin receptor kinase activity in liver and muscle from chronic uremic rats

International Nuclear Information System (INIS)

Cecchin, F.; Ittoop, O.; Sinha, M.K.; Caro, J.F.

1988-01-01

The authors have studied the structure and function of the partially purified insulin receptors from liver and skeletal muscle in a rat model of severe chronic uremia. 125 I-insulin binding was higher in the liver from uremic rats when compared with ad libitum- and pair-fed controls. Furthermore, the ability of insulin to stimulate the autophosphorylation of the β-subunit and insulin receptor kinase activity using Glu 80 , Tyr 20 as exogenous phosphoacceptor was increased in the liver of the uremic animals. The structural characteristics of the receptors, as determined by electrophoretic mobilities of affinity labeled α-subunit and the phosphorylated β-subunit, were normal in uremia. 125 I-insulin binding and insulin receptor kinase activity were similar in the skeletal muscle from uremic and pair- and ad libitum-fed animals. Thus the data are supportive of the hypothesis that in liver and muscle of chronic uremic rats, insulin resistance is due to a defect(s) distal to the insulin receptor kinase

IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils.

Science.gov (United States)

Esnault, Stephane; Kelly, Elizabeth A B; Shen, Zhong-Jian; Johansson, Mats W; Malter, James S; Jarjour, Nizar N

2015-09-15

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common β-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions. Copyright © 2015 by The American Association of Immunologists, Inc.

Effect of triiodothyronine on rat liver chromatin protein kinase

International Nuclear Information System (INIS)

Kruh, J.; Tichonicky, L.

1976-01-01

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

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Properties of Folate Binding Protein Purified from Cow’s Milk

Directory of Open Access Journals (Sweden)

SUBANDRATE

2012-09-01

Full Text Available Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway.

Science.gov (United States)

Shen, Hongyu; Li, Liangpeng; Yang, Sujin; Wang, Dandan; Zhou, Siying; Chen, Xiu; Tang, Jinhai

2017-08-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll‑like/interleukin‑1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription‑quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki‑67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β‑activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF‑7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6‑TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer.

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase.

Science.gov (United States)

Nakayama, Yuji; Yamaguchi, Naoto

2005-04-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation.

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase

International Nuclear Information System (INIS)

Nakayama, Yuji; Yamaguchi, Naoto

2005-01-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

OpenAIRE

Konisky, J; Michels, P C; Clark, D S

1995-01-01

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

Science.gov (United States)

López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

2018-03-01

The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Endurance Pump Test with MIL-PRF-83282 Hydraulic Fluid, Purified with Malabar Purifier

National Research Council Canada - National Science Library

Sharma, Shashi

2004-01-01

.... Endurance aircraft hydraulic pump tests under carefully controlled conditions were previously conducted using hydraulic fluid purified with a rotating-disk and vacuum type purifier, the portable...

IL2-dependent phosphorylation of 40S ribosomal protein S6 is controlled by PI-3K/mTOR signalling in CTLL2 cells

Czech Academy of Sciences Publication Activity Database

Tuháčková, Zdena; Šloncová, Eva; Vojtěchová, Martina; Sovová, Vlasta

2004-01-01

Roč. 13, č. 4 (2004), s. 601-605 ISSN 1107-3756 R&D Projects: GA ČR GA301/00/0269 Institutional research plan: CEZ:AV0Z5052915 Keywords : MTOR /PI3-K signalling, p70 S 6 kinase, interleukin Subject RIV: CE - Biochemistry Impact factor: 3.190, year: 2004

Phosphorylation of rat brain purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal kinase-3 modifies open-channel noise.

Science.gov (United States)

Gupta, Rajeev

2017-09-02

The drift kinetic energy of ionic flow through single ion channels cause vibrations of the pore walls which are observed as open-state current fluctuations (open-channel noise) during single-channel recordings. Vibration of the pore wall leads to transitions among different conformational sub-states of the channel protein in the open-state. Open-channel noise analysis can provide important information about the different conformational sub-state transitions and how biochemical modifications of ion channels would affect their transport properties. It has been shown that c-Jun N-terminal kinase-3 (JNK3) becomes activated by phosphorylation in various neurodegenerative diseases and phosphorylates outer mitochondrion associated proteins leading to neuronal apoptosis. In our earlier work, JNK3 has been reported to phosphorylate purified rat brain mitochondrial voltage-dependent anion channel (VDAC) in vitro and modify its conductance and opening probability. In this article we have compared the open-state noise profile of the native and the JNK3 phosphorylated VDAC using Power Spectral Density vs frequency plots. Power spectral density analysis of open-state noise indicated power law with average slope value α ≈1 for native VDAC at both positive and negative voltage whereas average α value open-state noise arises due to coupling of ionic transport and conformational sub-states transitions in open-state and this coupling is perturbed as a result of channel phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

Science.gov (United States)

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

2017-01-01

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

Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

Science.gov (United States)

de Bruijn, Marjolein J W; Rip, Jasper; van der Ploeg, Esmee K; van Greuningen, Lars W; Ta, Van T B; Kil, Laurens P; Langerak, Anton W; Rimmelzwaan, Guus F; Ellmeier, Wilfried; Hendriks, Rudi W; Corneth, Odilia B J

2017-04-15

The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca 2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase. Copyright © 2017 by The American Association of Immunologists, Inc.

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

NARCIS (Netherlands)

Etzkorn, M.; Kneuper, H.; Dünnwald, P.; Vijayan, V.; Krämer, J.; Griesinger, C.; Becker, S.; Unden, G.; Baldus, M.

2008-01-01

The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solidstate NMR, structural modeling and mutagenesis.

Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

DEFF Research Database (Denmark)

Riera, Marta; Pages, Montserrat; Issinger, Olaf Georg

2003-01-01

Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared...... to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio...

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

Energy Technology Data Exchange (ETDEWEB)

Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S. (Lodz - Poland); (UV)

2012-09-11

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

DEFF Research Database (Denmark)

Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

2011-01-01

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number...

Thymidine kinases in archaea

DEFF Research Database (Denmark)

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

2006-01-01

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

Handbook of purified gases

CERN Document Server

Schoen, Helmut

2015-01-01

Technical gases are used in almost every field of industry, science and medicine and also as a means of control by government authorities and institutions and are regarded as indispensable means of assistance. In this complete handbook of purified gases the physical foundations of purified gases and mixtures as well as their manufacturing, purification, analysis, storage, handling and transport are presented in a comprehensive way. This important reference work is accompanied with a large number of Data Sheets dedicated to the most important purified gases.  

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

Directory of Open Access Journals (Sweden)

Agarwala Usha

2011-06-01

Full Text Available Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6 are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP. An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with

Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain

International Nuclear Information System (INIS)

Duran, Joan; Navarro-Sabate, Aurea; Pujol, Anna; Perales, Jose C.; Manzano, Anna; Obach, Merce; Gomez, Marta; Bartrons, Ramon

2008-01-01

Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P 2 levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P 2 levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain

Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

Directory of Open Access Journals (Sweden)

Jagadeesh Kumar Dasappa

2013-01-01

Full Text Available Alzheimer’s disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A and neurofibrillary tangles (NFTs. The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB. The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6 exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

Insulin receptor binding and protein kinase activity in muscles of trained rats

International Nuclear Information System (INIS)

Dohm, G.L.; Sinha, M.K.; Caro, J.F.

1987-01-01

Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing ∼ 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise [ 125 I]. Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training

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.

Effect of heating and glycation on the allergenicity of 2S albumins (Ara h 2/6 from peanut.

Directory of Open Access Journals (Sweden)

Yvonne M Vissers

Full Text Available Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6 from peanut.Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition and functionality (basophil degranulation capacity of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6.Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

DEFF Research Database (Denmark)

Sapkota, Gopal P; Cummings, Lorna; Newell, Felicity S

2007-01-01

), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2......, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor......)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF...

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

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

2006-02-01

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chin-Soon Chee

2014-01-01

Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Structures of Staphylococcus aureus D-tagatose-6-phosphate kinase implicate domain motions in specificity and mechanism.

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Miallau, Linda; Hunter, William N; McSweeney, Sean M; Leonard, Gordon A

2007-07-06

High resolution structures of Staphylococcus aureus d-tagatose-6-phosphate kinase (LacC) in two crystal forms are herein reported. The structures define LacC in apoform, in binary complexes with ADP or the co-factor analogue AMP-PNP, and in a ternary complex with AMP-PNP and D-tagatose-6-phosphate. The tertiary structure of the LacC monomer, which is closely related to other members of the pfkB subfamily of carbohydrate kinases, is composed of a large alpha/beta core domain and a smaller, largely beta "lid." Four extended polypeptide segments connect these two domains. Dimerization of LacC occurs via interactions between lid domains, which come together to form a beta-clasp structure. Residues from both subunits contribute to substrate binding. LacC adopts a closed structure required for phosphoryl transfer only when both substrate and co-factor are bound. A reaction mechanism similar to that used by other phosphoryl transferases is proposed, although unusually, when both substrate and co-factor are bound to the enzyme two Mg(2+) ions are observed in the active site. A new motif of amino acid sequence conservation common to the pfkB subfamily of carbohydrate kinases is identified.

Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

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Yoo, Byung-Chun; Lee, Jung-Youn; Lucas, William J

2002-05-03

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

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Pieterse Corné MJ

2007-02-01

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

S6K1 is involved in polyploidization through its phosphorylation at Thr421/Ser424.

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Ma, Dongchu; Yu, Huiying; Lin, Di; Sun, Yinghui; Liu, Liping; Liu, Yage; Dai, Bing; Chen, Wei; Cao, Jianping

2009-04-01

Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole-induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole-induced Dami cell model, we found that 4E-BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M-phase in cytoplasm and oscillated in nocodazole-induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E-BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12-myristate 13-acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 --> Gln in PMA-induced Dami cells increased ploidy whereas overexpression of rapamycin-resistant form of S6K1 containing the mutations Thr421 --> Glu and Ser424 --> Asp significantly dephosphorylated 4E-BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA-induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin-resistant form of S6K1 significantly reversed polyploidization of nocodazole-induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole-induced Dami cells. Taken together, these data suggested that S6K1/4E-BP1 pathway may play an important role in polyploidization of megakaryocytes. (c) 2008 Wiley-Liss, Inc.

Role of adenosine 5'-monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle

DEFF Research Database (Denmark)

Glund, Stephan; Treebak, Jonas Thue; Long, Yun Chau

2009-01-01

IL-6 is released from skeletal muscle during exercise and has consequently been implicated to mediate beneficial effects on whole-body metabolism. Using 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), a pharmacological activator of 5'-AMP-activated protein kinase (AMPK), we tested......-type mice was also incubated with the AMPK activator A-769662. Incubation of mouse glycolytic extensor digitorum longus and oxidative soleus muscle for 2 h was associated with profound IL-6 mRNA production and protein release, which was suppressed by AICAR (P ... the hypothesis that AMPK modulates IL-6 release from isolated muscle. Skeletal muscle from AMPKalpha2 kinase-dead transgenic, AMPKalpha1 knockout (KO) and AMPKgamma3 KO mice and respective wild-type littermates was incubated in vitro, in the absence or presence of 2 mmol/liter AICAR. Skeletal muscle from wild...

Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli

International Nuclear Information System (INIS)

Bakali H, M. Amin; Nordlund, Pär; Hallberg, B. Martin

2006-01-01

The overexpression, crystallization and preliminary diffraction analysis of E. coli YegS are reported. yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2 1 , with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 Å, β = 96.97°. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (V M ) of 2.5 Å 3 Da −1 and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 Å using synchrotron radiation

Protein kinase activity associated with the corticosteroid binder IB

International Nuclear Information System (INIS)

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

1997-01-01

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

Activation of the interleukin-6/Janus kinase/STAT3 pathway in pleomorphic adenoma of the parotid gland

DEFF Research Database (Denmark)

Andreasen, Simon; Therkildsen, Marianne Hamilton; Grauslund, Morten

2015-01-01

The interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway is of crucial importance in promoting tumorigenesis in several malignant tumors but may also be active in benign tumors, e.g., of pleomorphic adenoma (PA). In this study we characterize ...

Phosphoglycerate kinase enhanced immunity of the whole cell of Streptococcus agalactiae in tilapia, Oreochromis niloticus.

Science.gov (United States)

Wang, Yi-Ting; Huang, Hsing-Yen; Tsai, Ming-An; Wang, Pei-Chi; Jiang, Bo-Huang; Chen, Shih-Chu

2014-12-01

Streptococcus agalactiae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of warmwater fish species. The outer-surface proteins in bacterial pathogens play an important role in pathogenesis. We evaluated the immunogenicity of two of the identified surface proteins namely phosphoglycerate kinase (PGK) and ornithine carbamoyl-transferase (OCT). PGK and OCT were over-expressed and purified from Escherichia coli and used as the subunit vaccines in tilapia. Tilapia immunized with the S. agalactiae modified bacteria vaccine (whole cell preparations with recombinant PGK and OCT proteins) individually were tested for the efficacy. OCT and PGK combined with WC had a higher survival rate. A high-level protection and significant specific antibody responses against S. agalactiae challenge was observed upon the vaccinated tilapia with the purified PGK protein and S. agalactiae whole cells. The specific antibody titer against S. agalactiae antigen suggested that increased antibody titers were correlated with post-challenge survival rate. Il-1β expression profile was higher in PGK + WC-treated group. Tnf-α expression in the PGK + WC group was significantly increased. Taken together, our results suggested the combinations of recombinant protein and whole cell may elicit immune responses that reach greater protection than that of individual S. agalactiae components. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

Science.gov (United States)

Kim, Han Ie; Jung, Jinwon; Lee, Eun-Saem; Kim, Yong-Chul; Lee, Weontae; Lee, Seung-Taek

2007-11-03

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

Science.gov (United States)

Chourashi, Rhishita; Das, Suman; Dhar, Debarpan; Okamoto, Keinosuke; Mukhopadhyay, Asish K; Chatterjee, Nabendu Sekhar

2018-05-01

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Cytotoxic Activity of 3,6-Dihydroxyflavone in Human Cervical Cancer Cells and Its Therapeutic Effect on c-Jun N-Terminal Kinase Inhibition

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Eunjung Lee

2014-08-01

Full Text Available Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR 4/CD14, p38 mitogen-activated protein kinase (MAPK, Jun-N terminal kinase (JNK, extracellular-signaling regulated kinase (ERK, and cyclooxygenase (COX-2 pathways in lipopolysaccharide (LPS-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M−1 and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

Impaired intracortical transmission in G2019S leucine rich-repeat kinase Parkinson patients.

Science.gov (United States)

Ponzo, Viviana; Di Lorenzo, Francesco; Brusa, Livia; Schirinzi, Tommaso; Battistini, Stefania; Ricci, Claudia; Sambucci, Manolo; Caltagirone, Carlo; Koch, Giacomo

2017-05-01

A mutation in leucine-rich repeat kinase 2 is the most common cause of hereditary Parkinson's disease (PD), yet the neural mechanisms and the circuitry potentially involved are poorly understood. We used different transcranial magnetic stimulation protocols to explore in the primary motor cortex the activity of intracortical circuits and cortical plasticity (long-term potentiation) in patients with the G2019S leucine-rich repeat kinase 2 gene mutation when compared with idiopathic PD patients and age-matched healthy subjects. Paired pulse transcranial magnetic stimulation was used to investigate short intracortical inhibition and facilitation and short afferent inhibition. Intermittent theta burst stimulation, a form of repetitive transcranial magnetic stimulation, was used to test long-term potentiation-like cortical plasticity. Leucine-rich repeat kinase 2 and idiopathic PD were tested both in ON and in OFF l-dopa therapy. When compared with idiopathic PD and healthy subjects, leucine-rich repeat kinase 2 PD patients showed a remarkable reduction of short intracortical inhibition in both ON and in OFF l-dopa therapy. This reduction was paralleled by an increase of intracortical facilitation in OFF l-dopa therapy. Leucine-rich repeat kinase 2 PD showed abnormal long-term potentiation-like cortical plasticity in ON l-dopa therapy. The motor cortex in leucine-rich repeat kinase 2 mutated PD patients is strongly disinhibited and hyperexcitable. These abnormalities could be a result of an impairment of inhibitory (gamma-Aminobutyric acid) transmission eventually related to altered neurotransmitter release. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways.

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Fang Xiao

Full Text Available p38 mitogen-activated protein kinase (MAPK is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.

Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei

International Nuclear Information System (INIS)

Zhuang, Ningning; Seo, Kyung Hye; Chen, Cong; Zhou, Jia; Kim, Seon Won; Lee, Kon Ho

2012-01-01

Recombinant mevalonate kinase from M. mazei has been crystallized. Diffraction data were collected to 2.08 Å resolution. Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl 2 , 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 97.11, b = 135.92, c = 46.03 Å. Diffraction data were collected to 2.08 Å resolution

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

OpenAIRE

Kilmury, Sara L. N.; Burrows, Lori L.

2016-01-01

Although two-component systems are a ubiquitous means of rapid bacterial adaptation to changing environments, identification of the specific signals detected by sensor kinases can be challenging. Also, little is known about the diverse, poorly characterized family of sensor kinases that detect intramembrane signals. We show that the major type IV pilin, PilA, is an inhibitory intramembrane ligand for the PilS sensor kinase that controls pilA expression and we characterize the mechanism of sig...

Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

International Nuclear Information System (INIS)

Senogles, S.E.; Caron, M.G.

1986-01-01

The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

Energy Technology Data Exchange (ETDEWEB)

Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

2015-09-18

The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

Detection of protein kinase activity by renaturation in sodium dodecyl sulfate-polyacrylamide gels

International Nuclear Information System (INIS)

Anostario, M. Jr.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

The authors have developed a procedure for identifying protein kinase activity in protein samples following electrophoresis on SDS-polyacrylamide gels. Proteins are allowed to renature directly in the gel by removal of detergent. The gel is then incubated with [γ- 32 P]ATP to allow renatured protein kinases to autophosphorylate or to phosphorylate various substrates which can be incorporated into the gel. The positions of the radiolabeled proteins can then be detected by autoradiography. With this technique, using purified catalytic subunit of cAMP-dependent protein kinase, enzyme concentrations as low as 0.01 μg can be detected on gels containing 1.0 mg/ml casein. The procedure is also applicable for the determination of active subunits of multisubunit protein kinases. For example, when the two subunits of casein kinase II are separated by SDS-polyacrylamide gel electrophoresis and allowed to renature, only the larger α subunit shows activity. This procedure can also be used to detect and distinguish kinases present in heterogeneous mixtures. Starting with a particulate fraction from LSTRA, a murine T cell lymphoma, several distinct enzymes were detected, including a 30,000 Dalton protein with protein-tyrosine kinase activity. This same enzyme has also been detected in T lymphocytes and other T lymphoid cell lines

2,6,9-Trisubstituted purines as CRK3 kinase inhibitors with antileishmanial activity in vitro

Czech Academy of Sciences Publication Activity Database

Řezníčková, Eva; Popa, Alexandr; Gucký, Tomáš; Zatloukal, Marek; Havlíček, Libor; Bazgier, Václav; Berka, K.; Jorda, Radek; Popa, Igor; Nasereddin, A.; Jaffe, Ch. L.; Kryštof, Vladimír; Strnad, Miroslav

2015-01-01

Roč. 25, č. 11 (2015), s. 2298-2301 ISSN 0960-894X R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Purine * Cyclin-dependent kinase * Leishmania Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.486, year: 2015

Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence

OpenAIRE

Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C.; Djordjevic, Julianne T.

2016-01-01

Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1?), and in combination with KCS1 (ipk1?kcs1?), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenua...

Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

DEFF Research Database (Denmark)

Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

1986-01-01

Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These e......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets...

Regulation of androgen receptor transactivity and mTOR-S6 kinase pathway by Rheb in prostate cancer cell proliferation.

Science.gov (United States)

Kobayashi, Takashi; Shimizu, Yosuke; Terada, Naoki; Yamasaki, Toshinari; Nakamura, Eijiro; Toda, Yoshinobu; Nishiyama, Hiroyuki; Kamoto, Toshiyuki; Ogawa, Osamu; Inoue, Takahiro

2010-06-01

Ras homolog-enriched in brain (Rheb), a small GTP-binding protein, is associated with prostate carcinogenesis through activating mammalian target of rapamycin (mTOR) signaling pathway. This study aimed to elucidate whether Rheb promotes proliferation of prostate cancer cells and can act as a potent therapeutic target in prostate cancer. Prostate cancer cell lines and human prostatic tissues were examined for the expression of Rheb. The effects of forced expression or knockdown of Rheb on cell proliferation were also examined. Semi-quantitative and quantitative RT-PCR were performed to evaluate mRNA expression. Western blotting was used to examine protein expression. Cell count and WST-1 assay were used to measure cell proliferation. Fluorescence-activated cell sorting was used to assess the cell cycle. Rheb mRNA and protein expression was higher in more aggressive, androgen-independent prostate cancer cell lines PC3, DU145, and C4-2, compared with the less aggressive LNCaP. Rheb expression was higher in cancer tissues than in benign prostatic epithelia. Forced expression of Rheb in LNCaP cells accelerated proliferation without enhancing androgen receptor transactivity. Attenuation of Rheb expression or treatment with the mTOR inhibitor rapamycin decreased proliferation of PC3 and DU145 cells, with a decrease in the activated form of p70S6 kinase, one of the main targets of mTOR. Rheb potentiates proliferation of prostate cancer cells and inhibition of Rheb or mTOR can lead to suppressed proliferation of aggressive prostate cancer cell lines in vitro. Rheb and the mTOR pathway are therefore probable targets for suppressing prostate cancer.

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.

Science.gov (United States)

Samuel, Marcus A; Mudgil, Yashwanti; Salt, Jennifer N; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R

2008-08-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

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

Science.gov (United States)

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

2016-07-29

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

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

International Nuclear Information System (INIS)

Tonoli, Celisa Caldana Costa; Vieira, Plinio Salmazo; Ward, Richard John; Arni, Raghuvir Krishnaswamy; Oliveira, Arthur Henrique Cavalcante de; Murakami, Mario Tyago

2009-01-01

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the nucleoside diphosphate kinase b from Leishmania major are reported. The crystals belonged to the trigonal space group P3 2 21 and diffracted to 2.18 Å resolution. Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host–parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 Å resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 Å. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3 2 21

Development of a biogas purifier for rural areas in Japan

Energy Technology Data Exchange (ETDEWEB)

Kimura, Y.; Hinata, T. [Hokkaido Central Agricultural Experiment Station, Hokkaido (Japan); Yasui, S. [Zukosha Co. Ltd., Obihiro, Hokkaido (Japan); Noguchi, N. [Hokkaido Univ., Sapporo, Hokkaido (Japan); Tsukamoto, T. [IHI Shibaura. Co. Ltd., Obihiro, Hokkaido (Japan); Imai, T. [Green Plan Co. Ltd., Sapporo, Hokkaido (Japan); Kanai, M. [Air Water Co. Ltd, Sakai, Osaka (Japan); Matsuda, Z. [Hokuren Agricultural Research Center, Sapporo, Hokkaido (Japan)

2010-07-01

Although the biogas that is currently produced for dairy farms in Japan is a carbon-neutral energy, its use is restricted to farming areas only because there is no effective method of transporting unused biogas. There is a need for establishing practical methods for biogas removal from operating systems. In this study, a gas separation membrane was used in order to modify biogas to city gas 12A specifications, and to develop a biogas purifier equipped with a device to fill high pressure purified gas into cylinders to be taken outside the farming area. The objective was to expand the use of biogas produced from stand-alone gas plants. The amount of purified gas produced at a newly created refining-compression-filling (RCF) facility was approximately 97.0 Nm{sup 3}/day, for a raw material amount of about 216.0 Nm{sup 3}/day. The heat quantity of the purified gas was 38.9 MJ/Nm{sup 3}, which was within city gas 12A specifications. A total of 14.3 cylinders were filled each day with the manufactured purified gas. Test calculations along with a simulation exercise revealed that it would be possible to provide purified gas to approximately 6 per cent of common residences in a town in northern Japan. It was concluded that the newly created RCF facility allowed the modification of carbon-neutral biogas to conform to city gas 12A specifications, and allowed the transport of this gas out of the farming area.

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

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Victoria Prieto-Echagüe

2011-04-01

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

Protein kinase CK2 structure-function relationship

DEFF Research Database (Denmark)

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

1994-01-01

Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations...

Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak’s extracts

International Nuclear Information System (INIS)

Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,; Suzery, Meiny

2015-01-01

Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak’s extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r 2 =0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak’s extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel

Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak’s extracts

Energy Technology Data Exchange (ETDEWEB)

Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,; Suzery, Meiny [Organic Chemistry Laboratory, Departement of Chemistry, Diponegoro University Jln Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia Tel / Fax: (024) 7460058 (Indonesia)

2015-12-29

Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak’s extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r{sup 2}=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak’s extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

Purifying hydrocarbons

Energy Technology Data Exchange (ETDEWEB)

Dunstan, A E

1918-06-03

Ligroin, kerosene, and other distillates from petroleum and shale oil, are purified by treatment with a solution of a hypochlorite containing an excess of alkali. The hydrocarbon may be poured into brine, the mixture stirred, and an electric current passed through. Heat may be applied.

Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes

International Nuclear Information System (INIS)

Gnemmi, Ilaria; Scotti, Claudia; Cappelletti, Donata; Canonico, Pier Luigi; Condorelli, Fabrizio; Rosano, Camillo

2006-01-01

The catalytic domain of human Fes tyrosine kinase has been cloned, expressed, purified and crystallized. The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell–cell and cell–matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described

Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

Science.gov (United States)

Lee, Bang Hyo; Yoon, Soo-Hyun; Kim, Yun-Sook; Kim, Sang Kook; Moon, Byong Jo; Bae, Young-Seuk

2008-01-01

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

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Zhang Rui-Wen

2011-05-01

Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

High Level of Soluble FMS-Like Tyrosine Kinase-1 (sFlt-1 Serum in Pregnancy as a Risk Factor of Preeclampsia

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I Gede Mega Putra

2016-07-01

Full Text Available Background: Recently, etiology and pathogenesis of preeclampsia remain unknown. One of the theory indicating that hypoxia and ischemic placenta caused by abnormal cytotrophoblast invasion in preeclampsia. Soluble Fms-like tyrosine kinase-1 (sFlt-1 serum as a laboratory marker of hypoxia condition that contributes to the occurrence of endothelial damage and clinical manifestations in preeclampsia. Objective: This study was aimed at proving that high level of soluble Fms-like tyrosine kinase-1 (sFlt-1 serum in pregnancy as a risk factor for preeclampsia. Methods: This study was a case control. Among 58 pregnant women studied, 29 women with preeclampsia as a case group and 29 women with normal pregnancy as a control group. Soluble Fms-like tyrosine kinase-1 (sFlt-1 serum was analyzed in the Prodia Laboratory. Collected data were tested for normality using Kolmogorov-Smirnov, then analyzed with independent sample test. Chi-Square test used to determine soluble Fms-like tyrosine kinase-1 (sFlt-1 serum level in preeclampsia. Results: This research concluded that the average level of soluble Fms-like tyrosine kinase-1 (sFlt-1 serum in preeclampsia were 11231.00 ± 8390.3 pg/mL and 3981.62 ± 4921.5 pg/mL in normal pregnancy. Analysis of significance with independent t-test concluded that the value of t = 4.01 and p = 0.001. This mean the average levels of soluble Fms-like tyrosine kinase-1 (sFlt-1serum levels in both groups were difference significantly (p <0.05. Based on the cut-off point of sFlt-1 serum levels was 4505.50 pg/mL with 79.3% sensitivity and 82.8% specificity, the relative risk of preeclampsia was 18 times (OR = 18.40, IK 95% = 4.93 to 68.70, p = 0.001. Conclusion:  Based on this research, high levels of soluble Fms-like tyrosine kinase-1 (sFlt-1 in pregnancy was proved as a risk factor for preeclampsia.

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

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Fabian König

Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

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Elena Nekhoroshkova

Full Text Available BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149 that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.

Proteomic Analysis of a Global Regulator GacS Sensor Kinase in the Rhizobacterium, Pseudomonas chlororaphis O6

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Chul Hong Kim

2014-06-01

Full Text Available The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulator of many traits relevant to the biocontrol function of this bacterium. Proteomic analysis revealed 12 proteins were down-regulated in a gacS mutant of P. chlororaphis O6. These GacS-regulated proteins functioned in combating oxidative stress, cell signaling, biosynthesis of secondary metabolism, and secretion. The extent of regulation was shown by real-time RT-PCR to vary between the genes. Mutants of P. chlororaphis O6 were generated in two GacS-regulated genes, trpE, encoding a protein involved in tryptophan synthesis, and prnA, required for conversion of tryptophan to the antimicrobial compound, pyrrolitrin. Failure of the trpE mutant to induce systemic resistance in tobacco against a foliar pathogen causing soft rot, Pectobacterium carotovorum SCCI, correlated with reduced colonization of root surfaces implying an inadequate supply of tryptophan to support growth. Although colonization was not affected by mutation in the prnA gene, induction of systemic resistance was reduced, suggesting that pyrrolnitrin was an activator of plant resistance as well as an antifungal agent. Study of mutants in the other GacS-regulated proteins will indicate further the features required for biocontrol-activity in this rhizobacterium.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

Science.gov (United States)

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

Protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with photosystem II

International Nuclear Information System (INIS)

Coughlan, S.J.; Hind, G.

1987-01-01

Thylakoid membranes were phosphorylated with [γ- 32 P]ATP and extracted with octyl glucoside and cholate. Among the radiolabeled phosphoproteins in the extract was a previously characterized protein kinase of 64-kDa apparent mass. The ability of this enzyme to undergo autophosphorylation in situ was used to monitor its distribution in the membrane. Fractionation studies showed that the kinase is confined to granal regions of the thylakoid, where it appears to be associated with the light-harvesting chlorophyll-protein complex of photosystem II. The kinetics of kinase autophosphorylation were investigated both in situ and in extracted, purified enzyme. In the membrane, autophosphorylation saturated within 20-30 min and was reversed with a half-time of 7-8 min upon removal of ATP or oxidative inactivation of the kinase; the accompanying dephosphorylation of light-harvesting complex was slower and kinetically complex. Fluoride (10 mM) inhibited these dephosphorylations. Autophosphorylation of the isolated kinase was independent of enzyme concentration, indicative of an intramolecular mechanism. A maximum of one serine residue per mole of kinase was esterified. Autophosphorylation was more rapid in the presence of histone IIIs, an exogenous substrate. Dephosphorylation of the isolated enzyme was not observed

Methods for Purifying Enzymes for Mycoremediation

Science.gov (United States)

Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

2014-01-01

A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

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.

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

Science.gov (United States)

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

2012-08-01

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

Hydrogen peroxide mediates Rac1 activation of S6K1

International Nuclear Information System (INIS)

Bae, Gyu-Un; Kim, Yong Kee; Kwon, Hyoung-Keun; Park, Jong Woo; Lee, Eun Kyung; Paek, Se Jin; Choi, Wahn Soo; Jung, In Duk; Lee, Hoi Young; Cho, Eun-Jung; Lee, Hyang Woo; Han, Jeung-Whan

2004-01-01

We previously reported that hydrogen peroxide (H 2 O 2 ) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H 2 O 2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H 2 O 2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H 2 O 2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H 2 O 2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H 2 O 2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H 2 O 2 as a mediator in the activation of S6K1 by Rac1. However, H 2 O 2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H 2 O 2 could be produced by arsenite, which has been shown to be a stimulator of H 2 O 2 production. Taken together, these results suggest that H 2 O 2 plays a pivotal role as a mediator in Rac1 activation of S6K1

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Science.gov (United States)

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

Metazoan-like signaling in a unicellular receptor tyrosine kinase

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Schultheiss Kira P

2013-02-01

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

Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

DEFF Research Database (Denmark)

Guerra, B; Niefind, K; Pinna, L A

1998-01-01

The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors

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Ting Xiao

2016-10-01

Full Text Available Eukaryotic elongation factor 2 kinase (eEF2K inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

Aurora kinase inhibitors: Progress towards the clinic

Czech Academy of Sciences Publication Activity Database

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

2012-01-01

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

Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells.

Science.gov (United States)

Ye, Xueting; Xie, Jing; Huang, Hang; Deng, Zhexian

2018-01-01

Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling. © 2018 The Author(s). Published by S. Karger AG, Basel.

Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation

International Nuclear Information System (INIS)

Vahedi, Shahrooz; Chueh, Fu-Yu; Chandran, Bala; Yu, Chao-Lan

2015-01-01

of intramitochondrial translational machinery, in Jurkat further supports the role of mitochondrial Lck as a negative regulator of CRIF1 through competitive binding. This is the first report demonstrating the role of mitochondrial Lck in metabolic reprogramming of leukemic cells. Mechanistically, it is distinct from other reported mitochondrial protein tyrosine kinases. In a kinase-independent manner, mitochondrial Lck interferes with mitochondrial translational machinery through competitive binding to CRIF1. These findings may reveal novel approaches in cancer therapy by targeting cancer cell metabolism. The online version of this article (doi:10.1186/s12885-015-1520-6) contains supplementary material, which is available to authorized users

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

Science.gov (United States)

Eguchi, Yoko; Utsumi, Ryutaro

2014-09-01

Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Simultaneous purifying of Hg0, SO2, and NOx from flue gas by Fe3+/H2O2: the performance and purifying mechanism.

Science.gov (United States)

Xing, Yi; Li, Liuliu; Lu, Pei; Cui, Jiansheng; Li, Qianli; Yan, Bojun; Jiang, Bo; Wang, Mengsi

2018-03-01

Hg 0 , SO 2 , and NOx result in heavily global environmental pollution and serious health hazards. Up to now, how to efficiently remove mercury with SO 2 and NOx from flue gas is still a tough task. In this study, series of high oxidizing Fenton systems were employed to purify the pollutants. The experimental results showed that Fe 3+ /H 2 O 2 was more suitable to purify Hg 0 than Fe 2+ /H 2 O 2 and Cu 2+ /H 2 O 2. The optimal condition includes Fe 3+ concentration of 0.008 mol/L, Hg 0 inlet concentration of 40 μg/m 3 , solution temperature of 50 °C, pH of 3, H 2 O 2 concentration of 0.7 mol/L, and O 2 percentage of 6%. When SO 2 and NOx were taken into account under the optimal condition, Hg 0 removal efficiency could be enhanced to 91.11% while the removal efficiency of both NOx and SO 2 was slightly declined, which was consistent to the analysis of purifying mechanism. The removal efficiency of Hg 0 was stimulated by accelerating the conversion of Fe 2+ to Fe 3+ , which resulted from the existence of SO 2 and NOx. The results of this study suggested that simultaneously purifying Hg 0 , SO 2 , and NOx from flue gas is feasible.

Inference of purifying and positive selection in three subspecies of chimpanzees (Pan troglodytes) from exome sequencing

DEFF Research Database (Denmark)

Bataillon, Thomas; Duan, Jinjie; Hvilsom, Christina

2015-01-01

of recent gene flow from Western into Eastern chimpanzees. The striking contrast in X-linked vs. autosomal polymorphism and divergence previously reported in Central chimpanzees is also found in Eastern and Western chimpanzees. We show that the direction of selection (DoS) statistic exhibits a strong non......-monotonic relationship with the strength of purifying selection S, making it inappropriate for estimating S. We instead use counts in synonymous vs. non-synonymous frequency classes to infer the distribution of S coefficients acting on non-synonymous mutations in each subspecies. The strength of purifying selection we...... infer is congruent with the differences in effective sizes of each subspecies: Central chimpanzees are undergoing the strongest purifying selection followed by Eastern and Western chimpanzees. Coding indels show stronger selection against indels changing the reading frame than observed in human...

Investigation of the Flexibility of Protein Kinases Implicated in the Pathology of Alzheimer’s Disease

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Michael P. Mazanetz

2014-06-01

Full Text Available The pathological characteristics of Alzheimer’s Disease (AD have been linked to the activity of three particular kinases—Glycogen Synthase Kinase 3β (GSK3β, Cyclin-Dependent Kinase 5 (CDK5 and Extracellular-signal Regulated Kinase 2 (ERK2. As a consequence, the design of selective, potent and drug-like inhibitors of these kinases is of particular interest. Structure-based design methods are well-established in the development of kinase inhibitors. However, progress in this field is limited by the difficulty in obtaining X-ray crystal structures suitable for drug design and by the inability of this method to resolve highly flexible regions of the protein that are crucial for ligand binding. To address this issue, we have undertaken a study of human protein kinases CDK5/p25, CDK5, ERK2 and GSK3β using both conventional molecular dynamics (MD and the new Active Site Pressurisation (ASP methodology, to look for kinase-specific patterns of flexibility that could be leveraged for the design of selective inhibitors. ASP was used to examine the intrinsic flexibility of the ATP-binding pocket for CDK5/p25, CDK5 and GSK3β where it is shown to be capable of inducing significant conformational changes when compared with X-ray crystal structures. The results from these experiments were used to quantify the dynamics of each protein, which supported the observations made from the conventional MD simulations. Additional information was also derived from the ASP simulations, including the shape of the ATP-binding site and the rigidity of the ATP-binding pocket. These observations may be exploited in the design of selective inhibitors of GSK3β, CDK5 and ERK2.

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor

Czech Academy of Sciences Publication Activity Database

Kryštof, Vladimír; McNae, I. W.; Walkinshaw, M. D.; Fischer, P.M.; Müller, P.; Vojtešek, B.; Orság, Martin; Havlíček, Libor; Strnad, Miroslav

2005-01-01

Roč. 62, č. 15 (2005), s. 1763-1771 ISSN 1420-682X R&D Projects: GA ČR GP204/03/D231 Institutional research plan: CEZ:AV0Z50380511 Keywords : olomoucine II * roscovitine * cyclin-dependent kinase inhibitor Subject RIV: CE - Biochemistry Impact factor: 4.582, year: 2005

Hydrogen purifier module with membrane support

Science.gov (United States)

A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

2012-07-24

A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

International Nuclear Information System (INIS)

Contreras-Paredes, Adriana; Cruz-Hernandez, Erick de la; Martinez-Ramirez, Imelda; Duenas-Gonzalez, Alfonso; Lizano, Marcela

2009-01-01

Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation

Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae.

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David E Garcia

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

Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase, extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun N-terminal kinase downregulation in obese rats.

Science.gov (United States)

Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal

2015-02-01

Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

Science.gov (United States)

Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

2018-05-01

The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.

Science.gov (United States)

Lebeau, Paul; Byun, Jae Hyun; Yousof, Tamana; Austin, Richard C

2018-04-22

Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity. Copyright © 2018. Published by Elsevier Inc.

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

Science.gov (United States)

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

DEFF Research Database (Denmark)

Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim

2005-01-01

characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly......, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372...

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture

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Sonia eGazzarrini

2014-04-01

Full Text Available Carbohydrates, or sugars, regulate various aspects of plant growth through modulation of cell division and expansion. Besides playing essential roles as sources of energy for growth and as structural components of cells, carbohydrates also regulate the timing of expression of developmental programs. The disaccharide trehalose is used as an energy source, as a storage and transport molecule for glucose, and as a stress-responsive compound important for cellular protection during stress in all kingdoms. Trehalose, however, is found in very low amounts in most plants, pointing to a signaling over metabolic role for this non-reducing disaccharide. In the last decade, trehalose-6-phosphate (T6P, an intermediate in trehalose metabolism, has been shown to regulate embryonic and vegetative development, flowering time, meristem determinacy and cell fate specification in plants. T6P acts as a global regulator of metabolism and transcription promoting plant growth and triggering developmental phase transitions in response to sugar availability. Among the T6P targets are members of the Sucrose-non-fermenting1-Related Kinase1 (SnRK1 family, which are sensors of energy availability and inhibit plant growth and development during metabolic stress to maintain energy homeostasis. In this review, we will discuss the opposite roles of the sugar metabolite T6P and the SnRK1 kinases in the regulation of developmental phase transitions in response to carbohydrate levels. We will focus on how these two global regulators of metabolic processes integrate environmental cues and interact with hormonal signaling pathways to modulate plant development.

Phosphoinositide-3-kinases p110alpha and p110beta mediate S phase entry in astroglial cells in the marginal zone of rat neocortex

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Rabea eMüller

2013-03-01

Full Text Available In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine. Only in astroglial cells harvested from the marginal zone of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with bromodeoxyuridine, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110 and p110were essential for S phase entry. p110 diminished the level of the p27Kip1 which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110phosphorylated and inhibited glycogen synthase kinase-3which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

Evaluation of a C57BL/6J × 129S1/SvImJ Hybrid Nestin-Thymidine Kinase Transgenic Mouse Model for Studying the Functional Significance of Exercise-Induced Adult Hippocampal Neurogenesis.

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Hamilton, G F; Majdak, P; Miller, D S; Bucko, P J; Merritt, J R; Krebs, C P; Rhodes, J S

2015-01-01

New neurons are continuously generated in the adult hippocampus but their function remains a mystery. The nestin thymidine kinase (nestin-TK) transgenic method has been used for selective and conditional reduction of neurogenesis for the purpose of testing the functional significance of new neurons in learning, memory and motor performance. Here we explored the nestin-TK model on a hybrid genetic background (to increase heterozygosity, and "hybrid vigor"). Transgenic C57BL/6J (B6) were crossed with 129S1/SvImJ (129) producing hybrid offspring (F1) with the B6 half of the genome carrying a herpes simplex virus thymidine kinase (TK) transgene regulated by a modified nestin promoter. In the presence of exogenously administered valganciclovir, new neurons expressing TK undergo apoptosis. Female B6 nestin-TK mice ( n = 80) were evaluated for neurogenesis reduction as a positive control. Male and female F1 nestin-TK mice ( n = 223) were used to determine the impact of neurogenesis reduction on the Morris water maze (MWM) and rotarod. All mice received BrdU injections to label dividing cells and either valganciclovir or control chow, with or without a running wheel for 30 days. Both the F1 and B6 background displayed approximately 50% reduction in neurogenesis, a difference that did not impair learning and memory on the MWM or rotarod performance. Running enhanced neurogenesis and performance on the rotarod but not MWM suggesting the F1 background may not be suitable for studying pro-cognitive effects of exercise on MWM. Greater reduction of neurogenesis may be required to observe behavioral impacts. Alternatively, new neurons may not play a critical role in learning, or compensatory mechanisms in pre-existing neurons could have masked the deficits. Further work using these and other models for selectively reducing neurogenesis are needed to establish the functional significance of adult hippocampal neurogenesis in behavior.

RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility

Czech Academy of Sciences Publication Activity Database

Vomastek, Tomáš; Iwanicki, M. P.; Schaeffer, J.; J.; Tarcsafalvi, A.; Parsons, J. T.; Weber, M. J.

2007-01-01

Roč. 27, č. 23 (2007), s. 8296-8305 ISSN 0270-7306 R&D Projects: GA AV ČR IAA500200716 Institutional research plan: CEZ:AV0Z50200510 Keywords : protein kinase * adhesion * cell Subject RIV: EE - Microbiology, Virology Impact factor: 6.420, year: 2007

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis.

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van der Meer, Jonathan H M; van der Poll, Tom; van 't Veer, Cornelis

2014-04-17

TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

Science.gov (United States)

Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

2006-06-01

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress

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Naomi Coulton

2017-12-01

Full Text Available While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10 and is epistatic with a mutation in DNA polymerase delta (cdc6.23 when DNA is alkylated by methyl-methanesulfate (MMS. The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50 is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

Science.gov (United States)

Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

2008-01-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

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

Science.gov (United States)

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

2001-01-01

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

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-01-01

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-05-06

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

21 CFR 880.6710 - Medical ultraviolet water purifier.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.

Science.gov (United States)

Yuan, Bifeng; Wang, Yinsheng

2008-08-29

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

Prolonged activation of S6K1 does not suppress IRS or PI-3 kinase signaling during muscle cell differentiation

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MacKenzie Matthew G

2010-05-01

Full Text Available Abstract Background Myogenesis in C2C12 cells requires the activation of the PI3K/mTOR signaling pathways. Since mTOR signaling can feedback through S6K1 to inhibit the activation of PI3K, the aim of this work was to assess whether feedback from S6K1 played a role in myogenesis and determine whether siRNA mediated knockdown of S6K1 would lead to an increased rate of myotube formation. Results S6K1 activity increased in a linear fashion following plating and was more than 3-fold higher after Day 3 of differentiation (subconfluent = 11.09 ± 3.05, Day 3 = 29.34 ± 3.58. IRS-1 levels tended to increase upon serum withdrawal but decreased approximately 2-fold (subconfluent = 0.88 ± 0.10, Day 3 = 0.42 ± 0.06 3 days following differentiation whereas IRS-2 protein remained stable. IRS-1 associated p85 was significantly reduced upon serum withdrawal (subconfluent = 0.86 ± 0.07, Day 0 = 0.31 ± 0.05, remaining low through day 1. IRS-2 associated p85 decreased following serum withdrawal (subconfluent = 0.96 ± 0.05, Day 1 = 0.56 ± 0.08 and remained suppressed up to Day 3 following differentiation (0.56 ± 0.05. Phospho-tyrosine associated p85 increased significantly from subconfluent to Day 0 and remained elevated throughout differentiation. siRNA directed against S6K1 and S6K2 did not result in changes in IRS-1 levels after either 48 or 96 hrs. Furthermore, neither 48 nor 96 hrs of S6K1 knockdown caused a change in myotube formation. Conclusions Even though S6K1 activity increases throughout muscle cell differentiation and IRS-1 levels decrease over this period, siRNA suggests that S6K1 is not mediating the decrease in IRS-1. The decrease in IRS-1/2 associated p85 together with the increase in phospho-tyrosine associated p85 suggests that PI3K associates primarily with scaffolds other than IRS-1/2 during muscle cell differentiation.

Mechanical performance of HMA-2 modified with purified and unpurified carbon nanotubes and nanofibers

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Mario Rodrigo Rubio

2017-05-01

Full Text Available The present study evaluates the mechanical performance of a Hot Mix Asphalt – Type II (HMA-2 modified with carbon nanotubes and carbon nanofibers (CNTF. CNTF were made by means the Catalytic Vapor Deposition (CVD technique at 700° C using a Nickel, Copper and Aluminum (NiCuAl catalyst with a Cu/Ni molar relation of 0,33. In order to properly assess HMA-2 performance, three different mixtures were analyzed: 1 HMA-2 modified with purified CNTF; 2 HMA-2 modified with non-purified CNTF and, 3 a Conventional HMA-2 (control. Samples manufactured in accordance with the Marshall Mix Design were tested in the laboratory to study rutting, resilient modulus (Mr and fatigue. In addition to the aforementioned dynamic characterization, the effect of CNTF purification on the asphalt mixture’s mechanical properties was analyzed. In short, a comparative study was designed to determine whether or not CNTF should be purified before introduction into the HMA-2. This investigation responds to the growing demand for economical materials capable of withstanding traffic loads while simultaneously enhancing pavement durability and mechanical properties. Although purified CNTF increased HMA-2 stiffness and elastic modulus, non-purified CNTF increased the asphalt mixture’s elastic modulus without considerable increases in stiffness. Thus, the latter modification is deemed to help address fatiguerelated issues and improve the long-term durability of flexible pavements.

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

Energy Technology Data Exchange (ETDEWEB)

Drygin, Denis, E-mail: ddrygin@cylenepharma.com; Ho, Caroline B.; Omori, Mayuko; Bliesath, Joshua; Proffitt, Chris; Rice, Rachel; Siddiqui-Jain, Adam; O' Brien, Sean; Padgett, Claire; Lim, John K.C.; Anderes, Kenna; Rice, William G.; Ryckman, David

2011-11-11

Highlights: Black-Right-Pointing-Pointer We examine the potential cross-talk between CK2 and IL-6. Black-Right-Pointing-Pointer Inhibition of CK2 by siRNA or CX-4945 inhibits expression of IL-6 in models of IBC. Black-Right-Pointing-Pointer Treatment of IBC patient in the clinic with CX-4945 reduces her IL-6 plasma levels. Black-Right-Pointing-Pointer We demonstrate that CK2 is a potential therapeutic target for IL-6 driven diseases. -- Abstract: Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

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

Science.gov (United States)

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

2007-01-11

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

Purification, crystallization and preliminary X-ray diffraction analysis of the non-ATPase subunit Nas6 in complex with the ATPase subunit Rpt3 of the 26S proteasome from Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Nakamura, Yoshihiro; Umehara, Takashi; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

2007-01-01

The complex of the non-ATPase subunit Nas6 with the C-terminal domain of the ATPase subunit Rpt3 of the 26S proteasome from S. cerevisiae was co-expressed in E. coli and purified to homogeneity. The crystals obtained from the protein complex diffracted to a resolution of 2.2 Å. The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P2 1 , with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 Å, β = 94.70° and with three Nas6–Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 Å resolution using synchrotron radiation

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

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

2017-01-01

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

Mutagenic and Cytotoxic Properties of 6-Thioguanine, S6-Methylthioguanine, and Guanine-S6-sulfonic Acid*S⃞

OpenAIRE

Yuan, Bifeng; Wang, Yinsheng

2008-01-01

Thiopurine drugs, including 6-thioguanine (SG), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of SG nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. SG in DNA can be methylated by S-adenosyl-l-methionine to give S6-methylthioguanine (S6mG) and oxidized by UVA light to render guanine-S6-sulfonic acid ...

Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

Science.gov (United States)

2015-12-01

AWARD NUMBER: W81XWH-14-1-0251 TITLE: Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase... Tyrosine Kinase Inhibitors 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0251 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kiran Mahajan 5d...ABSTRACT Central to all cycling cells-including prostate cancer stem cells- is the expression of WEE1 tyrosine kinase. WEE1 monitors duplication of

Comparative analysis of LytS/LytTR-type histidine kinase/response regulator systems in γ-proteobacteria.

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Stefan Behr

Full Text Available Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states. Escherichia coli contains two LytS/LytTR-type histidine kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT and YpdA/YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment.

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

DEFF Research Database (Denmark)

McCord, Ronald A; Michishita, Eriko; Hong, Tao

2009-01-01

-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

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Dhawan Gunjan

2012-07-01

Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

Expression, purification, crystallization and preliminary crystallographic analysis of the phosphoglycerate kinase from Acinetobacter baumannii

International Nuclear Information System (INIS)

Baretta, Kayla; Garen, Craig; Yin, Jiang; James, Michael N. G.

2012-01-01

Approximately five decades have passed with only one or two new antibiotics making it into clinical use. Phosphoglycerate kinase from A. baumanii has been selected as a potential target for antibiotic development; this paper presents the initial structural biological results from this research. Acinetobacter baumannii is a common multidrug-resistant clinical pathogen that is often found in hospitals. The A. baumannii phosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development. AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. The AbPGK crystals belonged to space group P222 1 . They diffracted to a resolution of 2.5 Å using synchrotron radiation at the Canadian Light Source

S6K1 in the central nervous system regulates energy expenditure via MC4R/CRH pathways in response to deprivation of an essential amino acid.

Science.gov (United States)

Xia, Tingting; Cheng, Ying; Zhang, Qian; Xiao, Fei; Liu, Bin; Chen, Shanghai; Guo, Feifan

2012-10-01

It is well established that the central nervous system (CNS), especially the hypothalamus, plays an important role in regulating energy homeostasis and lipid metabolism. We have previously shown that hypothalamic corticotropin-releasing hormone (CRH) is critical for stimulating fat loss in response to dietary leucine deprivation. The molecular mechanisms underlying the CNS regulation of leucine deprivation-stimulated fat loss are, however, still largely unknown. Here, we used intracerebroventricular injection of adenoviral vectors to identify a novel role for hypothalamic p70 S6 kinase 1 (S6K1), a major downstream effector of the kinase mammalian target of rapamycin, in leucine deprivation stimulation of energy expenditure. Furthermore, we show that the effect of hypothalamic S6K1 is mediated by modulation of Crh expression in a melanocortin-4 receptor-dependent manner. Taken together, our studies provide a new perspective for understanding the regulation of energy expenditure by the CNS and the importance of cross-talk between nutritional control and regulation of endocrine signals.

A chemical genetic approach to engineer phototropin kinases for substrate labeling.

Science.gov (United States)

Schnabel, Jonathan; Hombach, Peter; Waksman, Thomas; Giuriani, Giovanni; Petersen, Jan; Christie, John M

2018-04-13

Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N 6 -benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N 6 -benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions. © 2018 Schnabel et al.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Science.gov (United States)

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

Science.gov (United States)

Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

2002-02-01

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

Respirators: Air Purifying, Self-Study, Course 40723

Energy Technology Data Exchange (ETDEWEB)

Chochoms, Michael [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2017-12-21

Respirators: Air Purifying Self-Study (COURSE 40723) is designed for Los Alamos National Laboratory (LANL) workers, support services subcontractors, and other LANL subcontractors who work under the LANL Respiratory Protection Program (RPP). This course also meets the air-purifying respirators (APRs) retraining requirement.

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

Science.gov (United States)

Syed, Deeba N; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K; Adhami, Vaqar M; Mukhtar, Hasan

2014-06-01

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. Published by Elsevier Inc.

Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino- and carboxyl-terminal end domains.

Science.gov (United States)

Ando, S; Tokui, T; Yano, T; Inagaki, M

1996-04-05

We reported earlier that phosphorylation in vitro of keratin filaments reconstituted from rat type I keratin 18 and type II keratin 8 by cAPM-dependent protein kinase induces disassembly of the keratin filament structure. Keratin 8 rather than keratin 18 was the major target of the kinase. We have now identified the sites on rat keratin 8 for cAMP-dependent protein kinase. Sequential analysis of the purified phosphoropeptides, together with the known primary sequence, revealed that four major sites, Ser-12, Ser-23, Ser-36, and Ser-50, and three minor sites, Ser-8, Ser-33, Ser-42, are located in the amino-terminal head domain, while three minor sites, Ser-416, Ser-423 and Ser-425 locate in the carboxyl-terminal tail domain.

S6K1 and 4E-BP1 are independent regulated and control cellular growth in bladder cancer.

Directory of Open Access Journals (Sweden)

Roman Nawroth

Full Text Available Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K/Akt/mammalian target of rapamycin (mTOR and the mitogen activated protein kinase (MAPK signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC. However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.

Dendritic cells sensitize TCRs through self-MHC-mediated Src family kinase activation

Czech Academy of Sciences Publication Activity Database

Meraner, P.; Hořejší, Václav; Wolpl, A.; Fischer, G.F.; Stingl, G.; Maurer, D.

2007-01-01

Roč. 178, č. 4 (2007), s. 2262-2271 ISSN 0022-1767 Institutional research plan: CEZ:AV0Z50520514 Keywords : TCR * dendritic cells * Src kinases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.068, year: 2007

The effect of a photocatalytic air purifier on indoor air quality quantified using different measuring methods

DEFF Research Database (Denmark)

Kolarik, Barbara; Wargocki, Pawel; Skorek-Osikowska, A.

2010-01-01

The effect on indoor air quality of an air purifier based on photocatalytic oxidation (PCO) was determined by different measuring techniques: sensory assessments of air quality made by human subjects, Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) and chromatographic methods (Gas......, additional measurements were made with no pollution sources present in the office. All conditions were tested with the photocatalytic air purifier turned on and off. The results show that operation of the air purifier in the presence of pollutants emitted by building materials and furniture improves indoor...... Chromatography/Mass Spectrometry and High-Pressure Liquid Chromatography with UV detection). The experiment was conducted in a simulated office, ventilated with 0.6 h(-1), 2.5 h(-1) and 6 h(-1), in the presence of additional pollution sources (carpet, chipboard and linoleum). At the lowest air change rate...

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

Science.gov (United States)

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells*

Science.gov (United States)

Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-01-01

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. PMID:22669945

Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells.

Science.gov (United States)

Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-07-27

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

Experimental studies on removal of airborne haloanisoles by non-thermal plasma air purifiers

DEFF Research Database (Denmark)

Fang, Lei; Hallam, David; Bermúdez, Raúl

2016-01-01

A laboratory study was conducted to test the performance of non-thermal plasma air purifiers on its removal effectiveness of two haloanisoles – 2,4,6-trichloroanisole (TCA) and 2,4,6-Tribromoanisole (TBA). TCA and TBA are the two major compounds found in wine cellars that can contaminate wine to ...

A hepatic amino acid/mTOR/S6K-dependent signalling pathway modulates systemic lipid metabolism via neuronal signals.

Science.gov (United States)

Uno, Kenji; Yamada, Tetsuya; Ishigaki, Yasushi; Imai, Junta; Hasegawa, Yutaka; Sawada, Shojiro; Kaneko, Keizo; Ono, Hiraku; Asano, Tomoichiro; Oka, Yoshitomo; Katagiri, Hideki

2015-08-13

Metabolism is coordinated among tissues and organs via neuronal signals. Levels of circulating amino acids (AAs), which are elevated in obesity, activate the intracellular target of rapamycin complex-1 (mTORC1)/S6kinase (S6K) pathway in the liver. Here we demonstrate that hepatic AA/mTORC1/S6K signalling modulates systemic lipid metabolism via a mechanism involving neuronal inter-tissue communication. Hepatic expression of an AA transporter, SNAT2, activates the mTORC1/S6K pathway, and markedly elevates serum triglycerides (TGs), while downregulating adipose lipoprotein lipase (LPL). Hepatic Rheb or active-S6K expression have similar metabolic effects, whereas hepatic expression of dominant-negative-S6K inhibits TG elevation in SNAT2 mice. Denervation, pharmacological deafferentation and β-blocker administration suppress obesity-related hypertriglyceridemia with adipose LPL upregulation, suggesting that signals are transduced between liver and adipose tissue via a neuronal pathway consisting of afferent vagal and efferent sympathetic nerves. Thus, the neuronal mechanism uncovered here serves to coordinate amino acid and lipid levels and contributes to the development of obesity-related hypertriglyceridemia.

Inhibition of IGF-1 receptor kinase blocks the differentiation into cardiomyocyte-like cells of BMSCs induced by IGF-1.

Science.gov (United States)

Gong, Haibin; Wang, Xiuli; Wang, Lei; Liu, Ying; Wang, Jie; Lv, Qian; Pang, Hui; Zhang, Qinglin; Wang, Zhenquan

2017-07-01

Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into cardiomyocyte‑like cells (CLCs) if an appropriate cardiac environment is provided. Insulin‑like growth factor‑1 (IGF‑1) plays an important role in the cell migration, survival and differentiation of BMSCs. However, the effect of IGF‑1 on the cellular differentiation remains unclear. In the present study, BMSCs were isolated from rat femurs and tibias and the cells were purified at passage 6 (P6). IGF‑1 and IGF‑1 receptor (IGF‑1R) kinase inhibitor I‑OMe AG538 were added to detect if IGF‑1 could induce BMSCs to transdifferentiate into CLCs and if I‑OMe AG538 could inhibit IGF‑1‑mediated receptor activation and downstream signaling. Immunostaining demonstrated that all P6 BMSCs express CD29 and CD44 but not CD45. BMSCs induced by 15 ng/ml IGF‑1 revealed positivity for cardiac troponin‑T and cardiac troponin‑I. The optimal induction time was 14 days but the expression of these proteins were incompletely inhibited by 300 nmol/l I‑OMe AG538 and completely inhibited by 10 µmol/l I‑OMe AG538. Western blotting showed that the level of IGF‑1R autophosphorylation and the expression of cTnT and cTnI were higher when BMSCs were induced for 14 days. I‑OMe AG538 selectively inhibited IGF‑1‑mediated growth and signal transduction and the inhibitory effect of I‑OMe AG538 were not reverted in the presence of exogenous IGF‑1. In addition, when a time course analysis of the effects of I‑OMe AG538 on mitogen‑activated protein kinase kinase and phosphatidylinositol 3‑kinase signaling were done, we observed a transient inhibitory effect on Erk1/2 and Akt phosphorylation, in keeping with the inhibitory effects on cell growth. Taken together, these data indicate that I‑OMe AG538 could inhibit IGF-1-induced CLCs in BMSCs and this effect is time- and concentration-dependent.

S6Ks isoforms contribute to viability, migration, docetaxel resistance and tumor formation of prostate cancer cells

International Nuclear Information System (INIS)

Amaral, Camila L.; Freitas, Lidia B.; Tamura, Rodrigo E.; Tavares, Mariana R.; Pavan, Isadora C. B.; Bajgelman, Marcio C.; Simabuco, Fernando M.

2016-01-01

The S6 Kinase (S6K) proteins are some of the main downstream effectors of the mammalian Target Of Rapamycin (mTOR) and act as key regulators of protein synthesis and cell growth. S6K is overexpressed in a variety of human tumors and is correlated to poor prognosis in prostate cancer. Due to the current urgency to identify factors involved in prostate cancer progression, we aimed to reveal the cellular functions of three S6K isoforms–p70-S6K1, p85-S6K1 and p54-S6K2–in prostate cancer, as well as their potential as therapeutic targets. In this study we performed S6K knockdown and overexpression and investigated its role in prostate cancer cell proliferation, colony formation, viability, migration and resistance to docetaxel treatment. In addition, we measured tumor growth in Nude mice injected with PC3 cells overexpressing S6K isoforms and tested the efficacy of a new available S6K1 inhibitor in vitro. S6Ks overexpression enhanced PC3-luc cell line viability, migration, resistance to docetaxel and tumor formation in Nude mice. Only S6K2 knockdown rendered prostate cancer cells more sensitive to docetaxel. S6K1 inhibitor PF-4708671 was particularly effective for reducing migration and proliferation of PC3 cell line. These findings demonstrate that S6Ks play an important role in prostate cancer progression, enhancing cell viability, migration and chemotherapy resistance, and place both S6K1 and S6K2 as a potential targets in advanced prostate cancer. We also provide evidence that S6K1 inhibitor PF-4708671 may be considered as a potential drug for prostate cancer treatment. The online version of this article (doi:10.1186/s12885-016-2629-y) contains supplementary material, which is available to authorized users

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

Czech Academy of Sciences Publication Activity Database

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

2015-01-01

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

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.

A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation.

Directory of Open Access Journals (Sweden)

Angela Papageorgiou

Full Text Available mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

International Nuclear Information System (INIS)

Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

2009-01-01

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

Transcription factor Reb1p regulates DGK1-encoded diacylglycerol kinase and lipid metabolism in Saccharomyces cerevisiae.

Science.gov (United States)

Qiu, Yixuan; Fakas, Stylianos; Han, Gil-Soo; Barbosa, Antonio Daniel; Siniossoglou, Symeon; Carman, George M

2013-10-04

In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

DEFF Research Database (Denmark)

Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

2015-01-01

) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

Sangivamycin-Like Molecule 6 (SLM6) exhibits potent anti-multiple myeloma activity through inhibition of cyclin-dependent kinase-9 (CDK9)

Science.gov (United States)

Dolloff, Nathan G.; Allen, Joshua E.; Dicker, David T.; Aqui, Nicole; Vogl, Dan; Malysz, Jozef; Talamo, Giampaolo; El-Deiry, Wafik S.

2012-01-01

Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLMs). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or non-malignant cell lines at sub-micromolar concentrations. SLM6 was the most active compound in vivo, where it was well-tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (c-Maf, cyclin D1, and c-Myc). Furthermore, SLM6 demonstrated superior in vivo anti-MM activity over the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings demonstrate that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent. PMID:22964485

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

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Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

Methods for purifying carbon materials

Science.gov (United States)

Dailly, Anne [Pasadena, CA; Ahn, Channing [Pasadena, CA; Yazami, Rachid [Los Angeles, CA; Fultz, Brent T [Pasadena, CA

2009-05-26

Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

Science.gov (United States)

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Protocols for the Design of Kinase-focused Compound Libraries.

Science.gov (United States)

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

2018-05-01

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

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

Science.gov (United States)

Murungi, Edwin K; Kariithi, Henry M

2017-03-21

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

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

Directory of Open Access Journals (Sweden)

Edwin K. Murungi

2017-03-01

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

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

DEFF Research Database (Denmark)

Issinger, O G; Beier, H; Speichermann, N

1980-01-01

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

AGC kinases, mechanisms of regulation ‎and innovative drug development.

Science.gov (United States)

Leroux, Alejandro E; Schulze, Jörg O; Biondi, Ricardo M

2018-02-01

The group of AGC kinases consists of 63 evolutionarily related serine/threonine protein kinases comprising PDK1, PKB/Akt, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, and YANK, while two other families, Aurora and PLK, are the most closely related to the group. Eight of these families are physiologically activated downstream of growth factor signalling, while other AGC kinases are downstream effectors of a wide range of signals. The different AGC kinase families share aspects of their mechanisms of inhibition and activation. In the present review, we update the knowledge of the mechanisms of regulation of different AGC kinases. The conformation of the catalytic domain of many AGC kinases is regulated allosterically through the modulation of the conformation of a regulatory site on the small lobe of the kinase domain, the PIF-pocket. The PIF-pocket acts like an ON-OFF switch in AGC kinases with different modes of regulation, i.e. PDK1, PKB/Akt, LATS and Aurora kinases. In this review, we make emphasis on how the knowledge of the molecular mechanisms of regulation can guide the discovery and development of small allosteric modulators. Molecular probes stabilizing the PIF-pocket in the active conformation are activators, while compounds stabilizing the disrupted site are allosteric inhibitors. One challenge for the rational development of allosteric modulators is the lack of complete structural information of the inhibited forms of full-length AGC kinases. On the other hand, we suggest that the available information derived from molecular biology and biochemical studies can already guide screening strategies for the identification of innovative mode of action molecular probes and the development of selective allosteric drugs for the treatment of human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural studies of Schistosoma mansoni adenylate kinases

International Nuclear Information System (INIS)

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

2012-01-01

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

CHARACTERIZATION OF TIGHTLY-ASSOCIATED SMOOTH MUSCLE MYOSIN-MYOSIN LIGHT CHAIN KINASE-CALMODULIN COMPLEXES*

OpenAIRE

Hong, Feng; Haldeman, Brian D.; John, Olivia A.; Brewer, Paul D.; Wu, Yi-Ying; Ni, Shaowei; Wilson, David P.; Walsh, Michael P.; Baker, Jonathan E.; Cremo, Christine R.

2009-01-01

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by smooth muscle myosin light chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ~ 23–37% of that in gizzard tissue. Fifteen t...

Targeting Aberrant p70S6K Activation for Estrogen Receptor-Negative Breast Cancer Prevention.

Science.gov (United States)

Wang, Xiao; Yao, Jun; Wang, Jinyang; Zhang, Qingling; Brady, Samuel W; Arun, Banu; Seewaldt, Victoria L; Yu, Dihua

2017-11-01

The prevention of estrogen receptor-negative (ER-) breast cancer remains a major challenge in the cancer prevention field, although antiestrogen and aromatase inhibitors have shown adequate efficacy in preventing estrogen receptor-positive (ER + ) breast cancer. Lack of commonly expressed, druggable targets is a major obstacle for meeting this challenge. Previously, we detected the activation of Akt signaling pathway in atypical hyperplasic early-stage lesions of patients. In the current study, we found that Akt and the downstream 70 kDa ribosomal protein S6 kinase (p70S6K) signaling pathway was highly activated in ER - premalignant breast lesions and ER - breast cancer. In addition, p70S6K activation induced transformation of ER - human mammary epithelial cells (hMEC). Therefore, we explored the potential of targeting Akt/p70S6K in the p70S6K activated, ER - hMEC models and mouse mammary tumor models for the prevention of ER - breast cancer. We found that a clinically applicable Akt/p70S6K dual inhibitor, LY2780301, drastically decreased proliferation of hMECs with ErbB2-induced p70S6K activation via Cyclin B1 inhibition and cell-cycle blockade at G 0 -G 1 phase, while it did not significantly reverse the abnormal acinar morphology of these hMECs. In addition, a brief treatment of LY2780301 in MMTV- neu mice that developed atypical hyperplasia (ADH) and mammary intraepithelial neoplasia (MIN) lesions with activated p70S6K was sufficient to suppress S6 phosphorylation and decrease cell proliferation in hyperplasic MECs. In summary, targeting the aberrant Akt/p70S6K activation in ER - hMEC models in vitro and in the MMTV- neu transgenic mouse model in vivo effectively inhibited Akt/S6K signaling and reduced proliferation of hMECs in vitro and ADH/MIN lesions in vivo , indicating its potential in prevention of p70S6K activated ER - breast cancer. Cancer Prev Res; 10(11); 641-50. ©2017 AACR . ©2017 American Association for Cancer Research.

Study of hot corrosion of flakes of non purified graphite and of purified graphite

International Nuclear Information System (INIS)

Boule, Michel

1967-01-01

The author reports the study of hot corrosion of the Ticonderoga graphite. He reports the study of the defects of graphite flakes (structure defects due to impurities), the dosing of these impurities, and then their removal by purification. Flakes have then been oxidised by means of a specially designed apparatus. Based on photographs taken by optical and electronic microscopy, the author compares the oxidation features obtained in dry air and in humid air, between purified and non purified flakes. He also reports the study of the evolution of oxidation with respect to the initial rate of impurities, and the study of the evolution of oxidation features in humid air during oxidation. All these comparisons are made while taking the oxidation rate into account [fr

IL-3 maintains activation of the P90S6K/RPS6 pathway and increases translation in human eosinophils1

Science.gov (United States)

Esnault, Stephane; Kelly, Elizabeth A.B.; Shen, Zhong-Jian; Johansson, Mats W.; Malter, James S.; Jarjour, Nizar N.

2015-01-01

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines IL-5, IL-3, and GM-CSF are typically present in atopic diseases including allergic asthma. Due to the functional redundancy of these 3 cytokines on eosinophils and the loss of IL-5 receptor on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these 3 cytokines signal through a common β-chain receptor, and yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce production of proteins such as semaphorin-7A without affecting mRNA level suggests a unique influence by IL-3 on translation. The purpose of this study is to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared to IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein (RP) S6 and the upstream kinase, p90S6K. Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared to circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations place IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions. PMID:26276876

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

International Nuclear Information System (INIS)

Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

2012-01-01

Highlights: ► MG132 induces the phosphorylation of GSK3β Ser9 and, to a lesser extent, of GSK3β Thr390 . ► MG132 induces dephosphorylation of p70S6K Thr389 and phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 dephosphorylates GSK3β Ser9 and phosphorylates GSK3β Thr390 . ► Inactivation of p38 phosphorylates p70S6K Thr389 and increases the phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser 9 and, to a lesser extent, Thr 390 , the dephosphorylation of p70S6K at Thr 389 , and the phosphorylation of p70S6K at Thr 421 and Ser 424 . The specific p38 inhibitor SB203080 reduced the p-GSK3β Ser9 and autophagy through the phosphorylation of p70S6K Thr389 ; however, it augmented the levels of p-ERK, p-GSK3β Thr390 , and p-70S6K Thr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK Ser9 /p70S6K Thr380 and ERK/GSK3β Thr390 /p70S6K Thr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

RNAi Mediated Silencing of LRRK2G2019S in Parkinson’s Disease

Science.gov (United States)

2013-08-01

WT, but not LRRK2 mutant, protected 446 dopaminergic neurons against rotenone or paraquat toxicity, 447Q12 agents which compromise [52]. The...and animal models of G2019S-mediated neurotoxicity to establish a novel therapy for PD. To achieve this objective we proposed the following Specific...kinase domain (kinase dead mutants) diminishes neurotoxicity and basal kinase levels appear to be required for the toxicity of all LRRK2 mutants [6

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Science.gov (United States)

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Directory of Open Access Journals (Sweden)

Prerna Grover

Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity.

Science.gov (United States)

Recarte-Pelz, Pedro; Tàssies, Dolors; Espinosa, Gerard; Hurtado, Begoña; Sala, Núria; Cervera, Ricard; Reverter, Joan Carles; de Frutos, Pablo García

2013-03-12

Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples. Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients. The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9

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Susann Pohlers

2017-01-01

Full Text Available Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases—metalloenzymes that have been found in all domains of life—enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2. Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata. Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi.

Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells

Directory of Open Access Journals (Sweden)

Snyder Jeanne M

2002-10-01

Full Text Available Abstract Background It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A, the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression. Methods H441 cells, a human lung adenocarcinoma cell line, or human fetal lung explants were incubated with or without insulin. Transcription run-on assays were used to determine SP-A gene transcription rates. Northern blot analysis was used to examine the effect of various signal transduction inhibitors on SP-A gene expression. Immunoblot analysis was used to evaluate the levels and phosphorylation states of signal transduction protein kinases. Results Insulin decreased SP-A gene transcription in human lung epithelial cells within 1 hour. Insulin did not affect p44/42 mitogen-activated protein kinase (MAPK phosphorylation and the insulin inhibition of SP-A mRNA levels was not affected by PD98059, an inhibitor of the p44/42 MAPK pathway. In contrast, insulin increased p70 S6 kinase Thr389 phosphorylation within 15 minutes. Wortmannin or LY294002, both inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase, or rapamycin, an inhibitor of the activation of p70 S6 kinase, a downstream effector in the PI 3-kinase pathway, abolished or attenuated the insulin-induced inhibition of SP-A mRNA levels. Conclusion Insulin inhibition of SP-A gene expression in lung epithelial cells probably occurs via the rapamycin-sensitive PI 3-kinase signaling pathway.

Synthesis and characterization of highly purified nanosilica from pyrophyllite ores

Energy Technology Data Exchange (ETDEWEB)

Fuad, Abdulloh, E-mail: abdulloh.fuad.fmipa@um.ac.id; Mufti, Nandang; Diantoro, Markus; Subakti,; Septa Kurniawati, S. [Jurusan Fisika FMIPA Universitas Negeri Malang. Jl. Semarang No. 5 Malang, east Java (Indonesia)

2016-03-11

A simple method based on alkaline extraction followed by acid precipitation and acid dissolution has been developed to produce highly purified nanosilica from pyrophyllite materials. The reaction parameters such as molar ratio NaOH/SiO{sub 2}, reaction time and reaction temperature are varied for obtaining maximum nanosilica convertion. About 99,45% highly purified precipitated nanosilica measure with ICP, 259 m{sup 2}/gr measure with BET surface area, 97% whiteness and 3 ml/gr oil absorbtion from pyrophyllite materials has been achieved in closed system at 150°C within 180 min. The physicals and chemical properties (such as X-Ray Diffraction from PANalytical, X-Ray Fluorescence Minipal4 from PANanalytical, BET surface area, Forier Transform Infra Red Spectroscopy from Hitachi, and SEM-EDS Inspect-S50 from FEI Company) of the highly purity nanosilica were studied.

Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

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Rodriguez-del Valle Nuri

2007-11-01

Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

Isoforms of purified methyltransferase from human blood platelets ...

African Journals Online (AJOL)

... purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation ...

Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

International Nuclear Information System (INIS)

Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

2008-01-01

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) β. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKα1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-κB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-κB activation

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

African Journals Online (AJOL)

STORAGESEVER

2009-07-06

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

Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae*

Science.gov (United States)

Qiu, Yixuan; Fakas, Stylianos; Han, Gil-Soo; Barbosa, Antonio Daniel; Siniossoglou, Symeon; Carman, George M.

2013-01-01

In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, −166 to −160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism. PMID:23970552

Reproducible in vitro regeneration system for purifying sugarcane ...

African Journals Online (AJOL)

This procedure may be considered as one of the best ever published report on regeneration from in vitro grown plants to purify clones without subjecting the plants to field conditions and harvesting the mature cane. This technique was used to purify transgenic sugarcane plants carrying Bacillus thuringiensis gene.

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus.

Science.gov (United States)

Lehmacher, A; Vogt, A B; Hensel, R

1990-10-15

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

The interaction between beta 2-microglobulin (beta 2m) and purified class-I major histocompatibility (MHC) antigen

DEFF Research Database (Denmark)

Pedersen, L O; Hansen, A S; Olsen, A C

1994-01-01

been generated recently and this paper reports on a similar assay for the interaction between beta 2m and class I. As a model system human beta 2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined...... by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nM. The rate of association at 37 degrees C is very fast (the ka is around 5 x 10(4)/M/s) whereas the dissociation is slow (the kd is around 8 x 10(-6)/s); the ratio of dissociation...

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

International Nuclear Information System (INIS)

Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

1987-01-01

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis

NARCIS (Netherlands)

van der Meer, Jonathan H. M.; van der Poll, Tom; van 't Veer, Cornelis

2014-01-01

TAM receptors (Tyro3, Axl, andMer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein

The Daidzein Metabolite, 6,7,4'-Trihydroxyisoflavone, Is a Novel Inhibitor of PKCα in Suppressing Solar UV-Induced Matrix Metalloproteinase 1

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Tae-Gyu Lim

2014-11-01

Full Text Available Soy isoflavone is an attractive source of functional cosmetic materials with anti-wrinkle, whitening and skin hydration effects. After consumption, the majority of soy isoflavones are converted to their metabolites in the human gastrointestinal tract. To understand the physiological impact of soy isoflavone on the human body, it is necessary to evaluate and address the biological function of its metabolites. In this study, we investigated the effect of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF, a major metabolite of daidzein, against solar UV (sUV-induced matrix metalloproteinases (MMPs in normal human dermal fibroblasts. MMPs play a critical role in the degradation of collagen in skin, thereby accelerating the aging process of skin. The mitogen-activated protein/extracellular signal-regulated kinase (MEK/extracellular signal-regulated kinase (ERK, mitogen-activated protein kinase (MKK3/6/p38 and MKK4/c-Jun N-terminal kinases (JNK signaling pathways are known to modulate MMP-1 function, and their activation by sUV was significantly reduced by 6,7,4'-THIF pretreatment. Our results also indicated that the enzyme activity of protein kinase C (PKCα, an upstream regulator of MKKs signaling, is suppressed by 6,7,4'-THIF using the in vitro kinase assay. Furthermore, the direct interaction between 6,7,4'-THIF and endogenous PKCα was confirmed using the pull-down assay. Not only sUV-induced MMP-1 expression, but also sUV-induced signaling pathway activation were decreased in PKCα knockdown cells. Overall, we elucidated the inhibitory effect of 6,7,4'-THIF on sUV-induced MMPs and suggest PKCα as its direct molecular target.

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.

Purifying hydrocarbon oils

Energy Technology Data Exchange (ETDEWEB)

Rostin, H

1938-08-11

A process is described for continuously purifying hydrocarbon oils consisting in conducting the vapors of the same at a temperature of 300 to 400/sup 0/C over the oelitic ore minette together with reducing gases in presence of steam the proportion of the reducing gases and steam being such that the sulfur of the hydrocarbons escapes from the reaction chamber in the form of sulfuretted hydrogen without permanent sulfide of iron being formed.

Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence

Directory of Open Access Journals (Sweden)

Rajeev Gupta

2017-06-01

Full Text Available Voltage-Dependent Anion Channel (VDAC phosphorylated by c-Jun N-terminal Kinase-3 (JNK3 was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence.

Science.gov (United States)

Gupta, Rajeev; Ghosh, Subhendu

2017-06-01

Voltage-Dependent Anion Channel (VDAC) phosphorylated by c-Jun N-terminal Kinase-3 (JNK3) was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

Biochemical Properties and Mechanism of Action of Enterocin LD3 Purified from Enterococcus hirae LD3.

Science.gov (United States)

Gupta, Aabha; Tiwari, Santosh Kumar; Netrebov, Victoria; Chikindas, Michael L

2016-09-01

Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation's purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg(-1) with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2-6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ-COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.

Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

Science.gov (United States)

Zhang, Q-G; Han, D; Xu, J; Lv, Q; Wang, R; Yin, X-H; Xu, T-L; Zhang, G-Y

2006-12-01

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

Energy Technology Data Exchange (ETDEWEB)

Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

2012-02-24

Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

DEFF Research Database (Denmark)

Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

2008-01-01

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

Method and device for feeding purified water to a pressure vessel

International Nuclear Information System (INIS)

Hirato, Miharu.

1982-01-01

Purpose: To prevent thermal wear at the junction of feedwater pipes and purified water pipes, as well as maintain the function of the purified water feeding system by stopping the introduction of purified water to the heated water feeding system and introducing purified water to the recycling water system upon transient operation or start-up. Constitution: Since a feedwater heater does not function well during transient operation or upon start-up, the temperature of heated water flowing through the feedwater pipe is reduced to produce a temperature difference relative to the set temperature for the purified water feeding system. The temperature difference is detected by a temperature sensor and, when it arrives at a predetermined difference, an operation valve is switched to interrupt the feed of the purified water to the heated water feeding system and it is sent to a water recycling system. Then, the purified water is sent from the water recycling system by way of the discharge portion to the inside of a pressure vessel. Thus, since only the heated water flows to the junction between the cleaned water pipes and the heated water pipes, neither shocks are generated nor the performance of the purified water feeding system is impaired. (Moriyama, K.)

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

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Pinhero Reena

2004-01-01

Full Text Available We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14. Our protocol provides a novel systematic approach for the purification of these three (and possibly other recombinant CDKs.

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.

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

Science.gov (United States)

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

2016-01-01

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

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

DEFF Research Database (Denmark)

Massip, L; Garand, C; Labbé, A

2010-01-01

show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...... activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast...

AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Xi, Yu [Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Niu, Jianhua [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Shen, Yun [Department of Gastroenterology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Li, Dongmei [Department of Biochemistry and Molecular Biology, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Peng, Xinyu, E-mail: pppengxinyu@sina.com [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Wu, Xiangwei, E-mail: wuxiangweiys@126.com [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China)

2016-09-09

The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level, AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3β (GSK-3β) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate. - Highlights: • AT13148 is cytotoxic and anti-proliferative to human gastric cancer cells. • AT13148 induces gastric cancer cell apoptotic death, inhibited by Caspase inhibitors. • AT13148 inactivates multiple AGC kinases in human gastric cancer cells. • AT13148 oral administration suppresses HGC27 xenograft growth in nude mice. • AT13148 oral administration inhibits multiple AGC kinases in HGC27 xenograft tumors.

Assay of partially purified glutamate dehydrogenase isolated from ...

African Journals Online (AJOL)

Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

Proseek single-plex protein assay kit system to detect sAxl and Gas6 in serological material of brain tumor patients

Directory of Open Access Journals (Sweden)

Heidi Jaksch-Bogensperger

2018-06-01

Full Text Available • The receptor tyrosine kinase (RTK Axl and its ligand Gas6 are critically involved in the pathogenesis of high-grade glioma (HGG. Both proteins were found to be overexpressed e.g. in tumor cells, mediating cell proliferation and migration as well as tumor angiogenesis and neuroinflammation. The extracellular domain of Axl (sAxl and Gas6 were found in the peri-tumoral edema and blood of animals as well as in human glioma tissue. Therefore, we monitored the level of sAxl and Gas6 in human blood samples. To increase the sensitivity of protein detection beyond commonly used standard methods we preliminary tested the innovative Proseek Single-Plex Protein Assay Kit System from Olink Bioscience together with new antibodies against the soluble RTK sAxl and its ligand Gas6. We conclude that the Proseek method is a highly sensitive and fast procedure that can be used as a possible powerful tool compared to routinely used ELISA-methods.

ALK5 kinase inhibitory activity and synthesis of 2,3,4-substituted 5,5-dimethyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazoles

Czech Academy of Sciences Publication Activity Database

Řezníčková, Eva; Tenora, L.; Pospíšilová, Pavlína; Galeta, J.; Jorda, Radek; Berka, K.; Majer, Pavel; Potáček, M.; Kryštof, Vladimír

2017-01-01

Roč. 127, FEB 15 (2017), s. 632-642 ISSN 0223-5234 R&D Projects: GA MŠk(CZ) LO1204; GA ČR(CZ) GA15-15264S; GA MŠk(CZ) LM2015047 Institutional support: RVO:61389030 ; RVO:61388963 Keywords : i-receptor kinase * beta signaling pathway * small- molecule inhibitor * tgf-beta * domain inhibitors * growth * fibrosis * cancer * potent * series * Transforming growth factor beta receptor I * Protein kinase * Inhibitor * Substituted pyrrolo[1,2-b]pyrazoles Subject RIV: FR - Pharmacology ; Medidal Chemistry OBOR OECD: Pharmacology and pharmacy; Pharmacology and pharmacy (UOCHB-X) Impact factor: 4.519, year: 2016

Absence of the kinase S6k1 mimics the effect of chronic endurance exercise on glucose tolerance and muscle oxidative stress

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C. Binsch

2017-11-01

Conclusions: In high-fat fed mice, loss of S6K1 mimics endurance exercise training by reducing mitochondrial ROS production and upregulating oxidative utilization of ketone bodies. Pharmacological targeting of S6K1 may improve the outcome of exercise-based interventions in obesity and diabetes.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

Energy Technology Data Exchange (ETDEWEB)

Schmid, Stefan [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Seiler, Cornelia; Gerecke, Andreas C. [Swiss Federal Laboratories for Material Science and Technology (EMPA), CH-8600 Dübendorf (Switzerland); Hächler, Herbert [University of Zürich, Institute for Food Safety and Hygiene, National Centre for Enteropathogenic Bacteria and Listeria (NENT), CH-8057 Zürich (Switzerland); Hilbi, Hubert [Ludwig-Maximilians-Universität München Max von Pettenkofer-Institut, D-80336 München (Germany); Frey, Joachim [University of Bern, Institute for Veterinary Bacteriology, CH-3001 Bern (Switzerland); Weidmann, Simon; Meier, Lukas; Berchtold, Christian [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Zenobi, Renato, E-mail: zenobi@org.chem.ethz.ch [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland)

2013-07-15

Highlights: • Degradation of environmental toxins, a protein, and bioparticles were studied. • A commercial air purifier based on a cold plasma was used. • Passage through the device reduced the concentration of the compounds/particles. • Deposition inside the plasma air purifier was the main removal process. -- Abstract: Degradation of non-volatile organic compounds–environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)–in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0 m s{sup −1} (3200 L min{sup −1}), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10 L min{sup −1}. Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10 Hz or 50 Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative “degradation” efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

Gamma ray irradiation to semi-purified diet

International Nuclear Information System (INIS)

Takigawa, Akihiro; Danbara, Hiroshi; Ohyama, Yoshinobu

1976-01-01

Semi-purified diet containing 10% soybean oil was irradiated with gamma rays at levels of 0.6, 3 and 6 Mrad and was fed to chicks. Crude fat contents of the diets decreased and a considerable amount of peroxide was formed with high doses of irradiation. Feed consumption and feed efficiency of the highly irradiated diets were less than those of control. Metabolizable energy and digestibility of the diets, especially of fat, were decreased with the irradiation. The chicks fed with irradiated diets showed marked dilatation of the small intestine and the liver, and their erythrocytes were more fragile than those of control. The same phenomena were found with the chicks fed the diet containing the oil highly oxidized by autoxidation. Irradiation of the diet excluding oil showed little effect on the growth of chicks. It was considered that these phenomena were caused by the peroxide or other oxidation products of fat which were formed with gamma ray irradiation. (auth.)

Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

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Jessica W Luzwick

Full Text Available Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

Energy Technology Data Exchange (ETDEWEB)

Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri (Turkey); Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Mus Alparslan University Faculty of Sciences, Department of Moleculer Biology, 49250-Mus (Turkey); Özdemir, Hasan, E-mail: hozdemir@atauni.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey)

2016-04-18

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

Science.gov (United States)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-04-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

International Nuclear Information System (INIS)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-01-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC_5_0 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Phosphorylation of SU(VAR3-9 by the chromosomal kinase JIL-1.

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Joern Boeke

2010-04-01

Full Text Available The histone methyltransferase SU(VAR3-9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR3-9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR3-9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR3-9 N-terminus. Among several other proteins known to bind Su(VAR3-9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR3-9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR3-9 and JIL-1.

Purifying hydrocarbons

Energy Technology Data Exchange (ETDEWEB)

Demoulins, H D; Garner, F H

1923-02-07

Hydrocarbon distillates, including natural gases and vapors produced by cracking hydrocarbon oils, are desulfurized etc. by treating the vapor with an aqueous alkaline solution of an oxidizing agent. The hydrocarbons may be previously purified by sulfuric acid. In examples aqueous solutions of sodium or calcium hydrochlorite containing 1.5 to 5.0 grams per liter of available chlorine and sufficient alkali to give an excess of 0.1 percent in the spent reagent are preheated to the temperature of the vapor, and either sprayed or atomized into the vapors near the outlet of the dephlegmator or fractionating tower, or passed in countercurrent to the vapors through one or a series of scrubbers.

A phase i study of the cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) in patients with advanced solid tumors and lymphomas

NARCIS (Netherlands)

Infante, Jeffrey R.; Cassier, Philippe A.; Gerecitano, John F.; Witteveen, Petronella O.; Chugh, Rashmi; Ribrag, Vincent; Chakraborty, Abhijit; Matano, Alessandro; Dobson, Jason R.; Crystal, Adam S.; Parasuraman, Sudha; Shapiro, Geoffrey I.

2016-01-01

Purpose: Ribociclib (an oral, highly specific cyclin-dependent kinase 4/6 inhibitor) inhibits tumor growth in preclinical models with intact retinoblastoma protein (Rb+). This first-in-human study investigated the MTD, recommended dose for expansion (RDE), safety, preliminary activity,

Sphingosine kinase 1 deficiency confers protection against hyperoxia-induced bronchopulmonary dysplasia in a murine model: role of S1P signaling and Nox proteins.

Science.gov (United States)

Harijith, Anantha; Pendyala, Srikanth; Reddy, Narsa M; Bai, Tao; Usatyuk, Peter V; Berdyshev, Evgeny; Gorshkova, Irina; Huang, Long Shuang; Mohan, Vijay; Garzon, Steve; Kanteti, Prasad; Reddy, Sekhar P; Raj, J Usha; Natarajan, Viswanathan

2013-10-01

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

Science.gov (United States)

Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

2016-05-02

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Insulin receptor binding and tyrosine kinase activity in skeletal muscle from normal pregnant women and women with gestational diabetes

DEFF Research Database (Denmark)

Damm, P.; Handberg, A.; Kühl, C.

1993-01-01

OBJECTIVE: To ascertain whether the decreased glucose tolerance and insulin resistance found in normal and gestational diabetic pregnancy might be associated with changes in insulin receptor function. METHODS: Eight nonpregnant healthy women (nonpregnant controls), eight healthy pregnant women...... (pregnant controls), and eight women with gestational diabetes were investigated. All were non-obese. Muscle biopsies were obtained from the vastus lateralis muscle, and insulin binding and tyrosine kinase activities in partially purified skeletal muscle insulin receptors were studied. The pregnant controls...... with gestational diabetes compared to nonpregnant controls (P pregnant women did not differ from the other two groups. Postpartum, no differences in insulin binding were found between the groups. Basal and maximal tyrosine kinase activities toward the exogenous substrate poly(Glu4Tyr1) were...

Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

NARCIS (Netherlands)

Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

2012-01-01

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

Science.gov (United States)

de Sena, Amanda Reges; Barros Oliveira, Flávio Manoel; Campos Leite, Tonny Cley; Evaristo da Silva Nascimento, Talita Camila; Moreira, Keila Aparecida; de Assis, Sandra Aparecida

2017-10-21

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 2 4 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (M PEG 600; 4,000 and 8,000 g/ mol), and PEG (C PEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (C CIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) M PEG , 24% (w/w) C PEG , 15% (w/w) C CIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.

HPV8-E6 Interferes with Syntenin-2 Expression through Deregulation of Differentiation, Methylation and Phosphatidylinositide-Kinase Dependent Mechanisms

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Benjamin Marx

2017-09-01

Full Text Available The E6 oncoproteins of high-risk human papillomaviruses (HPV of genus alpha contain a short peptide sequence at the carboxy-terminus, the PDZ binding domain, with which they interact with the corresponding PDZ domain of cellular proteins. Interestingly, E6 proteins from papillomaviruses of genus beta (betaPV do not encode a comparable PDZ binding domain. Irrespective of this fact, we previously showed that the E6 protein of HPV8 (betaPV type could circumvent this deficit by targeting the PDZ protein Syntenin-2 through transcriptional repression (Lazic et al., 2012. Despite its high binding affinity to phosphatidylinositol-4,5-bisphosphate (PI(4,5P2, very little is known about Syntenin-2. This study aimed to extend the knowledge on Syntenin-2 and how its expression is controlled. We now identified that Syntenin-2 is expressed at high levels in differentiating and in lower amounts in keratinocytes cultured in serum-free media containing low calcium concentration. HPV8-E6 led to a further reduction of Syntenin-2 expression only in cells cultured in low calcium. In the skin of patients suffering from Epidermodysplasia verruciformis, who are predisposed to betaPV infection, Syntenin-2 was expressed in differentiating keratinocytes of non-lesional skin, but was absent in virus positive squamous tumors. Using 5-Aza-2′-deoxycytidine, which causes DNA demethylation, Syntenin-2 transcription was profoundly activated and fully restored in the absence and presence of HPV8-E6, implicating that E6 mediated repression of Syntenin-2 transcription is due to promoter hypermethylation. Since Syntenin-2 binds to PI(4,5P2, we further tested whether the PI(4,5P2 metabolic pathway might govern Syntenin-2 expression. PI(4,5P2 is generated by the activity of phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI or phosphatidylinositol-5-phosphate-4-kinase type II (PIP4KII isoforms α, β and γ. Phosphatidylinositide kinases have recently been identified as

The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿ †

OpenAIRE

Spangle, Jennifer M.; Münger, Karl

2010-01-01

The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y....

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

Science.gov (United States)

Schepetilnikov, Mikhail; Kobayashi, Kappei; Geldreich, Angèle; Caranta, Carole; Robaglia, Christophe; Keller, Mario; Ryabova, Lyubov A

2011-01-01

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. PMID:21343906

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

Science.gov (United States)

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

2017-10-13

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

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

Science.gov (United States)

Van de Werve, G; Hers, H G

1979-01-15

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli

International Nuclear Information System (INIS)

Zheng, Jimin; Lee, Daniel C.; Jia, Zongchao

2009-01-01

Isocitrate dehydrogenase kinase/phosphatase has been crystallized in three different crystal forms. Data were collected from each crystal form for structure determination. The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P4 1 2 1 2, P3 2 21 and P2 1 2 1 2 1 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively

Regulation of mTORC1 Signaling by Src Kinase Activity Is Akt1-Independent in RSV-Transformed Cells

Directory of Open Access Journals (Sweden)

Martina Vojtěchová

2008-02-01

Full Text Available Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB, phosphorylation of tuberin (TSC2, mammalian target of rapamycin (mTOR, S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

GIT1/beta PIX signaling proteins and PAK1 kinase regulate microtubule nucleation

Czech Academy of Sciences Publication Activity Database

Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-01-01

Roč. 1863, č. 6 (2016), s. 1282-1297 ISSN 0167-4889 R&D Projects: GA ČR GAP302/12/1673; GA ČR GA15-22194S; GA MŠk LH12050; GA MZd NT14467; GA ČR GA16-23702S Institutional support: RVO:68378050 Keywords : Centrosome * Microtubule nucleation * gamma-tubulin * GIT1/beta PIX signaling proteins * PAK1 kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.521, year: 2016

Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain α-ketoacid dehydrogenase (BCKDH)

International Nuclear Information System (INIS)

Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

1987-01-01

BCKDH is phosphorylated by a copurifying kinase at two serine residues on the Elα subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, α-chloroisocaproate (CIC) and branched chain α-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37 0 C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions

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

Science.gov (United States)

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

2012-02-01

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

RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

Science.gov (United States)

Chen, Jiwei; Zhang, Qian; Wang, Qigang; Feng, Ming; Li, Yang; Meng, Yonglu; Zhang, Yi; Liu, Guoqin; Ma, Zhimin; Wu, Hongzhi; Gao, Junping; Ma, Nan

2017-02-23

Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration. We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter. Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.

Altered Regulation of Contraction-Induced Akt/mTOR/p70S6k Pathway Signaling in Skeletal Muscle of the Obese Zucker Rat

Directory of Open Access Journals (Sweden)

Anjaiah Katta

2009-01-01

Full Text Available Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k, and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k (Thr389; 37.2% and Thr421/Ser424; 101.4%, Akt (Thr308; 25.1%, and mTOR (Ser2448; 63.0% was higher in obese animals. Contraction increased the phosphorylation of p70S6k (Thr389, Akt (Ser473, and mTOR (Ser2448 in both models however the magnitude and kinetics of activation differed between models. These results suggest that contraction-induced activation of p70S6k signaling is altered in the muscle of the insulin resistant obese Zucker rat.

An Experiment with Air Purifiers in Delhi during Winter 2015-2016.

Science.gov (United States)

Vyas, Sangita; Srivastav, Nikhil; Spears, Dean

2016-01-01

Particulate pollution has important consequences for human health, and is an issue of global concern. Outdoor air pollution has become a cause for alarm in India in particular because recent data suggest that ambient pollution levels in Indian cities are some of the highest in the world. We study the number of particles between 0.5μm and 2.5μm indoors while using affordable air purifiers in the highly polluted city of Delhi. Though substantial reductions in indoor number concentrations are observed during air purifier use, indoor air quality while using an air purifier is frequently worse than in cities with moderate pollution, and often worse than levels observed even in polluted cities. When outdoor pollution levels are higher, on average, indoor pollution levels while using an air purifier are also higher. Moreover, the ratio of indoor air quality during air purifier use to two comparison measures of air quality without an air purifier are also positively correlated with outdoor pollution levels, suggesting that as ambient air quality worsens there are diminishing returns to improvements in indoor air quality during air purifier use. The findings of this study indicate that although the most affordable air purifiers currently available are associated with significant improvements in the indoor environment, they are not a replacement for public action in regions like Delhi. Although private solutions may serve as a stopgap, reducing ambient air pollution must be a public health and policy priority in any region where air pollution is as high as Delhi's during the winter.

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

Science.gov (United States)

Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

2015-01-01

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo

KAUST Repository

Fujii, Hiroaki

2011-01-10

Osmotic stress associated with drought or salinity is a major factor that limits plant productivity. Protein kinases in the SNF1-related protein kinase 2 (SnRK2) family are activated by osmotic stress, suggesting that the kinases are involved in osmotic stress signaling. However, due to functional redundancy, their contribution to osmotic stress responses remained unclear. In this report, we constructed an Arabidopsis line carrying mutations in all 10 members of the SnRK2 family. The decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10 grew poorly under hyperosmotic stress conditions but was similar to the wild type in culture media in the absence of osmotic stress. The mutant was also defective in gene regulation and the accumulation of abscisic acid (ABA), proline, and inositol 1,4,5-trisphosphate under osmotic stress. In addition, analysis of mutants defective in the ABA-activated SnRK2s (snrk2.2/3/6) and mutants defective in the rest of the SnRK2s (snrk2.1/4/5/7/8/9/10) revealed that SnRK2s are a merging point of ABA-dependent and -independent pathways for osmotic stress responses. These results demonstrate critical functions of the SnRK2s in mediating osmotic stress signaling and tolerance.

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

International Nuclear Information System (INIS)

Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

2012-01-01

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A) + RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G 2 phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

Energy Technology Data Exchange (ETDEWEB)

Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

2012-10-01

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

Science.gov (United States)

Pascal, John M; Armen, Roger S

2012-01-01

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

A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

Science.gov (United States)

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

2009-01-01

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

IL6 induces TAM resistance via kinase-specific phosphorylation of ERα in OVCA cells.

Science.gov (United States)

Wang, Yue; Niu, Xiu Long; Guo, Xiao Qin; Yang, Jing; Li, Ling; Qu, Ye; Xiu Hu, Cun; Mao, Li Qun; Wang, Dan

2015-06-01

About 40-60% of ovarian cancer (OVCA) cases express ERα, but only a small proportion of patients respond clinically to anti-estrogen treatment with estrogen receptor (ER) antagonist tamoxifen (TAM). The mechanism of TAM resistance in the course of OVCA progression remains unclear. However, IL6 plays a critical role in the development and progression of OVCA. Our recent results indicated that IL6 secreted by OVCA cells may promote the resistance of these cells to TAM via ER isoforms and steroid hormone receptor coactivator-1. Here we demonstrate that both exogenous (a relatively short period of treatment with recombinant IL6) and endogenous IL6 (generated as a result of transfection with a plasmid encoding sense IL6) increases expression of pERα-Ser118 and pERα-Ser167 in non-IL6-expressing A2780 cells, while deleting endogenous IL6 expression in IL6-overexpressing CAOV-3 cells (by transfection with a plasmid encoding antisense IL6) reduces expression of pERα-Ser118 and pERα-Ser167, indicating that IL6-induced TAM resistance may also be associated with increased expression of pERα-Ser118 and pERα-Ser167 in OVCA cells. Results of further investigation indicate that IL6 phosphorylates ERα at Ser118 and Ser167 by triggering activation of MEK/ERK and phosphotidylinositol 3 kinase/Akt signaling, respectively, to activate the ER pathway and thereby induce OVCA cells resistance to TAM. These results indicate that IL6 secreted by OVCA cells may also contribute to the refractoriness of these cells to TAM via the crosstalk between ER and IL6-mediated intracellular signal transduction cascades. Overexpression of IL6 not only plays an important role in OVCA progression but also promotes TAM resistance. Our results indicate that TAM-IL6-targeted adjunctive therapy may lead to a more effective intervention than TAM alone. © 2015 Society for Endocrinology.

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

Science.gov (United States)

Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

[Investigation on the process of sapindus saponin purified with macroporous adsorption resin and screening of its bacteriostasis].

Science.gov (United States)

Fu, Yong; Lei, Peng; Han, Yu-mei; Yan, Dan

2010-02-01

To study the technological parameters of the purification process of saponins with macroporous adsorption resin. The adsorptive characteristics and elutive parameters of the process were studied by taking the elutive and purified ratio of saponins as markers. Bacteriostasis activity of each parts eluted was evaluated by the mean of cup-plate method. 13.6 mL of the extraction of sapindus saponin (crude drugs 0.01 g/mL) was purified with a column of macroporous adsorption resin (phi15 mm x H90 mm, dry weight 2.5 g) and washed with 3BV of distilled water, then eluted with 3BV of 30% ethanol and 3BV of 70% ethanol, most of saponins were collected in the 70% ethanol. With macroporous adsorption resin adsorbing and purifying, the elutive ratio of saponins was 93.8% and the purity reached 250.1%. So this process of applying macroporous adsorption resin to adsorb and purify saponins is feasible, and supplies reference to the purification of other types of saponin.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.

Science.gov (United States)

Schepetilnikov, Mikhail; Dimitrova, Maria; Mancera-Martínez, Eder; Geldreich, Angèle; Keller, Mario; Ryabova, Lyubov A

2013-04-17

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

Kinases and Cancer

OpenAIRE

Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

2018-01-01

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

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

Czech Academy of Sciences Publication Activity Database

Procházka, Radek; Blaha, Milan

2015-01-01

Roč. 61, č. 6 (2015), s. 495-502 ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.453, year: 2015

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

Directory of Open Access Journals (Sweden)

Anne Dettmann

2012-09-01

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

Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

Science.gov (United States)

Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

2017-01-01

Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

Black Tea High-Molecular-Weight Polyphenol-Rich Fraction Promotes Hypertrophy during Functional Overload in Mice

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Yuki Aoki

2017-03-01

Full Text Available Mitochondria activation factor (MAF is a high-molecular-weight polyphenol extracted from black tea that stimulates training-induced 5′ adenosine monophosphate-activated protein kinase (AMPK activation and improves endurance capacity. Originally, MAF was purified from black tea using butanol and acetone, making it unsuitable for food preparation. Hence, we extracted a MAF-rich sample “E80” from black tea, using ethanol and water only. Here, we examined the effects of E80 on resistance training. Eight-week old C57BL/6 mice were fed with a normal diet or a diet containing 0.5% E80 for 4, 7 and 14 days under conditions of functional overload. It was found that E80 administration promoted overload-induced hypertrophy and induced phosphorylation of the Akt/mammalian target of rapamycin (mTOR pathway proteins, such as Akt, P70 ribosomal protein S6 kinase (p70S6K, and S6 in the plantaris muscle. Therefore, functional overload and E80 administration accelerated mTOR signaling and increased protein synthesis in the muscle, thereby inducing hypertrophy.

A framework for classification of prokaryotic protein kinases.

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Nidhi Tyagi

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

Kinetic Characterisation of Phosphofructokinase Purified from Setaria cervi: A Bovine Filarial Parasite

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Bechan Sharma

2011-01-01

Full Text Available Phosphofructokinase (PFK, a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that Km values for F-6-P (1.05 mM and ATP (3 μM were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM, exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP competitively inhibited the filarial enzyme: Ki and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP also inhibited the enzyme competitively with the Ki value equal to 0.8 mM. The Hill coefficient values (>1.5 for F-6-P (at inhibitory concentration of ATP and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

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

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

2011-01-01

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 cryst...

HARP preferentially co-purifies with RPA bound to DNA-PK and blocks RPA phosphorylation.

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Quan, Jinhua; Yusufzai, Timur

2014-05-01

The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.

Inactivation of purified human recombinant monoamine oxidases A and B by rasagiline and its analogues.

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