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Sample records for facilitates efficient phosphorylation

  1. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    Science.gov (United States)

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

  2. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  3. KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity.

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    Caldas, Gina V; DeLuca, Keith F; DeLuca, Jennifer G

    2013-12-23

    Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore–microtubule (MT) turnover and preventing premature stabilization of kinetochore–MT attachments. Phosphorylation of kinetochore proteins during late mitosis is low, promoting attachment stabilization, which is required for anaphase onset. The kinetochore protein KNL1 recruits Aurora B–counteracting phosphatases and the Aurora B–targeting factor Bub1, yet the consequences of KNL1 depletion on Aurora B phospho-regulation remain unknown. Here, we demonstrate that the KNL1 N terminus is essential for Aurora B activity at kinetochores. This region of KNL1 is also required for Bub1 kinase activity at kinetochores, suggesting that KNL1 promotes Aurora B activity through Bub1-mediated Aurora B targeting. However, ectopic targeting of Aurora B to kinetochores does not fully rescue Aurora B activity in KNL1-depleted cells, suggesting KNL1 influences Aurora B activity through an additional pathway. Our findings establish KNL1 as a requirement for Aurora B activity at kinetochores and for wild-type kinetochore–MT attachment dynamics.

  4. Leptin in the hindbrain facilitates phosphorylation of STAT3 in the hypothalamus.

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    Desai, Bhavna N; Harris, Ruth B S

    2015-03-01

    Leptin receptors (ObRs) in the forebrain and hindbrain have been independently recognized as important mediators of leptin responses. We recently used low-dose leptin infusions to show that chronic activation of both hypothalamic and hindbrain ObRs is required to reduce body fat. The objective of the present study was to identify the brain nuclei that are selectively activated in rats that received chronic infusion of leptin in both the forebrain and hindbrain. Either saline or leptin was infused into third and fourth ventricles (0.1 μg/24 h in the third ventricle and 0.6 μg/24 h in the fourth ventricle) of male Sprague-Dawley rats for 6 days using Alzet pumps. Rats infused with leptin into both ventricles (LL rats) showed a significant increase in phosphorylated (p)STAT3 immunoreactivity in the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and posterior hypothalamus compared with other groups. No differences in pSTAT3 immunoreactivity were observed in midbrain or hindbrain nuclei despite a sixfold higher infusion of leptin into the fourth ventricle than the third ventricle. ΔFosB immunoreactivity, a marker of chronic neuronal activation, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and ventral tuberomamillary nucleus showed a significant increase in LL rats compared with other groups. These data demonstrate that low-dose leptin in the hindbrain increases pSTAT3 in areas of the hypothalamus known to respond to leptin, supporting the hypothesis that leptin-induced weight loss requires an integrated response from both the hindbrain and forebrain. Copyright © 2015 the American Physiological Society.

  5. A method to determine the ionization efficiency change of peptides caused by phosphorylation.

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    Gao, Yuan; Wang, Yinsheng

    2007-11-01

    Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation.

  6. Phosphorylation of h1 Calponin by PKC epsilon may contribute to facilitate the contraction of uterine myometrium in mice during pregnancy and labor

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    Li Lesai

    2012-05-01

    Full Text Available Abstract Background The timely onset of powerful uterine contractions during parturition occurs through thick and thin filament interactions, similar to other smooth muscle tissues. Calponin is one of the thin filament proteins. Phosphorylation of calponin induced by PKC-epsilon can promote the contraction of vascular smooth muscle. While the mechanism by which calponin regulates the contraction of pregnant myometrium has rarely been explored. Here, we explore whether PKC-epsilon/h1 calponin pathway contribute to regulation of myometrial contractility and development of parturition. Methods We detected the expression of h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon in the different stages of mice during pregnancy and in labor by the method of western blot and recorded the contraction activity of myometrium strips at the 19th day during pregnancy with different treatments by the organ bath experiments. Results The level of the four proteins including h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice. The ratios of phosphorylated h1 calponin/h1 calponin and phosphorylated PKC-epsilon/PKC-epsilon were reached the peak after the onset of labor in myometrium in the mice. After the treatment of more than 10(9- mol/L Psi-RACK (PKC-epsilon activator, the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1 calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-epsilon. Meanwhile, the change of the ratio of phosphorylated h1 calponin/h1 calponin was consistent with that of contraction force of mice myometrium strips. Conclusions These data suggest that in mice myometrium, phosphorylation of h1 calponin induced by the PKC-epsilon might facilitate the contraction of uterine in labor and regulate pregnant

  7. Warm body temperature facilitates energy efficient cortical action potentials.

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    Yuguo Yu

    Full Text Available The energy efficiency of neural signal transmission is important not only as a limiting factor in brain architecture, but it also influences the interpretation of functional brain imaging signals. Action potential generation in mammalian, versus invertebrate, axons is remarkably energy efficient. Here we demonstrate that this increase in energy efficiency is due largely to a warmer body temperature. Increases in temperature result in an exponential increase in energy efficiency for single action potentials by increasing the rate of Na(+ channel inactivation, resulting in a marked reduction in overlap of the inward Na(+, and outward K(+, currents and a shortening of action potential duration. This increase in single spike efficiency is, however, counterbalanced by a temperature-dependent decrease in the amplitude and duration of the spike afterhyperpolarization, resulting in a nonlinear increase in the spike firing rate, particularly at temperatures above approximately 35°C. Interestingly, the total energy cost, as measured by the multiplication of total Na(+ entry per spike and average firing rate in response to a constant input, reaches a global minimum between 37-42°C. Our results indicate that increases in temperature result in an unexpected increase in energy efficiency, especially near normal body temperature, thus allowing the brain to utilize an energy efficient neural code.

  8. Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers.

    Science.gov (United States)

    Lark, Daniel S; Torres, Maria J; Lin, Chien-Te; Ryan, Terence E; Anderson, Ethan J; Neufer, P Darrell

    2016-08-01

    Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P < 0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases. Copyright © 2016 the American Physiological Society.

  9. Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

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    Tripty A. Hirani

    2011-01-01

    Full Text Available We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana α2β2-heterotetrameric pyruvate dehydrogenase (E1 plus A. thaliana E1-kinase (AtPDK. Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

  10. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.

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    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang

    2016-04-01

    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.

  11. The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.

    OpenAIRE

    Kaufman, R J; Davies, M V; Pathak, V K; Hershey, J W

    1989-01-01

    Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases,...

  12. The FGFR4-G388R polymorphism promotes mitochondrial STAT3 serine phosphorylation to facilitate pituitary growth hormone cell tumorigenesis.

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    Toru Tateno

    2011-12-01

    Full Text Available Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP substituting an arginine (R for glycine (G in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL production, FGFR4-R388 cells express predominantly growth hormone (GH. Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3. In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders.

  13. The FGFR4-G388R polymorphism promotes mitochondrial STAT3 serine phosphorylation to facilitate pituitary growth hormone cell tumorigenesis.

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    Tateno, Toru; Asa, Sylvia L; Zheng, Lei; Mayr, Thomas; Ullrich, Axel; Ezzat, Shereen

    2011-12-01

    Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders.

  14. Facile Peptides Functionalization of Lanthanide-Based Nanocrystals through Phosphorylation Tethering for Efficient in Vivo NIR-to-NIR Bioimaging.

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    Yao, Chi; Wang, Peiyuan; Wang, Rui; Zhou, Lei; El-Toni, Ahmed Mohamed; Lu, Yiqing; Li, Xiaomin; Zhang, Fan

    2016-02-02

    Peptide modification of nanoparticles is a challenging task for bioapplications. Here, we show that noncovalent surface engineering based on ligand exchange of peptides for lanthanide based upconversion and downconversion near-infrared (NIR) luminescent nanoparticles can be efficiently realized by modifying the hydroxyl functional group of a side grafted serine of peptides into a phosphate group (phosphorylation). By using the phosphorylated peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the modification allows improving the selectivity, sensitivity, and signal-to-noise ratio for the cancer targeting and bioimaging and reducing the toxicity derived from nonspecific interactions of nanoparticles with cells. The in vivo NIR bioimaging signal could even be detected at low injection amounts down to 20 μg per animal.

  15. Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation

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    Li, Jasmine; Hardy, Kristine; Phetsouphanh, Chan; Tu, Wen Juan; Sutcliffe, Elissa L.; McCuaig, Robert; Sutton, Christopher R.; Zafar, Anjum; Munier, C. Mee Ling; Zaunders, John J.; Xu, Yin; Theodoratos, Angelo; Tan, Abel; Lim, Pek Siew; Knaute, Tobias; Masch, Antonia; Zerweck, Johannes; Brezar, Vedran; Milburn, Peter J.; Dunn, Jenny; Casarotto, Marco G.; Turner, Stephen J.; Seddiki, Nabila; Kelleher, Anthony D.

    2016-01-01

    ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. PMID:27149922

  16. Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

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    Makihara, Hiroyuki; Inaba, Hironori; Enomoto, Atsushi; Tanaka, Hiroki; Tomono, Yasuko; Ushida, Kaori; Goto, Mitsuo; Kurita, Kenichi; Nishida, Yoshihiro; Kasahara, Kousuke; Goto, Hidemasa; Inagaki, Masaki

    2016-09-23

    Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

  17. An efficient reagent for the phosphorylation of deoxyribonucleosides, DNA oligonucleotides, and their thermolytic analogues.

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    Ausín, Cristina; Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L

    2005-09-15

    [reaction: see text] The phosphoramidite 11 was prepared in three steps from methyl 2-mercaptoacetate and demonstrated efficiency in the synthesis of conventional 5'-/3'-phosphate/thiophosphate monoester derivatives of 2'-deoxyribonucleosides and DNA oligonucleotides. Moreover, the use of 11 has enabled the preparation of the dinucleoside phosphorothioate analogue 26 in high yields (>95%) with minimal cleavage (<2%) of the thermolytic thiophosphate protecting group.

  18. Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.

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    Hindriksen, Sanne; Bramer, Arne J; Truong, My Anh; Vromans, Martijn J M; Post, Jasmin B; Verlaan-Klink, Ingrid; Snippert, Hugo J; Lens, Susanne M A; Hadders, Michael A

    2017-01-01

    The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.

  19. DST, decision support tool to facilitate energy-efficient renovation of buildings

    Energy Technology Data Exchange (ETDEWEB)

    Kondratenko, I.; Koene, F.G.H.; Kaan, H.F. [ECN Energy in the Built Environment, Petten (Netherlands)

    2008-04-15

    The aim of the DEMOHOUSE project is to develop minimum standards and recommendations for energy-efficient and sustainable renovation of social housing estates. Within this, the Decision Support Tool is one of the final results and uses knowledge gathered throughout the DEMOHOUSE project. In many European countries, social housing is owned by housing associations, municipalities or housing co-operations. To facilitate the decision makers in these organisations a simple instrument which helps to select relevant information for making decisions is developed within the DEMOHOUSE project. There are several phases in the process of renovation of dwellings. Main decisions in relation to ambitions of the renovation in energy-efficiency, sustainability, economic feasibility and occupants participation take place in the first so called initiative phase. It is this phase that the DST is focusing on to guide the decision makers, hosing managers and home owners associations alike, through decision making process towards achieving energy-efficient and sustainable renovation of dwellings. As any building renovation is a complex process with many stages, the tool also contains practical information and offers guidance and links to further more in-depth information of relevance to other stakeholders for example: architects, energy experts, building contractors, and building users.

  20. LZAP inhibits p38 MAPK (p38 phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1.

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    Hanbing An

    Full Text Available LZAP (Cdk5rap3, C53 is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs. Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.

  1. LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1).

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    An, Hanbing; Lu, Xinyuan; Liu, Dan; Yarbrough, Wendell G

    2011-01-24

    LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.

  2. An RF-based wearable sensor system for indoor tracking to facilitate efficient healthcare management.

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    Yuzhe Ouyang; Shan, Kai; Bui, Francis Minhthang

    2016-08-01

    To understand the utilization of clinical resources and improve the efficiency of healthcare, it is often necessary to accurately locate patients and doctors in a healthcare facility. However, existing tracking methods, such as GPS, Wi-Fi and RFID, have technological drawbacks or impose significant costs, thus limiting their applications in many clinical environments, especially those with indoor enclosures. This paper proposes a low-cost and flexible tracking system that is well suited for operating in an indoor environment. Based on readily available RF transceivers and microcontrollers, our wearable sensor system can facilitate locating users (e.g., patients or doctors) or objects (e.g., medical devices) in a building. The strategic construction of the sensor system, along with a suitably designed tracking algorithm, together provide for reliability and dispatch in localization performance. For demonstration purposes, several simplified experiments, with different configurations of the system, are implemented in two testing rooms to assess the baseline performance. From the obtained results, our system exhibits immense promise in acquiring a user location and corresponding time-stamp, with high accuracy and rapid response. This capability is conducive to both short- and long-term data analytics, which are crucial for improving healthcare management.

  3. Establishment of HSV1 latency in immunodeficient mice facilitates efficient in vivo reactivation.

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    Chandran Ramakrishna

    2015-03-01

    Full Text Available The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1 infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD, but not low dose (LD, HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS, the majority of HD inoculated mice developed HSV1 encephalitis (HSE rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg. T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.

  4. H2O2 Treatment of HUVECs Facilitates PKC Mediated Thr495 Phosphorylation on eNOS when Pre-treated with High Glucose Levels

    DEFF Research Database (Denmark)

    Guterbaum, Thomas J; Braunstein, Thomas H; Fossum, Anna

    2015-01-01

    Objective: Metabolic syndrome entails hypertension, hyperglycemia, obesity and hypercholesterolemia. This syndrome increases the risk of cardiovascular disease and diabetes. Hyperglycemia during coronary reperfusion is associated with a poor prognosis. Contrastingly, targeting correction of hyper......Objective: Metabolic syndrome entails hypertension, hyperglycemia, obesity and hypercholesterolemia. This syndrome increases the risk of cardiovascular disease and diabetes. Hyperglycemia during coronary reperfusion is associated with a poor prognosis. Contrastingly, targeting correction...... -rich environment. Methods and results: HUVECs (human umbilical vein endothelial cells) were exposed to high glucose (20 mM) for either 20 or 72 hours co-incubated with or without H2 O2 (400 µM) for 30 minutes as models of increased oxidative stress during acute and prolonged hyperglycemia, respectively...... in an increase in Thr495 phosphorylation (to 425%, paffect phosphorylation of Thr495 and Ser1177. Stimulating HUVECs that were pre-incubated with 20 mM glucose for 72 hours with H2 O2...

  5. Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors.

    Science.gov (United States)

    Ort, D R; Izawa, S

    1974-03-01

    The rate of Hill reaction can be measured accurately as O(2) uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O(2) consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O(2) uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O(2) uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O(2)) through both photosystems II and I is practically independent of the donor used-catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e(2) ratio is 1.0 +/- 0.1. Only ascorbate gives a slightly lower value (P/e(2) = 0.9). (NH(2)OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O(2)) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e(2) ratio is 0.6 +/- 0.08.

  6. Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site

    DEFF Research Database (Denmark)

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

    1994-01-01

    -64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. The results obtained with the COOH-terminal-deleted mutants support the view that reconstitution of a functional holoenzyme must occur to allow efficient autophosphorylation. Polylysine prevents...... mutants reconstituting a tetrameric holoenzyme. Only with the three largest COOH-terminal deletion mutants beta delta 150-215, beta delta 171-215, and beta delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosphorylation of the beta-subunit mutants added in large molar...... excess to CK-2 holoenzyme (either native or recombinant) is also severely impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 region and by the deletion of the COOH-terminal segments 150-215 and 171-215. Such a phosphorylation is inhibited by polylysine, with the exception...

  7. Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites

    OpenAIRE

    Boris Prinz; Katherine L. Harvey; Louisa Wilcke; Ulrike Ruch; Klemens Engelberg; Laura Biller; Isabelle Lucet; Steffen Erkelenz; Dorothee Heincke; Tobias Spielmann; Christian Doerig; Conrad Kunick; Brendan S Crabb; Gilson, Paul R.; Gilberger, Tim W

    2016-01-01

    Central to the pathogenesis of malaria is the proliferation of Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of ...

  8. Promote Green Building, Facilitate Energy Efficiency and Emissions Reduction, and Improve Habitat Environment

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    @@ Green building here refers to the practice of creating structures able to best achieve efficient and environmentally friendly use of resources (energy, land, water, materials) throughout the life-cycle so as to offer healthy, convenient and pleasant buildings for people to live and work in in harmony with nature.It has become a worldwide trend in the development of the construction industry.

  9. Promote Green Building, Facilitate Energy Efficiency and Emissions Reduction, and Improve Habitat Environment

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Green building here refers to the practice of creating structures able to best achieve efficient and environmentally friendly use of resources(energy, land, water, materials) throughout the life-cycle so as to offer healthy, convenient and pleasant buildings for people to live and work in in harmony with nature. It has become a worldwide trend in the development of the construction industry.

  10. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    Science.gov (United States)

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  11. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    Science.gov (United States)

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

  12. Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression1[OPEN

    Science.gov (United States)

    Weimer, Annika K.; Stoppin-Mellet, Virginie; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin

    2017-01-01

    Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. PMID:27879390

  13. Oxidative phosphorylation efficiency, proton conductance and reactive oxygen species production of liver mitochondria correlates with body mass in frogs.

    Science.gov (United States)

    Roussel, Damien; Salin, Karine; Dumet, Adeline; Romestaing, Caroline; Rey, Benjamin; Voituron, Yann

    2015-10-01

    Body size is a central biological parameter affecting most biological processes (especially energetics) and the mitochondrion is a key organelle controlling metabolism and is also the cell's main source of chemical energy. However, the link between body size and mitochondrial function is still unclear, especially in ectotherms. In this study, we investigated several parameters of mitochondrial bioenergetics in the liver of three closely related species of frog (the common frog Rana temporaria, the marsh frog Pelophylax ridibundus and the bull frog Lithobates catesbeiana). These particular species were chosen because of their differences in adult body mass. We found that mitochondrial coupling efficiency was markedly increased with animal size, which led to a higher ATP production (+70%) in the larger frogs (L. catesbeiana) compared with the smaller frogs (R. temporaria). This was essentially driven by a strong negative dependence of mitochondrial proton conductance on body mass. Liver mitochondria from the larger frogs (L. catesbeiana) displayed 50% of the proton conductance of mitochondria from the smaller frogs (R. temporaria). Contrary to our prediction, the low mitochondrial proton conductance measured in L. catesbeiana was not associated with higher reactive oxygen species production. Instead, liver mitochondria from the larger individuals produced significantly lower levels of radical oxygen species than those from the smaller frogs. Collectively, the data show that key bioenergetics parameters of mitochondria (proton leak, ATP production efficiency and radical oxygen species production) are correlated with body mass in frogs. This research expands our understanding of the relationship between mitochondrial function and the evolution of allometric scaling in ectotherms.

  14. Coupling of metal-organic frameworks-containing monolithic capillary-based selective enrichment with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for efficient analysis of protein phosphorylation.

    Science.gov (United States)

    Li, Daojin; Yin, Danyang; Chen, Yang; Liu, Zhen

    2017-05-19

    Protein phosphorylation is a major post-translational modification, which plays a vital role in cellular signaling of numerous biological processes. Mass spectrometry (MS) has been an essential tool for the analysis of protein phosphorylation, for which it is a key step to selectively enrich phosphopeptides from complex biological samples. In this study, metal-organic frameworks (MOFs)-based monolithic capillary has been successfully prepared as an effective sorbent for the selective enrichment of phosphopeptides and has been off-line coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for efficient analysis of phosphopeptides. Using š-casein as a representative phosphoprotein, efficient phosphorylation analysis by this off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also demonstrated. Through introducing large surface areas and highly ordered pores of MOFs into monolithic column, the MOFs-based monolithic capillary exhibited several significant advantages, such as excellent selectivity toward phosphopeptides, superb tolerance to interference and simple operation procedure. Because of these highly desirable properties, the MOFs-based monolithic capillary could be a useful tool for protein phosphorylation analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

    Science.gov (United States)

    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  16. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  17. Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

    Science.gov (United States)

    Fukuhara, Takasuke; Kambara, Hiroto; Shiokawa, Mai; Ono, Chikako; Katoh, Hiroshi; Morita, Eiji; Okuzaki, Daisuke; Maehara, Yoshihiko; Koike, Kazuhiko

    2012-01-01

    Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly. PMID:22593164

  18. Efficient post-processing of aqueous generator eluates facilitates {sup 68}Ga-labelling under anhydrous conditions

    Energy Technology Data Exchange (ETDEWEB)

    Zoller, F.; Riss, P.J.; Roesch, F. [University of Mainz (Germany). Institute of Nuclear Chemistry; Montforts, F.P. [University of Bremen (Germany). Institute of Organic Chemistry

    2010-07-01

    The present study reports a convenient method for {sup 68}Ga-labelling under anhydrous conditions using solid-phase derived gallium-68-acetylacetonate ([ {sup 68}Ga]Ga(acac){sub 3}) in a microwave-enhanced radiosynthesis. Commercial {sup 68}Ge/{sup 68}Ga-radionuclide generators utilizing TiO{sub 2} to adsorb {sup 68}Ge(IV) (Obninsk-generators) were used. The initial aqueous generator eluate was transferred online onto a cation exchange resin and {sup 68}Ga was absorbed quantitatively. From this resin, {sup 68}Ga was eluted with different acetone-based, non-aqueous solvent systems. More than 95% of the generator-eluted {sup 68}Ga was obtained from the cation exchange resin with 600 {mu}L of a 98% acetone/2% acetylacetone mixture providing n.c.a. [ {sup 68}Ga]Ga(acac){sub 3} as labelling agent. Water-insoluble macrocyclic polypyrrole derivatives were chosen as model compounds for a proof-of-principle labelling of lipophilic compounds with {sup 68}Ga. Labelling of two different porphyrin derivatives, meso -tetraphenyl-porphyrin (Tpp) and 3-(1-hydroxyheptyl)deuteroporphyrin dimethylester (HHDPD) was performed in chloroform in a focused microwave synthesis system in yields of up to 90% within 5 min using phenol as co-ligand. Moreover, new co-ligands were investigated to be more effective and significantly less toxic than phenol. Among the phenol alternatives, gentisic acid (2,5-dihydroxy benzoic acid, DHB, 5 mg) emerged as the most useful, non-toxic phenol substitute. It facilitates reducing the load of co-ligand by 95%, while providing an increased labeling yield of 97%. {sup 68}Ga-labelled porphyrins may facilitate the medical application for molecular imaging via positron emission tomography. (orig.)

  19. A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti-apoptotic Bcl-2 family proteins, including phosphorylated Mcl-1.

    Science.gov (United States)

    Liu, Yubo; Xie, Mingzhou; Song, Ting; Sheng, Hongkun; Yu, Xiaoyan; Zhang, Zhichao

    2015-03-01

    The Bcl-2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl-1, a major anti-apoptotic protein in the Bcl-2 family, is extensively expressed in melanoma and contributes to melanoma's well-documented chemoresistance. Here, we provide the first evidence that Mcl-1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT-737, and a novel anti-apoptotic mechanism of phosphorylated Mcl-1 (pMcl-1) is revealed. pMcl-1 antagonized the known BH3 mimetics by sequestering pro-apoptotic proteins that were released from Bcl-2/Mcl-1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl-2, Mcl-1, and pMcl-1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro-apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl-1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl-1 in melanoma.

  20. An Efficient Solution-Processed Intermediate Layer for Facilitating Fabrication of Organic Multi-Junction Solar Cells

    DEFF Research Database (Denmark)

    Ning Li; Baran, Derya; Forberich, Karen

    2013-01-01

    ):poly(styrenesulfonate) (PEDOT:PSS) is demonstrated for series-connected multi-junction organic solar cells (OSCs). Drying at 80 °C in air is sufficient for this solution-processed IML to obtain excellent functionality and reliability, which allow the use of most of high performance donor materials in the tandem structure...... simulations, which are reliable and promising guidelines for the design and investigation of multi-junction OSCs, are discussed. The outcome of optical and electrical simulations is in excellent agreement with the experimental data, indicating the outstanding efficiency and functionality of this solution...

  1. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    Energy Technology Data Exchange (ETDEWEB)

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  2. Different mechanisms of homologous and heterologous μ-opioid receptor phosphorylation.

    Science.gov (United States)

    Mann, Anika; Illing, Susann; Miess, Elke; Schulz, Stefan

    2015-01-01

    The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence (370) TREHPSTANT(379) in the receptor's carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq -coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo. This article

  3. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    Science.gov (United States)

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  4. Robot-assisted intersphincteric resection facilitates an efficient sphincter-saving in patients with low rectal cancer.

    Science.gov (United States)

    Kim, Jin Cheon; Lee, Jong Lyul; Alotaibi, Abdulrahman Muaod; Yoon, Yong Sik; Kim, Chan Wook; Park, In Ja

    2017-08-01

    Few investigations of robot-assisted intersphincteric resection (ISR) are presently available to support this procedure as a safe and efficient procedure. We aimed to evaluate the utility of robot-assisted ISR by comparison between ISR and abdominoperineal resection (APR) using both robot-assisted and open approaches. The 558 patients with lower rectal cancer (LRC) who underwent curative operation was enrolled between July 2010 and June 2015 to perform either by robot-assisted (ISR vs. APR = 310 vs. 34) or open approaches (144 vs. 70). Perioperative and functional outcomes including urogenital and anorectal dysfunctions were measured. Recurrence and survival were examined in 216 patients in which >3 years had elapsed after the operation. The robot-assisted approach was the most significant parameter to determine ISR achievement among potent parameters (OR = 3.467, 95% CI = 2.095-5.738, p robot-assisted ISR at 12 and 24 months, respectively (p robot-assisted ISR replaced a significant portion of APR to achieve successful SSO via mostly transabdominal approach and double-stapled anastomosis. The robot-assisted ISR with minimal invasiveness might be a help to reduce anorectal and urogenital dysfunctions.

  5. Mathematical analysis and algorithms for efficiently and accurately implementing stochastic simulations of short-term synaptic depression and facilitation.

    Science.gov (United States)

    McDonnell, Mark D; Mohan, Ashutosh; Stricker, Christian

    2013-01-01

    The release of neurotransmitter vesicles after arrival of a pre-synaptic action potential (AP) at cortical synapses is known to be a stochastic process, as is the availability of vesicles for release. These processes are known to also depend on the recent history of AP arrivals, and this can be described in terms of time-varying probabilities of vesicle release. Mathematical models of such synaptic dynamics frequently are based only on the mean number of vesicles released by each pre-synaptic AP, since if it is assumed there are sufficiently many vesicle sites, then variance is small. However, it has been shown recently that variance across sites can be significant for neuron and network dynamics, and this suggests the potential importance of studying short-term plasticity using simulations that do generate trial-to-trial variability. Therefore, in this paper we study several well-known conceptual models for stochastic availability and release. We state explicitly the random variables that these models describe and propose efficient algorithms for accurately implementing stochastic simulations of these random variables in software or hardware. Our results are complemented by mathematical analysis and statement of pseudo-code algorithms.

  6. The perfect host: a mouse host embryo facilitating more efficient germ line transmission of genetically modified embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Robert A Taft

    Full Text Available There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs. We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium. ESC germline transmission was observed in 9/11 (82% lines using PH blastocysts, compared to 6/11 (55% when conventional host blastocysts were used. Furthermore, less than 35% (83/240 of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137 of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the

  7. The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Ribeiro, Mariana P.C.; Silva, Filomena S.G.; Santos, Armanda E. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal); Santos, Maria S. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal); Custódio, José B.A., E-mail: custodio@ci.uc.pt [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal)

    2013-02-15

    Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30 nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50 nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50 nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10 nmol/mg protein prevented and reversed the Ca{sup 2+}-induced depolarization of ΔΨ and the release of mitochondrial Ca{sup 2+}, similarly to cyclosporine A, indicating that EDX did not affect Ca{sup 2+} uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma. - Highlights: ► Mitochondria are important targets of Endoxifen. ► Endoxifen prevents mitochondrial permeability transition. ► Endoxifen prevents oxidative

  8. Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

    Science.gov (United States)

    Li, H; Haviv, Y S; Derdeyn, C A; Lam, J; Coolidge, C; Hunter, E; Curiel, D T; Blackwell, J L

    2001-12-10

    Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

  9. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    Science.gov (United States)

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

  10. Severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mRNA.

    Science.gov (United States)

    Tanaka, Tomohisa; Kamitani, Wataru; DeDiego, Marta L; Enjuanes, Luis; Matsuura, Yoshiharu

    2012-10-01

    Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is an enveloped virus containing a single-stranded, positive-sense RNA genome. Nine mRNAs carrying a set of common 5' and 3' untranslated regions (UTR) are synthesized from the incoming viral genomic RNA in cells infected with SCoV. A nonstructural SCoV nsp1 protein causes a severe translational shutoff by binding to the 40S ribosomal subunits. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNA. However, the mechanism by which SCoV viral proteins are efficiently produced in infected cells in which host protein synthesis is impaired by nsp1 is unknown. In this study, we investigated the role of the viral UTRs in evasion of the nsp1-mediated shutoff. Luciferase activities were significantly suppressed in cells expressing nsp1 together with the mRNA carrying a luciferase gene, while nsp1 failed to suppress luciferase activities of the mRNA flanked by the 5'UTR of SCoV. An RNA-protein binding assay and RNA decay assay revealed that nsp1 bound to stem-loop 1 (SL1) in the 5'UTR of SCoV RNA and that the specific interaction with nsp1 stabilized the mRNA carrying SL1. Furthermore, experiments using an SCoV replicon system showed that the specific interaction enhanced the SCoV replication. The specific interaction of nsp1 with SL1 is an important strategy to facilitate efficient viral gene expression in infected cells, in which nsp1 suppresses host gene expression. Our data indicate a novel mechanism of viral gene expression control by nsp1 and give new insight into understanding the pathogenesis of SARS.

  11. Efficiency

    NARCIS (Netherlands)

    I.P. van Staveren (Irene)

    2009-01-01

    textabstractThe dominant economic theory, neoclassical economics, employs a single economic evaluative criterion: efficiency. Moreover, it assigns this criterion a very specific meaning. Other – heterodox – schools of thought in economics tend to use more open concepts of efficiency, related to comm

  12. α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

    Science.gov (United States)

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-01-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N

  13. Protein phosphorylation and photorespiration.

    Science.gov (United States)

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  14. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    Directory of Open Access Journals (Sweden)

    John G Koland

    2014-01-01

    Full Text Available Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR, the intrinsic protein tyrosine kinase (PTK activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites in either of the two C-terminal (CT domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in

  15. Effects of Phosphorylation of β Subunits of Phycocyanins on State Transition in the Model Cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Chen, Zhuo; Zhan, Jiao; Chen, Ying; Yang, Mingkun; He, Chenliu; Ge, Feng; Wang, Qiang

    2015-10-01

    Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model cyanobacterium and has been used extensively for studies concerned with photosynthesis and environmental adaptation. Although dozens of protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted, only a few substrate proteins are known in Synechocystis. In this study, we report 194 in vivo phosphorylation sites from 149 proteins in Synechocystis, which were identified using a combination of peptide pre-fractionation, TiO(2) enrichment and liquid chromatograpy-tandem mass spectrometry (LC-MS/MS) analysis. These phosphorylated proteins are implicated in diverse biological processes, such as photosynthesis. Among all identified phosphoproteins involved in photosynthesis, the β subunits of phycocyanins (CpcBs) were found to be phosphorylated on Ser22, Ser49, Thr94 and Ser154. Four non-phosphorylated mutants were constructed by using site-directed mutagenesis. The in vivo characterization of the cpcB mutants showed a slower growth under high light irradiance and displayed fluorescence quenching to a lower level and less efficient energy transfer inside the phycobilisome (PBS). Notably, the non-phosphorylated mutants exhibited a slower state transition than the wild type. The current results demonstrated that the phosphorylation status of CpcBs affects the energy transfer and state transition of photosynthesis in Synechocystis. This study provides novel insights into the molecular mechanisms of protein phosphorylation in the regulation of photosynthesis in cyanobacteria and may facilitate the elucidation of the entire regulatory network by linking kinases to their physiological substrates. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Elaboration of a Program to Facilitate the Implementation of the Directive 2009/33/EC on the Promotion of Clean and Energy-Efficient Road Motor Vehicles

    Directory of Open Access Journals (Sweden)

    Krisztián Uhlik

    2012-09-01

    Full Text Available The energy consumption, carbon-dioxide and other air pollutant emissions of motor vehicles can be reduced substantially by various recently developed technical solutions. The use of these new technologies increases the price of the vehicles which causes an unwanted economic burden for the purchasers of such vehicles. The market competition between manufacturers requires low prices which delays the adaptation of the new, more efficient technologies. The recently enacted legislation, aimed at the promotion of purchasing clean and energy-efficient road transport vehicles, intends to remedy this problem.

  17. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    Science.gov (United States)

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-06-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

  18. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

    Science.gov (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2014-01-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

  19. Electrophysiological evidence for early perceptual facilitation and efficient categorization of self-related stimuli during an Implicit Association Test measuring neuroticism.

    Science.gov (United States)

    Fleischhauer, Monika; Strobel, Alexander; Diers, Kersten; Enge, Sören

    2014-02-01

    The Implicit Association Test (IAT) is a widely used latency-based categorization task that indirectly measures the strength of automatic associations between target and attribute concepts. So far, little is known about the perceptual and cognitive processes underlying personality IATs. Thus, the present study examined event-related potential indices during the execution of an IAT measuring neuroticism (N  =  70). The IAT effect was strongly modulated by the P1 component indicating early facilitation of relevant visual input and by a P3b-like late positive component reflecting the efficacy of stimulus categorization. Both components covaried, and larger amplitudes led to faster responses. The results suggest a relationship between early perceptual and semantic processes operating at a more automatic, implicit level and later decision-related categorization of self-relevant stimuli contributing to the IAT effect.

  20. Extracellular regulated kinase phosphorylates mitofusin 1 to control mitochondrial morphology and apoptosis.

    Science.gov (United States)

    Pyakurel, Aswin; Savoia, Claudia; Hess, Daniel; Scorrano, Luca

    2015-04-16

    Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.

  1. Extracellular Regulated Kinase Phosphorylates Mitofusin 1 to Control Mitochondrial Morphology and Apoptosis

    Science.gov (United States)

    Pyakurel, Aswin; Savoia, Claudia; Hess, Daniel; Scorrano, Luca

    2015-01-01

    Summary Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1T562. Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis. PMID:25801171

  2. RegPhos 2.0: an updated resource to explore protein kinase-substrate phosphorylation networks in mammals.

    Science.gov (United States)

    Huang, Kai-Yao; Wu, Hsin-Yi; Chen, Yi-Ju; Lu, Cheng-Tsung; Su, Min-Gang; Hsieh, Yun-Chung; Tsai, Chih-Ming; Lin, Kuo-I; Huang, Hsien-Da; Lee, Tzong-Yi; Chen, Yu-Ju

    2014-01-01

    Protein phosphorylation catalyzed by kinases plays crucial roles in regulating a variety of intracellular processes. Owing to an increasing number of in vivo phosphorylation sites that have been identified by mass spectrometry (MS)-based proteomics, the RegPhos, available online at http://csb.cse.yzu.edu.tw/RegPhos2/, was developed to explore protein phosphorylation networks in human. In this update, we not only enhance the data content in human but also investigate kinase-substrate phosphorylation networks in mouse and rat. The experimentally validated phosphorylation sites as well as their catalytic kinases were extracted from public resources, and MS/MS phosphopeptides were manually curated from research articles. RegPhos 2.0 aims to provide a more comprehensive view of intracellular signaling networks by integrating the information of metabolic pathways and protein-protein interactions. A case study shows that analyzing the phosphoproteome profile of time-dependent cell activation obtained from Liquid chromatography-mass spectrometry (LC-MS/MS) analysis, the RegPhos deciphered not only the consistent scheme in B cell receptor (BCR) signaling pathway but also novel regulatory molecules that may involve in it. With an attempt to help users efficiently identify the candidate biomarkers in cancers, 30 microarray experiments, including 39 cancerous versus normal cells, were analyzed for detecting cancer-specific expressed genes coding for kinases and their substrates. Furthermore, this update features an improved web interface to facilitate convenient access to the exploration of phosphorylation networks for a group of genes/proteins. Database URL: http://csb.cse.yzu.edu.tw/RegPhos2/

  3. Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding

    Directory of Open Access Journals (Sweden)

    Øystein Stakkestad

    2017-07-01

    Full Text Available Ameloblastin (AMBN, an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2 and protein kinase A (PKA and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

  4. Protein tyrosine phosphorylation in streptomycetes.

    Science.gov (United States)

    Waters, B; Vujaklija, D; Gold, M R; Davies, J

    1994-07-01

    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  5. Importance of protamine phosphorylation to histone displacement in spermatids: can the disruption of this process be used for male contraception

    Energy Technology Data Exchange (ETDEWEB)

    Balhorn, R.; Hud, N.V.; Corzett, M.; Mazrimas, J.

    1995-06-01

    Protamine is a small protein that packages DNA in the sperm of most vertebrates. Shortly after its synthesis, the serine and threonine residues in each protamine are phosphorylated and the modified proteins are deposited onto DNA, displacing the histones and other chromatin proteins. We have hypothesized that the phosphorylation of protamine 1 induces protamine dimerization and these dimers are required for efficient histone displacement. Histone displacement by protamines in late-step spermatids appears to be essential for the production of fertile sperm in man and other mammals, and the disruption of this process could provide a new approach for male contraception. As a first step towards testing this theory, we have initiated a set of in vitro experiments to determine whether of not protamine phosphorylation is essential for histone displacement. Thee results of these experiments, although incomplete, confirm that unphosphorylated protamine cannot effectively displace histone from DNA. Polyarginine molecules twice the size of a protamine molecule and salmine dimer were found to be more effective. These results are consistent with the theory that the disruption of protamine phosphorylation may prove to be a useful new approach for male contraception if it can be shown to facilitate or induce protamine dimerization.

  6. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

    Science.gov (United States)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-02

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  7. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

    Science.gov (United States)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  8. Very High Fuel Economy, Heavy Duty, Constant Speed, Truck Engine Optimized Via Unique Energy Recovery Turbines and Facilitated High Efficiency Continuously Variable Drivetrain

    Energy Technology Data Exchange (ETDEWEB)

    Bahman Habibzadeh

    2010-01-31

    The project began under a corporative agreement between Mack Trucks, Inc and the Department of Energy starting from September 1, 2005. The major objective of the four year project is to demonstrate a 10% efficiency gain by operating a Volvo 13 Litre heavy-duty diesel engine at a constant or narrow speed and coupled to a continuously variable transmission. The simulation work on the Constant Speed Engine started on October 1st. The initial simulations are aimed to give a basic engine model for the VTEC vehicle simulations. Compressor and turbine maps are based upon existing maps and/or qualified, realistic estimations. The reference engine is a MD 13 US07 475 Hp. Phase I was completed in May 2006 which determined that an increase in fuel efficiency for the engine of 10.5% over the OICA cycle, and 8.2% over a road cycle was possible. The net increase in fuel efficiency would be 5% when coupled to a CVT and operated over simulated highway conditions. In Phase II an economic analysis was performed on the engine with turbocompound (TC) and a Continuously Variable Transmission (CVT). The system was analyzed to determine the payback time needed for the added cost of the TC and CVT system. The analysis was performed by considering two different production scenarios of 10,000 and 60,000 units annually. The cost estimate includes the turbocharger, the turbocompound unit, the interstage duct diffuser and installation details, the modifications necessary on the engine and the CVT. Even with the cheapest fuel and the lowest improvement, the pay back time is only slightly more than 12 months. A gear train is necessary between the engine crankshaft and turbocompound unit. This is considered to be relatively straight forward with no design problems.

  9. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5

    OpenAIRE

    2015-01-01

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV (‘Berlin patient’). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by m...

  10. Phosphorylation of chicken growth hormone

    Energy Technology Data Exchange (ETDEWEB)

    Aramburo, C.; Montiel, J.L. (Universidad Nacional Autonoma de Mexico (Mexico)); Donoghue, D.; Scanes, C.G. (Rutgers Univ., New Brunswick, NJ (USA)); Berghman, L.R. (Laboratory for Neuroendocrinology and Immunological Biotechnology, Louvain (Belgium))

    1990-01-01

    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and {gamma}-{sup 32}P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of {sup 32}P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of {sup 32}P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.

  11. SecDF as part of the Sec-translocase facilitates efficient secretion of Bacillus cereus toxins and cell wall-associated proteins.

    Science.gov (United States)

    Vörös, Aniko; Simm, Roger; Slamti, Leyla; McKay, Matthew J; Hegna, Ida K; Nielsen-LeRoux, Christina; Hassan, Karl A; Paulsen, Ian T; Lereclus, Didier; Økstad, Ole Andreas; Molloy, Mark P; Kolstø, Anne-Brit

    2014-01-01

    The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

  12. Directional and quantitative phosphorylation networks

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Linding, Rune

    2008-01-01

    for unravelling phosphorylation-mediated cellular interaction networks. In particular, we will discuss how the combination of new quantitative mass-spectrometric technologies and computational algorithms together are enhancing mapping of these largely uncharted dynamic networks. By combining quantitative...

  13. Facilitering som styringsredskab

    OpenAIRE

    Jørgensen, Karen Overgaard

    2006-01-01

    #This thesis surveys facilitation as a new tool of steering within the public sector in Denmark. It is explored how facilitation is articulated and practiced among facilitators from the public, private and voluntary sector. Furthermore, the facilitator’s challenges by using facilitation are examined. The thesis is based on the presumption that facilitation is articulated by rationalities, which influence how facilitation is practiced and performed. Also, a facilitator is seen as a performer a...

  14. Chemistry of Phosphorylated Formaldehyde Derivatives. Part I

    Directory of Open Access Journals (Sweden)

    Vasily P. Morgalyuk

    2014-08-01

    Full Text Available The underinvestigated derivatives of unstable phosphorylated formaldehyde acetals and some of the structurally related compounds, such as thioacetals, aminonitriles, aminomethylphosphinoyl compounds, are considered. Separately considered are halogen aminals of phosphorylated formaldehyde, acetals of phosphorylated formaldehyde of H-phosphinate-type and a phosphorylated gem-diol of formaldehyde. Synthetic methods, chemical properties and examples of practical applications are given.

  15. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    Science.gov (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  16. Highly precise detection, discrimination, and removal of anionic surfactants over the full pH range via cationic conjugated polymer: an efficient strategy to facilitate illicit-drug analysis.

    Science.gov (United States)

    Hussain, Sameer; Malik, Akhtar H; Iyer, Parameswar K

    2015-02-11

    A water-soluble cationic conjugated polyelectrolyte (CPE), poly(1,4-bis(6-(1-methylimidazolium)-hexyloxy)-benzene bromide) (PMI) displays extraordinary stability over the full pH range of 1-14 as well as in seawater, brine, urine, and other solutions and carries out efficient detection, discrimination, and removal of moderately dissimilar anionic surfactants (viz., sodium dodecyl benzenesulfonate (SDBS) and sodium dodecyl sulfate (SDS)) at very low levels (31.7 and 17.3 parts per billion (ppb), respectively). PMI formed stable hydrogels in the presence of SDS that remained unaffected by strong acids/bases, heating, ultrasonication, or exposure to light, whereas SDBS formed precipitate with PMI as a result of its different interpolymer cofacial arrangement via Columbic attraction. The complex-forming ability of PMI with SDS and SDBS facilitated their elimination from water or drug-doped urine samples without the use of any organic solvent, chromatographic technique, or solid support. This protocol, the first of its kind for the removal of anionic surfactants at very low concentrations from any type of solution and competitive environments, demonstrates an original application using a CPE. The surfactant-free sample solutions could be precisely analyzed for the presence of illicit drugs by any standard methods. Using PMI, a newly developed CPE, a rapid and practical method for the efficient detection, discrimination, and removal of SDS and SDBS at ppb levels from water and urine, under harsh conditions, and in natural chemical environments is demonstrated.

  17. Adjusting ammonium uptake via phosphorylation.

    Science.gov (United States)

    Lanquar, Viviane; Frommer, Wolf B

    2010-06-01

    In plants, AMT/MEP/Rh superfamily mediates high affinity ammonium uptake. AMT/MEP transporters form a trimeric complex, which requires a productive interaction between subunits in order to be functional. The AMT/MEP C-terminal domain is highly conserved in more than 700 AMT homologs from cyanobacteria to higher plants with no cases found to be lacking this domain. AMT1;1 exists in active and inactive states, probably controlled by the spatial positioning of the C-terminus. Ammonium triggers the phosphorylation of a conserved threonine residue (T460) in the C-terminus of AMT1;1 in a time- and concentration-dependent manner. The T460 phosphorylation level correlates with a decrease of root ammonium uptake. We propose that ammonium-induced phosphorylation modulates ammonium uptake as a general mechanism to protect against ammonium toxicity.

  18. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  19. Nucleoside phosphorylation in amide solutions

    Science.gov (United States)

    Schoffstall, A. M.; Kokko, B.

    1978-01-01

    The paper deals with phosphorylation in possible prebiotic nonaqueous solvents. To this end, phosphorylation of nucleosides using inorganic phosphates in amide solutions is studied at room and elevated temperatures. Reaction proceeds most readily in formamide and N-methylformamide. Products obtained at elevated temperature are nucleotides, nucleoside 2',3'-cyclic phosphates, and when the phosphate concentration is high, nucleoside diphosphates. At room temperature, adenosine afforded a mixture of nucleotides, but none of the cyclic nucleotide. Conditions leading to the highest relative percentage of cyclic nucleotide involve the use of low concentrations of phosphate and an excess of nucleoside.

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  1. Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes

    NARCIS (Netherlands)

    Dhekne, Herschel S.; Hsiao, Nai-Hua; Roelofs, Pieter; Kumari, Meena; Slim, Christiaan L.; Rings, Edmond H. H. M.; van IJzendoorn, Sven C. D.

    2014-01-01

    Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset

  2. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

    2002-01-01

    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

  3. Thiamine phosphorylated derivatives and bioelectrogenesis.

    Science.gov (United States)

    Schoffeniels, E

    1983-09-01

    Kinetic as well as thermodynamic considerations favour the idea that the change in sodium conductance explaining the action potential, must result from a bimolecular reaction system. The fact that thiamine phosphorylated derivatives are associated with the specific protein forming the sodium channel could well mean that these thiamine derivatives and more specifically thiamine triphosphate are directly involved in the conductance change.

  4. Biocatalytic asymmetric phosphorylation of mevalonate

    NARCIS (Netherlands)

    Matsumi, R.; Hellriegel, C.; Schoenenberger, B.; Milesi, T.; Oost, van der J.; Wohlgemuth, R.

    2014-01-01

    The excellent selectivity of the mevalonate kinase-catalyzed phosphorylation of mevalonate simplifies lengthy multi-step routes to (R)-mevalonate-5-phosphate to a one-step biocatalytic reaction, because the phosphate group can be transferred directly and without any additional reaction steps

  5. Prebiotic Phosphorylation Reactions on the Early Earth

    Directory of Open Access Journals (Sweden)

    Maheen Gull

    2014-07-01

    Full Text Available Phosphorus (P is an essential element for life. It occurs in living beings in the form of phosphate, which is ubiquitous in biochemistry, chiefly in the form of C-O-P (carbon, oxygen and phosphorus, C-P, or P-O-P linkages to form life. Within prebiotic chemistry, several key questions concerning phosphorus chemistry have developed: what were the most likely sources of P on the early Earth? How did it become incorporated into the biological world to form the P compounds that life employs today? Can meteorites be responsible for the delivery of P? What were the most likely solvents on the early Earth and out of those which are favorable for phosphorylation? Or, alternatively, were P compounds most likely produced in relatively dry environments? What were the most suitable temperature conditions for phosphorylation? A route to efficient formation of biological P compounds is still a question that challenges astrobiologists. This article discusses these important issues related to the origin of biological P compounds.

  6. Phosphorylation of actopaxin regulates cell spreading and migration

    Science.gov (United States)

    Clarke, Dominic M.; Brown, Michael C.; LaLonde, David P.; Turner, Christopher E.

    2004-01-01

    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration. PMID:15353548

  7. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

    DEFF Research Database (Denmark)

    Erba, Elisabetta Boeri; Matthiesen, Rune; Bunkenborg, Jakob

    2007-01-01

    Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium p...

  8. Affinity chromatography of phosphorylated proteins.

    Science.gov (United States)

    Tchaga, Grigoriy S

    2008-01-01

    This chapter covers the use of immobilized metal ion affinity chromatography (IMAC) for enrichment of phosphorylated proteins. Some requirements for successful enrichment of these types of proteins are discussed. An experimental protocol and a set of application data are included to enable the scientist to obtain high-yield results in a very short time with pre-packed phospho-specific metal ion affinity resin (PMAC).

  9. Learning facilitating leadership

    DEFF Research Database (Denmark)

    Rasmussen, Lauge Baungaard; Hansen, Mette Sanne

    2016-01-01

    in teaching facilitation and the literature. These types of skills are most effectively acquired by combining conceptual lectures, classroom exercises and the facilitation of groups in a real-life context. The paper also reflects certain ‘shadow sides’ related to facilitation observed by the students...

  10. Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1.

    Science.gov (United States)

    Sarg, Bettina; Helliger, Wilfried; Talasz, Heribert; Förg, Barbara; Lindner, Herbert H

    2006-03-10

    H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.

  11. Phosphorylation of brain proteins in generalized convulsions

    Energy Technology Data Exchange (ETDEWEB)

    Horan, M.P.

    1986-01-01

    Phosphorylation of neuronal proteins is being proposed as a modulating influence on several aspects of neuronal function. By labeling proteins with radioactive phosphorus (/sup 32/P) and then separating these proteins by polyacrylamide gel electrophoresis, the author can determine what factors change the phosphorylation of these proteins. They have used such a system to analyze the effects of generalized convulsions on protein phosphorylation. Electroshock (ES) and pentylenetetrazol (PTZ) were utilized to produce generalized convulsions. Brain membranes, taken from rats immediately after a convulsion, exhibited an increase in protein phosphorylation in vitro. The most noticeable change took place in proteins in the 18,000-20,000 MW range. They have designated these proteins as the low molecular weight (LMW) proteins. The change in phosphorylation was basically the same after one convulsions as after six daily convulsions. Twenty-four hours after a single convulsion no change in phosphorylation was observed. When rat membranes are exposed to PTZ in vitro, phosphorylation is increased at 20 sec but has returned to control level at 90 sec of incubation. This effect is produced without a convulsion. In general, as the concentration of magnesium is increased from 5 mM to 10 mM phosphorylation is increased. Increasing the incubation time from 20 sec to 90 sec and increasing the calcium concentration to 10 mM both decrease phosphorylation of the LMW proteins. Human temporal cortex samples present with phosphorylated proteins having patterns very similar to those in rat membranes.

  12. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. S.; Qian, Weijun

    2009-08-01

    resource for facilitating future hypothesis-driven or targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.

  13. Learning facilitating leadership

    DEFF Research Database (Denmark)

    Rasmussen, Lauge Baungaard; Hansen, Mette Sanne

    2016-01-01

    This paper explains how engineering students at a Danish university acquired the necessary skills to become emergent facilitators of organisational development. The implications of this approach are discussed and related to relevant viewpoints and findings in the literature. The methodology deplo....... By connecting the literature, the authors’ and engineering students’ reflections on facilitator skills, this paper adds value to existing academic and practical discussions on learning facilitating leadership....

  14. Visual explorer facilitator's guide

    CERN Document Server

    Palus, Charles J

    2010-01-01

    Grounded in research and practice, the Visual Explorer™ Facilitator's Guide provides a method for supporting collaborative, creative conversations about complex issues through the power of images. The guide is available as a component in the Visual Explorer Facilitator's Letter-sized Set, Visual Explorer Facilitator's Post card-sized Set, Visual Explorer Playing Card-sized Set, and is also available as a stand-alone title for purchase to assist multiple tool users in an organization.

  15. PPARy phosphorylation mediated by JNK MAPK: a potential role in macrophage-derived foam cell formation

    Institute of Scientific and Technical Information of China (English)

    Ran YIN; Yu-gang DONG; Hong-lang LI

    2006-01-01

    Aim: To investigate whether oxidized low-density lipoprotein (ox-LDL) modulates peroxisome proliferator-activated receptor γ (PPARγ) activity through phosphorylation in macrophages, and the effect of PPARy phosphorylation on macrophages-derived foam cell formation. Methods: After exposing the cultured THP-1 cells to ox-LDL in the presence or absence of different mitogen-activated protein kinase (MAPK) inhibitors, PPARγ and phosphorylated PPARγ protein levels were detected by Western blot. MAPK activity was analyzed using MAP Kinase Assay Kit. Intracellular cholesterol accumulation was assessed by Oil red O staining and cholesterol oxidase enzymatic method. The Mrna level of PPARγ target gene was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results: ox-LDL evaluated PPARγ phosphorylation status and subsequently decreased PPARγ target gene expression in a dose-dependent manner. Ox-LDL also induced MAPK activation. Treatment of THP-1 cells with c-Jun N-terminal kinase-, but not p38- or extracellular signal-regulated kinase-MAPK inhibitor, significantly suppressed PPARγ phosphorylation induced by ox-LDL, which in turn inhibited foam cell formation. Conclusion: In addition to its ligand-dependent activation, ox-LDL modulates PPARγ activity through phosphorylation, which is mediated by MAPK activation. PPARγ phosphorylation mediated by MAPK facilitates foam cell formation from macrophages exposed to ox-LDL.

  16. Analysis of mitotic phosphorylation of Borealin

    Directory of Open Access Journals (Sweden)

    Date Dipali A

    2007-01-01

    Full Text Available Abstract Background The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro. Results Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis. Conclusion Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.

  17. Phosphorylation impact on Spleen Tyrosine kinase conformation by Surface Enhanced Raman Spectroscopy

    Science.gov (United States)

    Cottat, Maximilien; Yasukuni, Ryohei; Homma, Yo; Lidgi-Guigui, Nathalie; Varin-Blank, Nadine; Lamy de La Chapelle, Marc; Le Roy, Christine

    2017-01-01

    Spleen Tyrosine Kinase (Syk) plays a crucial role in immune cell signalling and its altered expression or activation are involved in several cancers. Syk activity relies on its phosphorylation status and its multiple phosphorylation sites predict several Syk conformations. In this report, we characterized Syk structural changes according to its phosphorylation/activation status by Surface Enhanced Raman Spectroscopy (SERS). Unphosphorylated/inactive and phosphorylated/active Syk forms were produced into two expression systems with different phosphorylation capability. Syk forms were then analysed by SERS that was carried out in liquid condition on a lithographically designed gold nanocylinders array. Our study demonstrated that SERS signatures of the two Syk forms were drastically distinct, indicating structural modifications related to their phosphorylation status. By comparison with the atomic structure of the unphosphorylated Syk, the SERS peak assignments of the phosphorylated Syk nearest gold nanostructures revealed a differential interaction with the gold surface. We finally described a model for Syk conformational variations according to its phosphorylation status. In conclusion, SERS is an efficient technical approach for studying in vitro protein conformational changes and might be a powerful tool to determine protein functions in tumour cells.

  18. The challenges of facilitation

    DEFF Research Database (Denmark)

    Agger, Annika

    and at the same time make closures in order to secure progress in the process? The analysis draws upon theoretical perspectives on deliberative democracy and facilitation. Whereas, the scholarly literature on deliberative democracy is rich in describing potential outcomes and criteria for deliberative processes...... hours transcriptions of three table deliberations; questionnaires of 91 participants, 2 focus group interviews with participants and facilitators....

  19. Training facilitators and supervisors

    DEFF Research Database (Denmark)

    Kjær, Louise Binow; O Connor, Maja; Krogh, Kristian;

    At the Master’s program in Medicine at Aarhus University, Denmark, we have developed a faculty development program for facilitators and supervisors in 4 progressing student modules in communication, cooperation, and leadership. 1) A course for module 1 and 3 facilitators inspired by the apprentic...

  20. Phosphorylation of Intrinsically Disordered Regions in Remorin Proteins

    Directory of Open Access Journals (Sweden)

    Macarena eMarín

    2012-05-01

    Full Text Available Plant-specific remorin proteins reside in subdomains of plasma membranes, originally termed membrane rafts. They probably facilitate cellular signal transduction by direct interaction with signalling proteins such as receptor-like kinases (RLKs and may dynamically modulate their lateral segregation within plasma membranes. Recent evidence suggests such functions of remorins during plant-microbe interactions and innate immune responses, where differential phosphorylation of some of these proteins has been described to be dependent on the perception of the microbe-associated molecular pattern (MAMP flg22 and the presence of the NBS-LRR resistance protein RPM1. A number of specifically phosphorylated residues in their highly variable and intrinsically disordered N-terminal regions have been identified. Sequence diversity of these evolutionary distinct domains suggests that remorins may serve a wide range of biological functions. Here, we describe patterns and features of intrinsic disorder in remorin protein and discuss possible functional implications of phosphorylation within these rapidly evolving domains.

  1. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  2. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

  3. Case study: using sequence homology to identify putative phosphorylation sites in an evolutionarily distant species (honeybee).

    Science.gov (United States)

    Trost, Brett; Napper, Scott; Kusalik, Anthony

    2015-09-01

    The majority of scientific resources are devoted to studying a relatively small number of model species, meaning that the ability to translate knowledge across species is of considerable importance. Obtaining species-specific knowledge enables targeted investigations of the biology and pathobiology of a particular species, and facilitates comparative analyses. Phosphorylation is the most widespread posttranslational modification in eukaryotes, and although many phosphorylation sites have been experimentally identified for some species, little or no data are available for others. Using the honeybee as a test organism, this case study illustrates the process of using protein sequence homology to identify putative phosphorylation sites in a species of interest using experimentally determined sites from other species. A number of issues associated with this process are examined and discussed. Several databases of experimentally determined phosphorylation sites exist; however, it can be difficult for the nonspecialist to ascertain how their contents compare. Thus, this case study assesses the content and comparability of several phosphorylation site databases. Additional issues examined include the efficacy of homology-based phosphorylation site prediction, the impact of the level of evolutionary relatedness between species in making these predictions, the ability to translate knowledge of phosphorylation sites across large evolutionary distances and the criteria that should be used in selecting probable phosphorylation sites in the species of interest. Although focusing on phosphorylation, the issues discussed here also apply to the homology-based cross-species prediction of other posttranslational modifications, as well as to sequence motifs in general. © The Author 2014. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  4. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  5. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

    Science.gov (United States)

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan

    2013-05-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

  6. Characterizing the Microenvironment Surrounding Phosphorylated Protein Sites

    Institute of Scientific and Technical Information of China (English)

    Shi-Cai Fan; Xue-Gong Zhang

    2005-01-01

    Protein phosphorylation plays an important role in various cellular processes. Due to its high complexity, the mechanism needs to be further studied. In the last few years, many methods have been contributed to this field, but almost all of them investigated the mechanism based on protein sequences around protein sites. In this study, we implement an exploration by characterizing the microenvironment surrounding phosphorylated protein sites with a modified shell model, and obtain some significant properties by the rank-sum test, such as the lack of some classes of residues, atoms, and secondary structures. Furthermore, we find that the depletion of some properties affects protein phosphorylation remarkably. Our results suggest that it is a meaningful direction to explore the mechanism of protein phosphorylation from microenvironment and we expect further findings along with the increasing size of phosphorylation and protein structure data.

  7. Facilitating Understandings of Geometry.

    Science.gov (United States)

    Pappas, Christine C.; Bush, Sara

    1989-01-01

    Illustrates some learning encounters for facilitating first graders' understanding of geometry. Describes some of children's approaches using Cuisenaire rods and teacher's intervening. Presents six problems involving various combinations of Cuisenaire rods and cubes. (YP)

  8. Prediction of PK-specific phosphorylation site based on information entropy

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Phosphorylation is a crucial way to control the activity of proteins in many eukaryotic organisms in vivo. Experimental methods to determine phosphorylation sites in substrates are usually restricted by the in vitro condition of enzymes and very intensive in time and labor. Although some in silico methods and web servers have been introduced for automatic detection of phosphorylation sites, sophisticated methods are still in urgent demand to further improve prediction performances. Protein primary se-quences can help predict phosphorylation sites catalyzed by different protein kinase and most com-putational approaches use a short local peptide to make prediction. However, the useful information may be lost if only the conservative residues that are not close to the phosphorylation site are consid-ered in prediction, which would hamper the prediction results. A novel prediction method named IEPP (Information-Entropy based Phosphorylation Prediction) is presented in this paper for automatic de-tection of potential phosphorylation sites. In prediction, the sites around the phosphorylation sites are selected or excluded by their entropy values. The algorithm was compared with other methods such as GSP and PPSP on the ABL, MAPK and PKA PK families. The superior prediction accuracies were ob-tained in various measurements such as sensitivity (Sn) and specificity (Sp). Furthermore, compared with some online prediction web servers on the new discovered phosphorylation sites, IEPP also yielded the best performance. IEPP is another useful computational resource for identification of PK-specific phosphorylation sites and it also has the advantages of simpleness, efficiency and con-venience.

  9. Facilitating Knowledge Sharing

    DEFF Research Database (Denmark)

    Holdt Christensen, Peter

    Abstract This paper argues that knowledge sharing can be conceptualized as different situations of exchange in which individuals relate to each other in different ways, involving different rules, norms and traditions of reciprocity regulating the exchange. The main challenge for facilitating...... and the intermediaries regulating the exchange, and facilitating knowledge sharing should therefore be viewed as a continuum of practices under the influence of opportunistic behaviour, obedience or organizational citizenship behaviour. Keywords: Knowledge sharing, motivation, organizational settings, situations...

  10. Oxidative phosphorylation in cancer cells.

    Science.gov (United States)

    Solaini, Giancarlo; Sgarbi, Gianluca; Baracca, Alessandra

    2011-06-01

    Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

  11. Phosphorylated STAT3 physically interacts with NPM and transcriptionally enhances its expression in cancer.

    Science.gov (United States)

    Ren, Z; Aerts, J L; Pen, J J; Heirman, C; Breckpot, K; De Grève, J

    2015-03-26

    The signal transducer and activator of transcription 3 (STAT3) can be activated by the tyrosine kinase domain of the chimeric protein nucleophosmin/anaplastic lymphoma kinase (NPM/ALK), and has a pivotal role in mediating NPM/ALK-related malignant cell transformation. Although the role of STAT3 and wild-type NPM in oncogenesis has been extensively investigated, the relationship between both molecules in cancer remains poorly understood. In the present study, we first demonstrate that STAT3 phosphorylation at tyrosine 705 is accompanied by a concomitant increase in the expression level of NPM. Nuclear co-translocation of phosphorylated STAT3 with NPM can be triggered by interferon-alpha (IFN-α) stimulation of Jurkat cells and phosphorylated STAT3 co-localizes with NPM in cancer cells showing constitutive STAT3 activation. We further demonstrate that STAT3 phosphorylation can transcriptionally mediate NPM upregulation in IFN-α-stimulated Jurkat cells and is responsible for maintaining its expression in cancer cells showing constitutive STAT3 activation. Inhibition of STAT3 phosphorylation or knockdown of NPM expression abrogates their simultaneous transnuclear movements. Finally, we found evidence for a physical interaction between NPM and STAT3 in conditions of STAT3 activation. In conclusion, NPM is a downstream effector of the STAT3 signaling, and can facilitate the nuclear entry of phosphorylated STAT3. These observations might open novel opportunities for targeting the STAT3 pathway in cancer.

  12. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    Science.gov (United States)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  13. In vivo analysis of Yorkie phosphorylation sites.

    Science.gov (United States)

    Oh, H; Irvine, K D

    2009-04-30

    The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.

  14. Consistent wind Facilitates Vection

    Directory of Open Access Journals (Sweden)

    Masaki Ogawa

    2011-10-01

    Full Text Available We examined whether a consistent haptic cue suggesting forward self-motion facilitated vection. We used a fan with no blades (Dyson, AM01 providing a wind of constant strength and direction (wind speed was 6.37 m/s to the subjects' faces with the visual stimuli visible through the fan. We used an optic flow of expansion or contraction created by positioning 16,000 dots at random inside a simulated cube (length 20 m, and moving the observer's viewpoint to simulate forward or backward self-motion of 16 m/s. we tested three conditions for fan operation, which were normal operation, normal operation with the fan reversed (ie, no wind, and no operation (no wind and no sound. Vection was facilitated by the wind (shorter latency, longer duration and larger magnitude values with the expansion stimuli. The fan noise did not facilitate vection. The wind neither facilitated nor inhibited vection with the contraction stimuli, perhaps because a headwind is not consistent with backward self-motion. We speculate that the consistency between multi modalities is a key factor in facilitating vection.

  15. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  16. Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

    Directory of Open Access Journals (Sweden)

    Ward Michael

    2009-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

  17. Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.

    LENUS (Irish Health Repository)

    Stephan, Holger

    2009-10-01

    The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.

  18. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  19. Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice.

    Directory of Open Access Journals (Sweden)

    Joeli Marrero

    2013-01-01

    Full Text Available Mycobacterium tuberculosis (Mtb is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. (13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

  20. Tyrosine Phosphorylation Pattern in Sperm Proteins Isolated from Normospermic and Teratospermic Men

    Science.gov (United States)

    Jabbari, Sepideh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mahdi; Ebrahim Habibi, Azadeh; Amirjanati, Naser; Lakpour, Niknam; Asgharpour, Lima; Ardekani, Ali M.

    2009-01-01

    tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates. PMID:23926467

  1. Tyrosyl phosphorylation toggles a Runx1 switch

    OpenAIRE

    Benjamin G. Neel; Speck, Nancy A.

    2012-01-01

    The Runx1 transcription factor is post-translationally modified by seryl/threonyl phosphorylation, acetylation, and methylation that control its interactions with transcription factor partners and epigenetic coregulators. In this Perspective, the study by Huang et al. (in this issue), which describes how the regulation of Runx1 tyrosyl phosphorylation by Src family kinases and the Shp2 phosphatase toggle Runx1's interactions between different coregulatory molecules, is discussed.

  2. Facilitation skills for trainers

    Directory of Open Access Journals (Sweden)

    F. Cilliers

    2000-06-01

    Full Text Available This research aims to develop the facilitation skills of trainers. Facilitation is defined form the Person-Centered approach, as providing an opportunity for the trainee to experience personal growth and learning. A facilitation skills workshop was presented to 40 trainers, focussing on enhancing selfactualisation, its intra and inter personal characteristics, and attending and responding behaviour. Measurement with the Personal Orientation Inventory and Carkhuff scales, indicate enhanced cognitive, affective and conative sensitivity and interpersonal skills. A post-interview indicates the trainers experienced empowerment in dealing with the providing of opportunities for growth amongst trainees, in all kinds of training situations. Recommendations are made to enhance facilitation development amongst trainers. Opsomming Hierdie navorsing poog om die fasiliteringsvaardighede van opieiers te ontwikkel. Fasilitering word gedefinieer vanuit die Persoonsgesentreerde benadering as die beskikbaarstelling van 'n geleentheid om persoonlike groei en leer te ervaar. 'n Fasiliteringsvaardighede werkswinkel is aangebied vir 40 opieiers, met die fokus op die stimulering van selfaktualisering, die intra en interpersoonlike kenmerke daarvan, en aandagskenk- en responderings- gedrag. Meting met die Persoonlike Orientasievraelys en die Carkhuff skale, dui op n toename in kognitiewe, affektiewe en konatiewe sensitiwiteit en interpersoonlike vaardighede. n Post-onderhoud dui op die opleier se ervaarde bemagtiging in die beskikbaarstelling van groeigeleenthede vir opleidelinge, in all tipe opleidingsituasies. Aanbevelings word gemaak om die ontwikkeling van fasiliteringsvaardighede by opleiers te verhoog.

  3. From Teaching to Facilitation

    DEFF Research Database (Denmark)

    de Graaff, Erik

    2013-01-01

    A shift from teaching to learning is characteristic of the introduction of Problem Based Learning (PBL) in an existing school. As a consequence the teaching staff has to be trained in skills like facilitating group work and writing cases. Most importantly a change in thinking about teaching...

  4. Facilitation of Adult Development

    Science.gov (United States)

    Boydell, Tom

    2016-01-01

    Taking an autobiographical approach, I tell the story of my experiences facilitating adult development, in a polytechnic and as a management consultant. I relate these to a developmental framework of Modes of Being and Learning that I created and elaborated with colleagues. I connect this picture with a number of related models, theories,…

  5. Facilitation skills for nurses

    Directory of Open Access Journals (Sweden)

    F Cilliers

    2000-09-01

    Full Text Available Using the pcrson-centered approach, facilitation in this study was conceptualised as providing opportunities for personal growth in the patient, and operationalised in a skills workshop for 40 nurses from different hospitals in Gauteng. The first objective was to evaluate the workshop and the second to ascertain its effect on the participant’s experienced performance. A combined quantitative and qualitative research design was used. The quantitative measurement (Personal Orientation Inventory, Carkhuff scales indicated that the workshop stimulated self-actualisation in terms of intrapersonal awareness, and the interpersonal skills of respect, realness, concreteness, empathy, as well as in terms of attending and responding behaviour. The qualitative measurement (a semi-structured interview indicated that the participants were able to empower patients to find their own answers to difficult personal questions. The alternative hypothesis was accepted, namely that this workshop in facilitations skills significantly enhanced the intra- and interpersonal characteristics associated with self-actualisation and the facilitation of growth in patients. The findings highlighted the difference between the two roles of instructor and facilitator, and recommendations to this effect were formulated.

  6. Facilitating leadership team communication

    OpenAIRE

    Hedman, Eerika

    2015-01-01

    The purpose of this study is to understand and describe how to facilitate competent communication in leadership teamwork. Grounded in the premises of social constructionism and informed by such theoretical frameworks as coordinated management of meaning theory (CMM), dialogic organization development (OD), systemic-constructionist leadership, communication competence, and reflexivity, this study seeks to produce further insights into understanding leadership team communicati...

  7. Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.

    Directory of Open Access Journals (Sweden)

    Yongbin Chen

    2011-06-01

    Full Text Available Hedgehog (Hh signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo, but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.

  8. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    Science.gov (United States)

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied.

  9. Fibronectin phosphorylation by ecto-protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru (Meiji Institute of Health Science, Odawara (Japan))

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  10. Protein phosphorylation: Localization in regenerating optic axons

    Energy Technology Data Exchange (ETDEWEB)

    Larrivee, D. (Cornell Univ. Medical College, New York, NY (USA))

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  11. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: suryeonseo@kangwon.ac.kr [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  12. Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation.

    Directory of Open Access Journals (Sweden)

    Abbey P Theiss

    Full Text Available Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR, but shorter ischemic intervals (less than 17 minutes facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

  13. Mindfulness for group facilitation

    DEFF Research Database (Denmark)

    Adriansen, Hanne Kirstine; Krohn, Simon

    2014-01-01

    In this paper, we argue that mindfulness techniques can be used for enhancing the outcome of group performance. The word mindfulness has different connotations in the academic literature. Broadly speaking there is ‘mindfulness without meditation’ or ‘Western’ mindfulness which involves active...... thinking and ‘Eastern’ mindfulness which refers to an open, accepting state of mind, as intended with Buddhist-inspired techniques such as meditation. In this paper, we are interested in the latter type of mindfulness and demonstrate how Eastern mindfulness techniques can be used as a tool for facilitation....... A brief introduction to the physiology and philosophy of Eastern mindfulness constitutes the basis for the arguments of the effect of mindfulness techniques. The use of mindfulness techniques for group facilitation is novel as it changes the focus from individuals’ mindfulness practice...

  14. Facilitating Learning at Conferences

    DEFF Research Database (Denmark)

    Ravn, Ib; Elsborg, Steen

    2011-01-01

    and facilitate a variety of simple learning techniques at thirty one- and two-day conferences of up to 300 participants each. We present ten of these techniques and data evaluating them. We conclude that if conference organizers allocate a fraction of the total conference time to facilitated processes......The typical conference consists of a series of PowerPoint presentations that tend to render participants passive. Students of learning have long abandoned the transfer model that underlies such one-way communication. We propose an al-ternative theory of conferences that sees them as a forum...... for learning, mutual inspiration and human flourishing. We offer five design principles that specify how conferences may engage participants more and hence increase their learning. In the research-and-development effort reported here, our team collaborated with conference organizers in Denmark to introduce...

  15. Program Facilitates Distributed Computing

    Science.gov (United States)

    Hui, Joseph

    1993-01-01

    KNET computer program facilitates distribution of computing between UNIX-compatible local host computer and remote host computer, which may or may not be UNIX-compatible. Capable of automatic remote log-in. User communicates interactively with remote host computer. Data output from remote host computer directed to local screen, to local file, and/or to local process. Conversely, data input from keyboard, local file, or local process directed to remote host computer. Written in ANSI standard C language.

  16. Facilitating Knowledge Sharing

    OpenAIRE

    Holdt Christensen, Peter

    2005-01-01

    Abstract This paper argues that knowledge sharing can be conceptualized as different situations of exchange in which individuals relate to each other in different ways, involving different rules, norms and traditions of reciprocity regulating the exchange. The main challenge for facilitating knowledge sharing is to ensure that the exchange is seen as equitable for the parties involved, and by viewing the problems of knowledge sharing as motivational problems situated in different organization...

  17. Synthesis and Characterization of a Novel Soluble Diethoxy Phosphoryl Chitosan%Synthesis and Characterization of a Novel Soluble Diethoxy Phosphoryl Chitosan

    Institute of Scientific and Technical Information of China (English)

    Ma, Li; Li, Kerang; Li, Limin; Liu, Pu

    2012-01-01

    A simple and efficient method for the preparation of a novel soluble chitosan derivative, diethoxy phosphoryl chitosan (PH-chitosan), has been developed. Ph-chitosan was characterized by elemental analysis, FT-IR, NMR, ICP, XRD, TG and SEM, respectively. The chemical identity of PH-chitosan was determined by FT-IR and confirmed by NMR, and those results unequivocally demonstrated that diethoxy phosphoryl groups were grafted onto the amino and hydroxyl groups of chitosan. The results of XRD indicated that the crystalline structure of chitosan was destroyed due to the incorporation of diethoxy phosphoryl group resulting in loss of hydrogen bond. The analysis of TG demonstrated that PH-chitosan was less thermal stable than chitosan. This simple synthetic method provided a new and available approach to prepare a soluble high molecule weight chitosan derivative.

  18. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation.

    Science.gov (United States)

    Karim, Christine B; Espinoza-Fonseca, L Michel; James, Zachary M; Hanse, Eric A; Gaynes, Jeffrey S; Thomas, David D; Kelekar, Ameeta

    2015-09-28

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  19. Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation.

    Science.gov (United States)

    Aguirre, Jacob D; Dunkerley, Karen M; Mercier, Pascal; Shaw, Gary S

    2017-01-10

    Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential.

  20. Phosphorylation of Cdc5 regulates its accumulation

    Directory of Open Access Journals (Sweden)

    Simpson-Lavy Kobi J

    2011-12-01

    Full Text Available Abstract Background Cdc5 (polo kinase/Plk1 is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28 phosphorylation site present. Findings Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans and drastically reduces Clb2 levels. Conclusions Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1 caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

  1. Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions.

    Science.gov (United States)

    Woyda-Ploszczyca, Andrzej; Jarmuszkiewicz, Wieslawa

    2013-05-01

    The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μM), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

  2. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

    Directory of Open Access Journals (Sweden)

    Jun Sumaoka

    2016-01-01

    Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

  3. Apoptotic phosphorylation of histone H3 on Ser-10 by protein kinase Cδ.

    Directory of Open Access Journals (Sweden)

    Choon-Ho Park

    Full Text Available Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new 'apoptotic histone code' mediated by PKCδ.

  4. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  5. Insulin stimulation regulates AS160 and TBC1D1 phosphorylation sites in human skeletal muscle

    DEFF Research Database (Denmark)

    Middelbeek, R J W; Chambers, M A; Tantiwong, P

    2013-01-01

    Individuals with obesity and type 2 diabetes (T2D) are typically insulin resistant, exhibiting impaired skeletal muscle glucose uptake. Animal and cell culture experiments have shown that site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 and TBC1D1 is critical for GLUT4 tr...... translocation facilitating glucose uptake, but their regulation in human skeletal muscle is not well understood....

  6. Facilitative root interactions in intercrops

    DEFF Research Database (Denmark)

    Hauggaard-Nielsen, H.; Jensen, E.S.

    2005-01-01

    Facilitation takes place when plants ameliorate the environment of their neighbours, and increase their growth and survival. Facilitation occurs in natural ecosystems as well as in agroecosystems. We discuss examples of facilitative root interactions in intercropped agroecosystems; including...... intensified cropping systems using chemical and mechanical inputs also show that facilitative interactions definitely can be of significance. It is concluded that a better understanding of the mechanisms behind facilitative interactions may allow us to benefit more from these phenomena in agriculture...

  7. Facilitation as a teaching strategy : experiences of facilitators

    Directory of Open Access Journals (Sweden)

    E Lekalakala-Mokgele

    2006-09-01

    Full Text Available Changes in nursing education involve the move from traditional teaching approaches that are teacher-centred to facilitation, a student centred approach. The studentcentred approach is based on a philosophy of teaching and learning that puts the learner on centre-stage. The aim of this study was to identify the challenges of facilitators of learning using facilitation as a teaching method and recommend strategies for their (facilitators development and support. A qualitative, explorative and contextual design was used. Four (4 universities in South Africa which utilize facilitation as a teaching/ learning process were identified and the facilitators were selected to be the sample of the study. The main question posed during in-depth group interviews was: How do you experience facilitation as a teaching/learning method?. Facilitators indicated different experiences and emotions when they first had to facilitate learning. All of them indicated that it was difficult to facilitate at the beginning as they were trained to lecture and that no format for facilitation was available. They experienced frustrations and anxieties as a result. The lack of knowledge of facilitation instilled fear in them. However they indicated that facilitation had many benefits for them and for the students. Amongst the ones mentioned were personal and professional growth. Challenges mentioned were the fear that they waste time and that they do not cover the content. It is therefore important that facilitation be included in the training of nurse educators.

  8. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation

    Science.gov (United States)

    Rigatti, Marc; Le, Andrew V.; Gerber, Claire; Moraru, Ion I.; Dodge-Kafka, Kimberly L.

    2016-01-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations. PMID:26027516

  9. Essence: Facilitating Software Innovation

    DEFF Research Database (Denmark)

    Aaen, Ivan

    2008-01-01

      This paper suggests ways to facilitate creativity and innovation in software development. The paper applies four perspectives – Product, Project, Process, and People –to identify an outlook for software innovation. The paper then describes a new facility–Software Innovation Research Lab (SIRL......) – and a new method concept for software innovation – Essence – based on views, modes, and team roles. Finally, the paper reports from an early experiment using SIRL and Essence and identifies further research....

  10. TBK1 controls autophagosomal engulfment of polyubiquitinated mitochondria through p62/SQSTM1 phosphorylation.

    Science.gov (United States)

    Matsumoto, Gen; Shimogori, Tomomi; Hattori, Nobutaka; Nukina, Nobuyuki

    2015-08-01

    Selective autophagy adaptor proteins, including p62/SQSTM1, play pivotal roles in the targeted degradation of ubiquitinated proteins or organelles through the autophagy-lysosome system. However, how autophagy adaptors promote the autophagosomal engulfment of selected substrates is poorly understood. Here, we show that p62 phosphorylation at S403 is required for the efficient autophagosomal engulfment of polyubiquitinated mitochondria during Parkin-dependent mitophagy. p62 is able to interact with Parkin-recruited mitochondria without S403 phosphorylation under mitophagy-inducing conditions, but those mitochondria are not enclosed by autophagosomes. Intriguingly, the S403 phosphorylation occurs only in the early period of mitochondrial depolarization. Optineurin and TANK-binding kinase 1 (TBK1) are transiently recruited to the polyubiquitinated mitochondria, and the activated TBK1 phosphorylates p62 at S403. TBK1 inhibitor, BX795, prevents the p62-mediated autophagosomal engulfment of Parkin-recruited mitochondria. Our results suggest that TBK1-mediated S403 phosphorylation regulates the efficient autophagosomal engulfment of ubiquitinated mitochondria as an immediate response to the mitochondrial depolarization.

  11. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks.

    Science.gov (United States)

    Van Meter, Michael; Simon, Matthew; Tombline, Gregory; May, Alfred; Morello, Timothy D; Hubbard, Basil P; Bredbenner, Katie; Park, Rosa; Sinclair, David A; Bohr, Vilhelm A; Gorbunova, Vera; Seluanov, Andrei

    2016-09-06

    The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  12. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  13. Accurate determination of peptide phosphorylation stoichiometry via automated diagonal capillary electrophoresis coupled with mass spectrometry: proof of principle.

    Science.gov (United States)

    Mou, Si; Sun, Liangliang; Dovichi, Norman J

    2013-11-19

    While reversible protein phosphorylation plays an important role in many cellular processes, simple and reliable measurement of the stoichiometry of phosphorylation can be challenging. This measurement is confounded by differences in the ionization efficiency of phosphorylated and unphosphorylated sites during analysis by mass spectrometry. Here, we demonstrate diagonal capillary electrophoresis-mass spectrometry for the accurate determination of this stoichiometry. Diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized alkaline phosphatase microreactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. The first dimension is used to separate a mixture of the phosphorylated and unphosphorylated forms of a peptide. Fractions are parked in the reactor where they undergo complete dephosphorylation. The products are then periodically transferred to the second capillary and analyzed by mass spectrometry (MS). Because the phosphorylated and unphosphorylated forms differ in charge, they are well resolved in the first dimension separation. Because the unphosphorylated and dephosphorylated peptides are identical, there is no bias in ionization efficiency, and phosphorylation stoichiometry can be determined by the ratio of the signal of the two forms. A calibration curve was generated from mixtures of a phosphorylated standard peptide and its unphosphorylated form, prepared in a bovine serum albumin tryptic digest. This proof of principle experiment demonstrated a linear response across nearly 2 orders of magnitude in stoichiometry.

  14. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

    Science.gov (United States)

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

  15. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.

    Science.gov (United States)

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J; Eckford, Paul D W; Molinski, Steven; Wallace, B A; Bear, Christine E

    2017-02-03

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.

  16. Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production.

    Science.gov (United States)

    Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen; Lübeck, Mette

    2014-10-01

    Co-cultivation of fungi may be an excellent system for on-site production of cellulolytic enzymes in a single bioreactor. Enzyme supernatants from mixed cultures of Trichoderma reesei RutC30, with either the novel Aspergillus saccharolyticus AP, Aspergillus carbonarius ITEM 5010 or Aspergillus niger CBS 554.65 cultivated in solid-state fermentation were tested for avicelase, FPase, endoglucanase and beta-glucosidase activity as well as in hydrolysis of pretreated wheat straw. Around 30% more avicelase activity was produced in co-cultivation of T. reesei and A. saccharolyticus than in T. reesei monoculture, suggesting synergistic interaction between those fungi. Fermentation broths of mixed cultures of T. reesei with different Aspergillus strains resulted in approx. 80% efficiency of hydrolysis which was comparable to results obtained using blended supernatants from parallel monocultures. This indicates that co-cultivation of T. reesei with A. saccharolyticus or A. carbonarius could be a competitive alternative for monoculture enzyme production and a cheaper alternative to commercial enzymes.

  17. Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system.

    Science.gov (United States)

    Tudor, Catalina O; Ross, Karen E; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

    2015-01-01

    Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein-protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation-dependent interaction

  18. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death.

    Science.gov (United States)

    Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B

    2007-03-28

    Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.

  19. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...

  20. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

    2013-01-01

    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosp...

  1. Phosphorylation sites within Ebola virus nucleoprotein

    Institute of Scientific and Technical Information of China (English)

    Sora; Yasri; Viroj; Wiwanitkit

    2015-01-01

    To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

  2. Ion channels, phosphorylation and mammalian sperm capacitation.

    Science.gov (United States)

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-05-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  3. Ion channels, phosphorylation and mammalian sperm capacitation

    Institute of Scientific and Technical Information of China (English)

    Pablo E Visconti; Dario Krapf; José Luis de la Vega-Beltrán; Juan José Acevedo; Alberto Darszon

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  4. Phosphorylated α-synuclein in Parkinson's disease

    DEFF Research Database (Denmark)

    Stewart, Tessandra; Sossi, Vesna; Aasly, Jan O;

    2015-01-01

    INTRODUCTION: α-Synuclein (α-syn) is a key protein in Parkinson's disease (PD), and one of its phosphorylated forms, pS129, is higher in PD patients than healthy controls. However, few studies have examined its levels in longitudinally collected cerebrospinal fluid (CSF) or in preclinical cases. ...

  5. Transferases for alkylation, glycosylation and phosphorylation

    NARCIS (Netherlands)

    Auriol, D.; ter Halle, R.; Lefèvre, F.; Visser, D.F.; Gordon, G.E.R.; Bode, M.L.; Mathiba, K.; Brady, D.; De Winter, K.; Desmet, T.; Cerdobbel, A.; Soetaert, W.; van Herk, T.; Hartog, A.F.; Wever, R.; Brzezińska-rodak, M.; Klimek-Ochab, M.; Żymańczyk-Duda, E.; Mukherjee, J.; Gupta, M.N.; Yin, W.B.; Li, S.M.; Gruber-Khadjawi, M.; Whittall, J.; Sutton, P.W.

    2012-01-01

    This chapter contains sections titled: Industrial Production of Caffeic Acid-α-D-O-Glucoside Enzymatic Synthesis of 5-Methyluridine by Transglycosylation of Guanosine and Thymine Preparation and Use of Sucrose Phosphorylase as Cross-Linked Enzyme Aggregate (CLEA) Enzymatic Synthesis of Phosphorylate

  6. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  7. Expert and novice facilitated modelling

    DEFF Research Database (Denmark)

    Tavella, Elena; Papadopoulos, Thanos

    2015-01-01

    This paper provides an empirical study based on action research in which expert and novice facilitators in facilitated modelling workshops are compared. There is limited empirical research analysing the differences between expert and novice facilitators. Aiming to address this gap we study...

  8. Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.

    Science.gov (United States)

    Makrantoni, Vasso; Corbishley, Stephen J; Rachidi, Najma; Morrice, Nicholas A; Robinson, David A; Stark, Michael J R

    2014-01-01

    The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

  9. Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Vasso Makrantoni

    Full Text Available The chromosomal passenger complex (CPC is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15 and two additional proteins (Survivin/Bir1 and Borealin/Nbl1. Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A or to acidic residues to mimic constitutive phosphorylation (sli15-20D. Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

  10. Integrating phosphorylation network with transcriptional network reveals novel functional relationships.

    Directory of Open Access Journals (Sweden)

    Lin Wang

    Full Text Available Phosphorylation and transcriptional regulation events are critical for cells to transmit and respond to signals. In spite of its importance, systems-level strategies that couple these two networks have yet to be presented. Here we introduce a novel approach that integrates the physical and functional aspects of phosphorylation network together with the transcription network in S.cerevisiae, and demonstrate that different network motifs are involved in these networks, which should be considered in interpreting and integrating large scale datasets. Based on this understanding, we introduce a HeRS score (hetero-regulatory similarity score to systematically characterize the functional relevance of kinase/phosphatase involvement with transcription factor, and present an algorithm that predicts hetero-regulatory modules. When extended to signaling network, this approach confirmed the structure and cross talk of MAPK pathways, inferred a novel functional transcription factor Sok2 in high osmolarity glycerol pathway, and explained the mechanism of reduced mating efficiency upon Fus3 deletion. This strategy is applicable to other organisms as large-scale datasets become available, providing a means to identify the functional relationships between kinases/phosphatases and transcription factors.

  11. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins.

    Science.gov (United States)

    Matsumoto, Gen; Wada, Koji; Okuno, Misako; Kurosawa, Masaru; Nukina, Nobuyuki

    2011-10-21

    Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 at S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.

  12. Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

    Directory of Open Access Journals (Sweden)

    Jeffrey M. Dale

    2012-01-01

    Full Text Available Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs. NFs are type IV intermediate filaments (IFs that can be composed of four subunits, neurofilament heavy (NF-H, neurofilament medium (NF-M, neurofilament light (NF-L, and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

  13. Program Facilitates CMMI Appraisals

    Science.gov (United States)

    Sweetser, Wesley

    2005-01-01

    A computer program has been written to facilitate appraisals according to the methodology of Capability Maturity Model Integration (CMMI). [CMMI is a government/industry standard, maintained by the Software Engineering Institute at Carnegie Mellon University, for objectively assessing the engineering capability and maturity of an organization (especially, an organization that produces software)]. The program assists in preparation for a CMMI appraisal by providing drop-down lists suggesting required artifacts or evidence. It identifies process areas for which similar evidence is required and includes a copy feature that reduces or eliminates repetitive data entry. It generates reports to show the entire framework for reference, the appraisal artifacts to determine readiness for an appraisal, and lists of interviewees and questions to ask them during the appraisal. During an appraisal, the program provides screens for entering observations and ratings, and reviewing evidence provided thus far. Findings concerning strengths and weaknesses can be exported for use in a report or a graphical presentation. The program generates a chart showing capability level ratings of the organization. A context-sensitive Windows help system enables a novice to use the program and learn about the CMMI appraisal process.

  14. Facilitating post traumatic growth

    Directory of Open Access Journals (Sweden)

    Cox Helen

    2004-07-01

    Full Text Available Abstract Background Whilst negative responses to traumatic injury have been well documented in the literature, there is a small but growing body of work that identifies posttraumatic growth as a salient feature of this experience. We contribute to this discourse by reporting on the experiences of 13 individuals who were traumatically injured, had undergone extensive rehabilitation and were discharged from formal care. All participants were injured through involvement in a motor vehicle accident, with the exception of one, who was injured through falling off the roof of a house. Methods In this qualitative study, we used an audio-taped in-depth interview with each participant as the means of data collection. Interviews were transcribed verbatim and analysed thematically to determine the participants' unique perspectives on the experience of recovery from traumatic injury. In reporting the findings, all participants' were given a pseudonym to assure their anonymity. Results Most participants indicated that their involvement in a traumatic occurrence was a springboard for growth that enabled them to develop new perspectives on life and living. Conclusion There are a number of contributions that health providers may make to the recovery of individuals who have been traumatically injured to assist them to develop new views of vulnerability and strength, make changes in relationships, and facilitate philosophical, physical and spiritual growth.

  15. Facilitating post traumatic growth

    Science.gov (United States)

    Turner, de Sales; Cox, Helen

    2004-01-01

    Background Whilst negative responses to traumatic injury have been well documented in the literature, there is a small but growing body of work that identifies posttraumatic growth as a salient feature of this experience. We contribute to this discourse by reporting on the experiences of 13 individuals who were traumatically injured, had undergone extensive rehabilitation and were discharged from formal care. All participants were injured through involvement in a motor vehicle accident, with the exception of one, who was injured through falling off the roof of a house. Methods In this qualitative study, we used an audio-taped in-depth interview with each participant as the means of data collection. Interviews were transcribed verbatim and analysed thematically to determine the participants' unique perspectives on the experience of recovery from traumatic injury. In reporting the findings, all participants' were given a pseudonym to assure their anonymity. Results Most participants indicated that their involvement in a traumatic occurrence was a springboard for growth that enabled them to develop new perspectives on life and living. Conclusion There are a number of contributions that health providers may make to the recovery of individuals who have been traumatically injured to assist them to develop new views of vulnerability and strength, make changes in relationships, and facilitate philosophical, physical and spiritual growth. PMID:15248894

  16. In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

    Institute of Scientific and Technical Information of China (English)

    Stuart Sullivan; Catriona E. Thomson; Douglas J.Lamont; Matthew A. Jones; John M.Christie

    2008-01-01

    Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2,located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser58, Ser85, Ser350, and Ser410), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

  17. BRCA1 and its phosphorylation involved in caffeine-inhibitable event upstream of G2 checkpoint

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Caffeine,which specifically inhibits ATM/ATR kinases,efficiently abrogates the ionizing radiation(IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR.Mechanisms for the effect of caffeine remain to be elucidated.As a target of ATM/ATR kinases,BRCA1 becomes activated and phosphorylated in response to IR.Thus,in this work,we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process.For these purposes,the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine.The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524,which was followed by an override of G2 arrest by caffeine.In addition,the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine.These data suggest that BRCA1 may be a potential target of caffeine.BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.

  18. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

    Science.gov (United States)

    Matsushita, Masaki; Kitoh, Hiroshi; Ohkawara, Bisei; Mishima, Kenichi; Kaneko, Hiroshi; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2013-01-01

    Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

  19. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

    Directory of Open Access Journals (Sweden)

    Masaki Matsushita

    Full Text Available Achondroplasia (ACH is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8 cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

  20. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin.

    Science.gov (United States)

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey

    2014-12-01

    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  1. Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar; Chattopadhyay, Samit, E-mail: samit@nccs.res.in

    2010-01-08

    CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thus releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.

  2. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  3. MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

    Directory of Open Access Journals (Sweden)

    Arun Gupta

    2014-07-01

    Full Text Available Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ or homologous recombination (HR. Here, we report that double-strand breaks (DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase phosphorylation (p-T392-MOF and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  4. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  5. BAD Phosphorylation: A Novel Link between Apoptosis and Cancer

    OpenAIRE

    Polzien, Lisa

    2011-01-01

    BAD (Bcl-2 antagonist of cell death, Bcl-2 associated death promoter) is a pro-apoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD (mBAD), little data are available with respect to phosphorylation of human BAD (hBAD) protein. In this work, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating se...

  6. Flux control through protein phosphorylation in yeast

    DEFF Research Database (Denmark)

    Chen, Yu; Nielsen, Jens

    2016-01-01

    describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies......Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...

  7. Phosphorylated 5-ethynyl-2'-deoxyuridine for advanced DNA labeling.

    Science.gov (United States)

    Seo, Siyoong; Onizuka, Kazumitsu; Nishioka, Chieko; Takahashi, Eiki; Tsuneda, Satoshi; Abe, Hiroshi; Ito, Yoshihiro

    2015-04-21

    The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.

  8. Phosphorylation sites within Ebola virus nucleoprotein

    Directory of Open Access Journals (Sweden)

    Sora Yasri

    2015-07-01

    Full Text Available To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

  9. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  10. Solid polymer electrolyte from phosphorylated chitosan

    Energy Technology Data Exchange (ETDEWEB)

    Fauzi, Iqbal, E-mail: arcana@chem.itb.ac.id; Arcana, I Made, E-mail: arcana@chem.itb.ac.id [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)

    2014-03-24

    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  11. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F;

    2008-01-01

    Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently...... sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info)....

  12. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV

    2010-01-01

    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  13. Phosphorylation of erythrocyte membrane liberates calcium

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, V.P.S.; Brockerhoff, H.

    1986-05-01

    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca/sup 2 +/ electrode and /sup 45/Ca. This effect could not be observed in the presence of p/sup -/ chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP/sub 2/). These results support the proposal that an inositol shuttle, PI in equilibrium PIP in equilibrium PIP/sub 2/, operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released.

  14. Phosphorylation of Astrin Regulates Its Kinetochore Function.

    Science.gov (United States)

    Chung, Hee Jin; Park, Ji Eun; Lee, Nam Soo; Kim, Hongtae; Jang, Chang-Young

    2016-08-19

    The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

  15. Control mechanisms in mitochondrial oxidative phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jana Hroudová; Zdeněk Fi(s)ar

    2013-01-01

    Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

  16. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah

    2000-12-01

    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  17. Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1

    Science.gov (United States)

    Suriyanarayanan, Tanujaa; Periasamy, Saravanan; Lin, Miao-Hsia; Ishihama, Yasushi; Swarup, Sanjay

    2016-01-01

    Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal. PMID:27701473

  18. A phosphorylation cascade controls the degradation of active SREBP1.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Ericsson, Johan

    2009-02-27

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

  19. A strategy to quantitate global phosphorylation of bone matrix proteins.

    Science.gov (United States)

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  20. Multistep phosphorylation systems: tunable components of biological signaling circuits.

    Science.gov (United States)

    Valk, Evin; Venta, Rainis; Ord, Mihkel; Faustova, Ilona; Kõivomägi, Mardo; Loog, Mart

    2014-11-05

    Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications.

  1. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria.

    Science.gov (United States)

    Kavanagh, N I; Ainscow, E K; Brand, M D

    2000-02-24

    Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

  2. Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117.

    Science.gov (United States)

    Fu, Guolong; Wei, Youheng; Wang, Xiaoli; Yu, Long

    2016-06-01

    We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117.

  3. Facilitated Communication in Mainstream Schools.

    Science.gov (United States)

    Remington-Gurney, Jane; Crossley, Rosemary

    Facilitated communication is described as a method of training communication partners or facilitators to provide physical assistance to communication aid users, to help them overcome physical and emotional problems in using their aids. In Melbourne (Victoria, Australia), the DEAL (Dignity, Education and Language) Centre has identified 96 people…

  4. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    Science.gov (United States)

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  5. Genetic Manipulation of Neurofilament Protein Phosphorylation.

    Science.gov (United States)

    Jones, Maria R; Villalón, Eric; Garcia, Michael L

    2016-01-01

    Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

  6. PPM1A regulates antiviral signaling by antagonizing TBK1-mediated STING phosphorylation and aggregation.

    Directory of Open Access Journals (Sweden)

    Zexing Li

    2015-03-01

    Full Text Available Stimulator of interferon genes (STING, also known as MITA and ERIS is critical in protecting the host against DNA pathogen invasion. However, the molecular mechanism underlying the regulation of STING remains unclear. Here, we show that PPM1A negatively regulates antiviral signaling by targeting STING in its phosphatase activity-dependent manner, and in a line with this, PPM1A catalytically dephosphorylates STING and TBK1 in vitro. Importantly, we provide evidence that whereas TBK1 promotes STING aggregation in a phosphorylation-dependent manner, PPM1A antagonizes STING aggregation by dephosphorylating both STING and TBK1, emphasizing that phosphorylation is crucial for the efficient activation of STING. Our findings demonstrate a novel regulatory circuit in which STING and TBK1 reciprocally regulate each other to enable efficient antiviral signaling activation, and PPM1A dephosphorylates STING and TBK1, thereby balancing this antiviral signal transduction.

  7. Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Yan-Chu Chen

    2017-07-01

    Full Text Available Galectin-3, a member of the galectin protein family, has been found to regulate cell proliferation, inhibit apoptosis and promote inflammatory responses. Galectin-3 is also expressed in the adult rat hippocampus, but its role in learning and memory function is not known. Here, we found that contextual fear-conditioning training, spatial training or injection of NMDA into the rat CA1 area each dramatically decreased the level of endogenous galectin-3 expression. Overexpression of galectin-3 impaired fear memory, whereas galectin-3 knockout (KO enhanced fear retention, spatial memory and hippocampal long-term potentiation. Galectin-3 was further found to associate with integrin α3, an association that was decreased after fear-conditioning training. Transfection of the rat CA1 area with small interfering RNA against galectin-3 facilitated fear memory and increased phosphorylated focal adhesion kinase (FAK levels, effects that were blocked by co-transfection of the FAK phosphorylation-defective mutant Flag-FAKY397F. Notably, levels of serine-phosphorylated galectin-3 were decreased by fear conditioning training. In addition, blockade of galectin-3 phosphorylation at Ser-6 facilitated fear memory, whereas constitutive activation of galectin-3 at Ser-6 impaired fear memory. Interestingly galectin-1 plays a role in fear-memory formation similar to that of galectin-3. Collectively, our data provide the first demonstration that galectin-3 is a novel negative regulator of memory formation that exerts its effects through both extracellular and intracellular mechanisms.

  8. Phenobarbital Meets Phosphorylation of Nuclear Receptors.

    Science.gov (United States)

    Negishi, Masahiko

    2017-05-01

    Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

  9. Modelling the Krebs cycle and oxidative phosphorylation.

    Science.gov (United States)

    Korla, Kalyani; Mitra, Chanchal K

    2014-01-01

    The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

  10. Phosphorylation regulates coilin activity and RNA association

    Directory of Open Access Journals (Sweden)

    Hanna J. Broome

    2013-02-01

    The Cajal body (CB is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.

  11. Learning to Facilitate (Online) Meetings

    DEFF Research Database (Denmark)

    Reimann, Peter; Bull, Susan; Vatrapu, Ravi

    2013-01-01

    We describe an approach to teaching collaboration skills directly by building on competences for meeting facilitation. (Online) meetings provide a rich arena to practice collaboration since they can serve multiple purposes: learning, problem solving, decision making, idea generation and advancement......, etc.. We argue that facilitating meetings is a competence worth developing in students and describe the main knowledge and skill components that pertain to this competence. We then describe some implemented software tools that can be used in schools and colleges to provide opportunities for practicing...... and developing group facilitation skills....

  12. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Guo [Los Alamos National Laboratory; Mcmahon, Benjamin H [Los Alamos National Laboratory; Tung, Chang - Shung [Los Alamos National Laboratory

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  13. Phosphorylation of chromosome core components may serve as axis marks for the status of chromosomal events during mammalian meiosis.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Fukuda

    2012-02-01

    Full Text Available Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.

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

    Science.gov (United States)

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

    2014-03-28

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

  15. Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation.

    Science.gov (United States)

    Temmei, Yusuke; Uchida, Shinichi; Hoshino, Daisuke; Kanzawa, Nobuyuki; Kuwahara, Michio; Sasaki, Sei; Tsuchiya, Takahide

    2005-08-15

    cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

  16. Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant

    Science.gov (United States)

    Daugherty, Ashley B.; Yang, Zunyi; Shaw, Ryan; Dong, Mengxing; Lutz, Stefan; Benner, Steven A.

    2017-01-01

    One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1’-b-d-2’-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo. PMID:28323896

  17. Kinetic analyses of phosphorylated and non-phosphorylated eIFiso4E binding to mRNA cap analogues.

    Science.gov (United States)

    Khan, Mateen A; Goss, Dixie J

    2017-08-08

    Phosphorylation of eukaryotic initiation factors was previously shown to interact with m(7)G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m(7)G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m(7)-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22°C. Phosphorylated and non-phosphorylated eIFiso4E-m(7)G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m(7)-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m(7)-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m(7)-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    Science.gov (United States)

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  19. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

    Science.gov (United States)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

    2016-09-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

  20. Phosphorylation modifies the molecular stability of β-amyloid deposits

    Science.gov (United States)

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

    2016-04-01

    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain.

  1. Control of Collagen Triple Helix Stability by Phosphorylation.

    Science.gov (United States)

    Acevedo-Jake, Amanda M; Ngo, Daniel H; Hartgerink, Jeffrey D

    2017-03-10

    The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix.

  2. Chemical Approaches to Studying Labile Amino Acid Phosphorylation.

    Science.gov (United States)

    Marmelstein, Alan M; Moreno, Javier; Fiedler, Dorothea

    2017-04-01

    Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

  3. Den gode facilitator af refleksionsarbejde

    DEFF Research Database (Denmark)

    Jørgensen, Pia

    2009-01-01

    tværfaglig lektorgruppe fra social og sundhedssektoren.’Learning by doing’, selvevaluering og sparring følger herefter som bud på, hvordan man kan leve op til de tilsyneladende ret utopiske krav til en god facilitator. At kunne skabe det tillidsfulde refleksionsrum og at kunne stille gode...... præsenteres i det følgende afsnit, og forfatteren argumenterer for begrebet facilitator af refleksionsarbejde. Herefter udfoldes rollen som facilitator ifølge Ghay og Lillyman. De har fokus på positive praksisoplevelser og tillidsfulde relationer. Gillie Boltons teoretiske og praktiske referenceramme...... for facilitatorrollen beskrives herefter. Bolton beskriver refleksionsarbejde som en fysisk (ikke ren kognitiv), passioneret (ikke ren intellektuel) kontekstbunden kunstnerisk proces, som kræver flair, stil og intuition. I de følgende afsnit beskrives den gode facilitator af refleksionsarbejde detaljeret af en...

  4. Facilitating Value Co-Creation

    DEFF Research Database (Denmark)

    Veith, Anne; Assaf, Albert; Josiassen, Alexander

    2013-01-01

    ) introduced a new dominant logic in the marketing literature, the Service-Dominant Logic (S-D Logic), in which service, interactions, and enhanced experiences help create value, and this potential for value is what attracts consumers. Therefore organizations must be customer-centric in order to facilitate...... unique, positive experiences. As the name indicates, both organizations and consumers (should) obtain value when co-creating, which is why both parties are willing to increase their degree of involvement, e.g. spending more resources, sharing tacit knowledge, etc., because a high degree of involvement....... Through an exploratory qualitative study, 9 facilitators for B2C value co-creation were uncovered. The study was set in the creative industries. The 9 facilitators are a combination of the main facilitators found in the literature review and the ones found through the empirical research. The 9...

  5. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Science.gov (United States)

    Kondapalli, Chandana; Kazlauskaite, Agne; Zhang, Ning; Woodroof, Helen I.; Campbell, David G.; Gourlay, Robert; Burchell, Lynn; Walden, Helen; Macartney, Thomas J.; Deak, Maria; Knebel, Axel; Alessi, Dario R.; Muqit, Miratul M. K.

    2012-01-01

    Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD. PMID:22724072

  6. Pin1-mediated Sp1 phosphorylation by CDK1 increases Sp1 stability and decreases its DNA-binding activity during mitosis.

    Science.gov (United States)

    Yang, Hang-Che; Chuang, Jian-Ying; Jeng, Wen-Yih; Liu, Chia-I; Wang, Andrew H-J; Lu, Pei-Jung; Chang, Wen-Chang; Hung, Jan-Jong

    2014-12-16

    We have shown that Sp1 phosphorylation at Thr739 decreases its DNA-binding activity. In this study, we found that phosphorylation of Sp1 at Thr739 alone is necessary, but not sufficient for the inhibition of its DNA-binding activity during mitosis. We demonstrated that Pin1 could be recruited to the Thr739(p)-Pro motif of Sp1 to modulate the interaction between phospho-Sp1 and CDK1, thereby facilitating CDK1-mediated phosphorylation of Sp1 at Ser720, Thr723 and Thr737 during mitosis. Loss of the C-terminal end of Sp1 (amino acids 741-785) significantly increased Sp1 phosphorylation, implying that the C-terminus inhibits CDK1-mediated Sp1 phosphorylation. Binding analysis of Sp1 peptides to Pin1 by isothermal titration calorimetry indicated that Pin1 interacts with Thr739(p)-Sp1 peptide but not with Thr739-Sp1 peptide. X-ray crystallography data showed that the Thr739(p)-Sp1 peptide occupies the active site of Pin1. Increased Sp1 phosphorylation by CDK1 during mitosis not only stabilized Sp1 levels by decreasing interaction with ubiquitin E3-ligase RNF4 but also caused Sp1 to move out of the chromosomes completely by decreasing its DNA-binding activity, thereby facilitating cell cycle progression. Thus, Pin1-mediated conformational changes in the C-terminal region of Sp1 are critical for increased CDK1-mediated Sp1 phosphorylation to facilitate cell cycle progression during mitosis.

  7. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

    Science.gov (United States)

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias

    2010-12-01

    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  8. Synthesis of O-Phosphorylated Oligopeptides Using Phosphoramidite

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Reversible protein phosphorylation is of great importance in the regulation of many cellular processes. Structurally well-defined compounds are needed for the study of the roles of the phospho-proteins in biological processes. In this paper, O-phosphorylated oligopeptides were synthesized using bis-alkyloxy-N,N-dialkylphosphoramidite reacting with the oligopeptide followed by oxidation. Many hydroxyl groups in oligopeptides can be phosphorylated in one step.

  9. Constitutive phosphorylation of Shc proteins in human tumors

    DEFF Research Database (Denmark)

    Pelicci, G; Lanfrancone, L; Salcini, A E

    1995-01-01

    cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins...... activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation....

  10. A New Intermolecular Phosphoryl Transfer between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    SU,Yu-Qian; NIU,Ming-Yu; CAO,Shu-Xia; ZHANG,Jian-Chen; QU,Ling-Bo; LIAO,Xin-Cheng; ZHAO,Yu-Fen

    2004-01-01

    @@ Phosphoryl transfer constitutes one of the most important reactions in functionalized molecules, bioorganic chemistry and biochemistry.[1] The transformations are involved in diverse processes, such as activated state change of phosphorus, DNA/RNA synthesis, energy metabolism and signal transduction. So, phosphoryl transfer reaction which can be performed by either intramolecular or intermolecular phosphorylation and dephosphorylation mechanism has been investigated by many scientists in wide fields.

  11. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M

    2008-01-01

    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  12. Myeloid differentiation factor-2 interacts with Lyn kinase and is tyrosine phosphorylated following lipopolysaccharide-induced activation of the TLR4 signaling pathway.

    Science.gov (United States)

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R; Arditi, Moshe

    2011-10-15

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.

  13. Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

    Science.gov (United States)

    Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

    2013-01-01

    A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

  14. Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

    Directory of Open Access Journals (Sweden)

    Yuming Lu

    Full Text Available A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments based transient expression system (PCR-TES for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

  15. Role of Ser7 phosphorylation of the CTD during transcription of snRNA genes

    Science.gov (United States)

    Egloff, Sylvain

    2012-01-01

    The largest subunit of RNA polymerase (pol) II, Rpb1, contains an unusual carboxyl-terminal domain (CTD) composed of consecutive repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). During transcription, Ser2, Ser5 and Ser7 are subjected to dynamic phosphorylation and dephosphorylation by CTD kinases and phosphatases, creating a characteristic CTD phosphorylation pattern along genes. This CTD “code” allows the coupling of transcription with co-transcriptional RNA processing, through the timely recruitment of the appropriate factors at the right point of the transcription cycle. In mammals, phosphorylation of Ser7 (Ser7P) is detected on all pol II-transcribed genes, but is only essential for expression of a sub-class of genes encoding small nuclear (sn)RNAs. The molecular mechanisms by which Ser7P influences expression of these particular genes are becoming clearer. Here, I discuss our recent findings clarifying how Ser7P facilitates transcription of these genes and 3′end processing of the transcripts, through recruitment of the RPAP2 phosphatase and the snRNA gene-specific Integrator complex. PMID:22858677

  16. Specific threonine-4 phosphorylation and function of RNA polymerase II CTD during M phase progression.

    Science.gov (United States)

    Hintermair, Corinna; Voß, Kirsten; Forné, Ignasi; Heidemann, Martin; Flatley, Andrew; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2016-06-06

    Dynamic phosphorylation of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 heptad-repeats in the C-terminal domain (CTD) of the large subunit coordinates progression of RNA polymerase (Pol) II through the transcription cycle. Here, we describe an M phase-specific form of Pol II phosphorylated at Thr4, but not at Tyr1, Ser2, Ser5, and Ser7 residues. Thr4 phosphorylated Pol II binds to centrosomes and midbody and interacts with the Thr4-specific Polo-like kinase 1. Binding of Pol II to centrosomes does not require the CTD but may involve subunits of the non-canonical R2TP-Prefoldin-like complex, which bind to and co-localize with Pol II at centrosomes. CTD Thr4 mutants, but not Ser2 and Ser5 mutants, display severe mitosis and cytokinesis defects characterized by multipolar spindles and polyploid cells. We conclude that proper M phase progression of cells requires binding of Pol II to centrosomes to facilitate regulation of mitosis and cytokinesis in a CTD Thr4-P dependent manner.

  17. Visual analytics of phosphorylation time-series data on insulin response

    Science.gov (United States)

    Ma, David K. G.; Stolte, Christian; Kaur, Sandeep; Bain, Michael; O'Donoghue, Seán I.

    2013-10-01

    Visual analysis of time-series data on protein phosphorylation presents a particular challenge: bioinformatics tools currently available for visualising 'omics' data in time series have been developed primarily to study gene expression, and cannot easily be adopted to phosphorylation data, where a single protein typically has multiple phosphosites. In this study, we worked with an experimental research group that is applying very recent methods in high-throughput experimental proteomics to study the time course of protein phosphorylation events in human cells in vitro following stimulation by insulin, as part of a broader study of diabetes and obesity. We applied several existing visual analytics approaches with the goal of organising the data to facilitate new insight into underlying molecular processes. We developed a novel layout strategy called 'Minardo' that is loosely based on cell topology and ordered by time and causality. This layout utilises a frame of reference familiar to life scientists and helpful for organising and interpreting time-series data. This strategy proved to be useful, leading to new insights into the insulin response pathway. We are working on generalising the Minardo layout to accommodate similar datasets related to other signalling pathways, which should be straightforward.

  18. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  19. A grammar inference approach for predicting kinase specific phosphorylation sites.

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  20. Metadherin facilitates podocyte apoptosis in diabetic nephropathy

    Science.gov (United States)

    Liu, Wen-Ting; Peng, Fen-Fen; Li, Hong-Yu; Chen, Xiao-Wen; Gong, Wang-Qiu; Chen, Wen-Jing; Chen, Yi-Hua; Li, Pei-Lin; Li, Shu-Ting; Xu, Zhao-Zhong; Long, Hai-Bo

    2016-01-01

    Apoptosis, one of the major causes of podocyte loss, has been reported to have a vital role in diabetic nephropathy (DN) pathogenesis, and understanding the mechanisms underlying the regulation of podocyte apoptosis is crucial. Metadherin (MTDH) is an important oncogene, which is overexpressed in most cancers and responsible for apoptosis, metastasis, and poor patient survival. Here we show that the expression levels of Mtdh and phosphorylated p38 mitogen-activated protein kinase (MAPK) are significantly increased, whereas those of the microRNA-30 family members (miR-30s) are considerably reduced in the glomeruli of DN rat model and in high glucose (HG)-induced conditionally immortalized mouse podocytes (MPC5). These levels are positively correlated with podocyte apoptosis rate. The inhibition of Mtdh expression, using small interfering RNA, but not Mtdh overexpression, was shown to inhibit HG-induced MPC5 apoptosis and p38 MAPK pathway, and Bax and cleaved caspase 3 expression. This was shown to be similar to the effects of p38 MAPK inhibitor (SB203580). Furthermore, luciferase assay results demonstrated that Mtdh represents the target of miR-30s. Transient transfection experiments, using miR-30 microRNA (miRNA) inhibitors, led to the increase in Mtdh expression and induced the apoptosis of MPC5, whereas the treatment with miR-30 miRNA mimics led to the reduction in Mtdh expression and apoptosis of HG-induced MPC5 cells in comparison with their respective controls. Our results demonstrate that Mtdh is a potent modulator of podocyte apoptosis, and that it represents the target of miR-30 miRNAs, facilitating podocyte apoptosis through the activation of HG-induced p38 MAPK-dependent pathway. PMID:27882943

  1. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  2. Radioactive phosphorylation of alcohols to monitor biocatalytic Diels-Alder reactions.

    Directory of Open Access Journals (Sweden)

    Alexander Nierth

    Full Text Available Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope (32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme-an RNA sequence that catalyzes the eponymous reaction. We used the (32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the (32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.

  3. Phosphorylated Peptide Functionalization of Lanthanide Upconversion Nanoparticles for Tuning Nanomaterial-Cell Interactions.

    Science.gov (United States)

    Yao, Chi; Wei, Caiyi; Huang, Zhi; Lu, Yiqing; El-Toni, Ahmed Mohamed; Ju, Dianwen; Zhang, Xiangmin; Wang, Wenning; Zhang, Fan

    2016-03-23

    Peptide modification of nanoparticles with high efficiency is critical in determining the properties and bioapplications of nanoparticles, but the methodology remains a challenging task. Here, by using the phosphorylated linear and cyclic peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the peptides binding efficiency for the inorganic metal compound nanoparticles was increased significantly after the phosphorylation treatment, and the modification allowed for improving the selectivity and signal-to-noise ratio for cancer targeting and reduced the toxicity derived from nonspecific interactions of nanoparticles with cells owing to the higher amount of phosphopeptide binding. In addition, molecular dynamics (MD) simulations of various peptides on inorganic metal compound surfaces revealed that the peptide adsorption on the surface is mainly driven by electrostatic interactions between phosphate oxygen and the polarized interfacial water layer, consistent with the experimental observation of the strong binding propensity of phosphorylated peptides. Significantly, with the RGD phosphopeptide surface modification, these nanoparticles provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy and may prove useful for various diagnostic and therapeutic applications.

  4. Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sato

    2009-07-01

    Full Text Available p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

  5. Facilitation as a Governance Strategy: Unravelling Governments’ Facilitation Frames

    NARCIS (Netherlands)

    S. Grotenbreg (Sanne); M.W. van Buuren (Arwin)

    2017-01-01

    textabstractGovernments increasingly choose facilitation as a strategy to entice others to produce public goods and services, including in relation to the realisation of sustainable energy innovations. An important instrument to implement this governance strategy is discursive framing. To learn how

  6. Facilitation as a governance strategy: Unravelling governments' facilitation frames

    NARCIS (Netherlands)

    Grotenbreg, S. (Sanne); M.W. van Buuren (Arwin)

    2017-01-01

    textabstractGovernments increasingly choose facilitation as a strategy to entice others to produce public goods and services, including in relation to the realisation of sustainable energy innovations. An important instrument to implement this governance strategy is discursive framing. To learn how

  7. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef; (Pitt); (CNRS-CRMD)

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of

  8. Regulation of endothelial permeability and transendothelial migration of cancer cells by tropomyosin-1 phosphorylation

    Directory of Open Access Journals (Sweden)

    Simoneau Bryan

    2012-11-01

    Full Text Available Abstract Background Loss of endothelial cell integrity and selective permeability barrier is an early event in the sequence of oxidant-mediated injury and may result in atherosclerosis, hypertension and facilitation of transendothelial migration of cancer cells during metastasis. We already reported that endothelial cell integrity is tightly regulated by the balanced co-activation of p38 and ERK pathways. In particular, we showed that phosphorylation of tropomyosin-1 (tropomyosin alpha-1 chain = Tm1 at Ser283 by DAP kinase, downstream of the ERK pathway might be a key event required to maintain the integrity and normal functions of the endothelium in response to oxidative stress. Methods Endothelial permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of HUVECs grown to confluence in Boyden chambers. Actin and Tm1 dynamics and distribution were evaluated by immunofluorescence. We modulated the expression of Tm1 by siRNA and lentiviral-mediated expression of wild type and mutated forms of Tm1 insensitive to the siRNA. Transendothelial migration of HT-29 colon cancer cells was monitored in Boyden chambers similarly as for permeability. Results We provide evidence indicating that Tm1 phosphorylation at Ser283 is essential to regulate endothelial permeability under oxidative stress by modulating actin dynamics. Moreover, the transendothelial migration of colon cancer cells is also regulated by the phosphorylation of Tm1 at Ser283. Conclusion Our finding strongly support the role for the phosphorylation of endothelial Tm1 at Ser283 to prevent endothelial barrier dysfunction associated with oxidative stress injury.

  9. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

    Science.gov (United States)

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F G; Sørensen, Jakob B; Verhage, Matthijs

    2016-05-03

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

  10. Bidirectional regulation of eEF2 phosphorylation controls synaptic plasticity by decoding neuronal activity patterns.

    Science.gov (United States)

    McCamphill, Patrick K; Farah, Carole A; Anadolu, Mina N; Hoque, Sanjida; Sossin, Wayne S

    2015-03-11

    At the sensory-motor neuron synapse of Aplysia, either spaced or continuous (massed) exposure to serotonin (5-HT) induces a form of intermediate-term facilitation (ITF) that requires new protein synthesis but not gene transcription. However, spaced and massed ITF use distinct molecular mechanisms to maintain increased synaptic strength. Synapses activated by spaced applications of 5-HT generate an ITF that depends on persistent protein kinase A (PKA) activity, whereas an ITF produced by massed 5-HT depends on persistent protein kinase C (PKC) activity. In this study, we demonstrate that eukaryotic elongation factor 2 (eEF2), which catalyzes the GTP-dependent translocation of the ribosome during protein synthesis, acts as a biochemical sensor that is tuned to the pattern of neuronal stimulation. Specifically, we find that massed training leads to a PKC-dependent increase in phosphorylation of eEF2, whereas spaced training results in a PKA-dependent decrease in phosphorylation of eEF2. Importantly, by using either pharmacological or dominant-negative strategies to inhibit eEF2 kinase (eEF2K), we were able to block massed 5-HT-dependent increases in eEF2 phosphorylation and subsequent PKC-dependent ITF. In contrast, pharmacological inhibition of eEF2K during the longer period of time required for spaced training was sufficient to reduce eEF2 phosphorylation and induce ITF. Finally, we find that the massed 5-HT-dependent increase in synaptic strength requires translation elongation, but not translation initiation, whereas the spaced 5-HT-dependent increase in synaptic strength is partially dependent on translation initiation. Thus, bidirectional regulation of eEF2 is critical for decoding distinct activity patterns at synapses by activating distinct modes of translation regulation. Copyright © 2015 the authors 0270-6474/15/354403-15$15.00/0.

  11. Radix Astragali Stimulates p38 MARK Phosphorylation in Pediatric Patients with β-Thalassemia

    Directory of Open Access Journals (Sweden)

    Zhuoming Lu

    2016-01-01

    Full Text Available A previous study conducted by our group demonstrated that Radix Astragali compounded with Codonopsis pilosula and Plastrum testudinis was effective in treating pediatric β-thalassemia in a randomized, controlled clinical trial. However, the mechanism of action that underpins this treatment remains to be elucidated. Blood was collected from patients participating in this clinical trial and nucleated red blood cell-enriched mononuclear cells were isolated to facilitate the extraction of RNA and protein. RT-PCR was used to monitor the expression of globin genes and p38 MAPK, and total and phosphorylated p38 MAPK expression was assessed using Western blot analysis. Expression of α-, β-, and Aγ-globin mRNAs was not significantly affected following treatment with R. Astragali or the compounded formulation. However, Gγ-globin mRNA levels increased significantly in both treatment groups (when compared with pretreatment levels following 12 weeks of treatment. Moreover, posttreatment Gγ-globin expression was significantly higher in both treatment groups compared with the control group. Although neither p38 MAPK mRNA nor protein levels were affected by the treatments, posttreatment phosphorylation of p38 MAPK was significantly increased in the R. Astragali and compounded formulation groups compared with the control group. These data suggest that the molecular mechanisms that underpin the efficacious use of R. Astragali (and its compounded formulation in pediatric β-thalassemia treatment facilitate the induction of Gγ-globin expression following activation of p38 MAPK.

  12. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

    OpenAIRE

    Kijas, Amanda W.; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F.

    2015-01-01

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the ...

  13. The in vivo phosphorylation sites of rat brain dynamin I

    DEFF Research Database (Denmark)

    Graham, Mark E; Anggono, Victor; Bache, Nicolai

    2007-01-01

    tail splice variant dynIxa and was not hierarchical. Co-purified, (32)P-labeled dynIII was phosphorylated at Ser(759), Ser(763), and Ser(853). Ser(853) is homologous to Ser(851) in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure...

  14. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    Science.gov (United States)

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  15. Intermolecular Phosphoryl Transfer Between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    Yu Qian SU; Ming Yu NIU; Shu Xia CAO; Jian Chen ZHANG; Yu Fen ZHAO

    2004-01-01

    A novel intermolecular phosphoryl transfer from O-trimethylsilyl-N-(O, O-diisopropyl) phosphoryl serine trimethylsilyl ester to N, N'-bis(trimethylsilyl) histidine trimethylsilyl ester was studied through electrospray ionization mass spectrometry (ESI-MS). It was proposed that the transfer reaction went through penta-coordinated phosphorus intermediate.

  16. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK...

  17. Phosphorylation of the Goodpasture antigen by type A protein kinases.

    Science.gov (United States)

    Revert, F; Penadés, J R; Plana, M; Bernal, D; Johansson, C; Itarte, E; Cervera, J; Wieslander, J; Quinones, S; Saus, J

    1995-06-02

    Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

  18. Facilitation of learning: part 1.

    Science.gov (United States)

    Warburton, Tyler; Trish, Houghton; Barry, Debbie

    2016-04-06

    This article, the fourth in a series of 11, discusses the context for the facilitation of learning. It outlines the main principles and theories for understanding the process of learning, including examples which link these concepts to practice. The practical aspects of using these theories in a practice setting will be discussed in the fifth article of this series. Together, these two articles will provide mentors and practice teachers with knowledge of the learning process, which will enable them to meet the second domain of the Nursing and Midwifery Council's Standards to Support Learning and Assessment in Practice on facilitation of learning.

  19. DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1

    OpenAIRE

    Xie, Binhui; Lin, Weihao; Ye, Junming; Wang, Xiaonong; Zhang, Bing; Xiong, Shiqiu; Li, Heping; Tan, Guosheng

    2015-01-01

    Background Several studies have found that DDR2 is up-regulated in many tumor types and facilitates tumor progression. However, the role of DDR2 in hepatocellular carcinoma (HCC) progression and its downstream signaling pathways remain unclear. Methods DDR2 expression was assessed in several cell lines and 112 pairs of HCC and matched adjacent noncancerous liver tissues. Clinical significance of DDR2 in HCC was analyzed. Phosphorylated DDR2 (p-DDR2) expression was detected by immunoblotting t...

  20. Methylglyoxal mediates adipocyte proliferation by increasing phosphorylation of Akt1.

    Directory of Open Access Journals (Sweden)

    Xuming Jia

    Full Text Available Methylglyoxal (MG is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5-20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity.

  1. Exportability of the mitochondrial oxidative phosphorylation machinery into myelin sheath.

    Science.gov (United States)

    Morelli, Alessandro; Ravera, Silvia; Calzia, Daniela; Panfoli, Isabella

    2011-01-01

    White matter comprises over half of the brain, and its role in axonal survival is being reconsidered, consistently with the observation that axonal degeneration follows demyelination. The recent evidence of an extra-mitochondrial aerobic ATP production in isolated myelin vesicles, thanks to the expression therein of the mitochondrial Oxydative Phosphorylation (OXPHOS) machinery, stands in for myelin playing a functional bioenergetic role in ATP supply for the axon. The observation that subunits of the OXPHOS encoded by the mitochondrial genome are expressed in myelin, suggests that they can be the same as those of the inner mitochondrial membrane. This would mean that the OXPHOS is exportable. Here the hypothesis is exposed that the mitochondrion is the unique site of the assembly of the OXPHOS, so that this is exported to those sub cellular districts displaying high energy demand, such as myelin sheath. There the OXPHOS would display a higher efficiency in oxidative ATP production than inside the mitochondrion itself In this respect, the role of the glia in the nervous conduction is shed new light and the oligodendrocyte mitochondrial OXPHOS are hypothesized to be delivered to nascent myelin.

  2. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    that has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial...... physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...

  3. Systematic inference of functional phosphorylation events in yeast metabolism

    DEFF Research Database (Denmark)

    Chen, Yu; Wang, Yonghong; Nielsen, Jens

    2017-01-01

    Motivation: Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification...... of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics...... has received increased attention.Results: We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution...

  4. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  5. The participation of calponin in the cross talk between 20-hydroxyecdysone and juvenile hormone signaling pathways by phosphorylation variation.

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Liu

    Full Text Available 20-hydroxyecdysone (20E and juvenile hormone (JH signaling pathways interact to mediate insect development, but the mechanism of this interaction is poorly understood. Here, a calponin homologue domain (Chd containing protein (HaCal is reported to play a key role in the cross talk between 20E and JH signaling by varying its phosphorylation. Chd is known as an actin binding domain present in many proteins including some signaling proteins. Using an epidermal cell line (HaEpi, HaCal was found to be up-regulated by either 20E or the JH analog methoprene (JHA. 20E induced rapid phosphorylation of HaCal whereas no phosphorylation occurred with JHA. HaCal could be quickly translocated into the nuclei through 20E or JH signaling but interacted with USP1 only under the mediation of JHA. Knockdown of HaCal by RNAi blocked the 20E inducibility of USP1, PKC and HR3, and also blocked the JHA inducibility of USP1, PKC and JHi. After gene silencing of HaCal by ingestion of dsHaCal expressed by Escherichia coli, the larval development was arrested and the gene expression of USP1, PKC, HR3 and JHi were blocked. These composite data suggest that HaCal plays roles in hormonal signaling by quickly transferring into nucleus to function as a phosphorylated form in the 20E pathway and as a non-phosphorylated form interacting with USP1 in the JH pathway to facilitate 20E or JH signaling cascade, in short, by switching its phosphorylation status to regulate insect development.

  6. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

    Directory of Open Access Journals (Sweden)

    Danming Tang

    2012-09-01

    GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

  7. Corpus Linguistics Facilitates English Teaching

    Institute of Scientific and Technical Information of China (English)

    朱思亲

    2014-01-01

    Corpus linguistics has been widely applied in English teaching. Corpus linguistics has changed the way to teach English. The essay discusses two approaches in English teaching based on corpus, corpus-driven approach and corpus-based approach. It finds out that both corpus-driven approach and corpus-based approach facilitate English teaching in their own ways.

  8. Brug af mindfulness til facilitering

    DEFF Research Database (Denmark)

    Adriansen, Hanne Kirstine; Krohn, Simon

    2011-01-01

    Gennem de senere år er mindfulness gået fra udelukkende at være en eksistentiel praksis til også at være en behandlingsform og senest til også at blive brugt som et praktisk redskab i erhvervslivet. Denne artikel viser, at mindfulness også kan anvendes i forbindelse med facilitering. Facilitering...... er et værktøj, som bruges i arbejdslivet fx til møder og konferencer, hvor en gruppe mennesker er samlet for at lære eller udrette noget sammen. Det nye ved at kombinere mindfulness med facilitering er, at fokus hermed ændres fra individet, som er centrum for den eksistentielle fordybelse eller det...... terapeutiske forløb, til gruppen, som er udgangspunktet i facilitering. Artiklen viser, hvordan mindfulness konkret kan bruges på gruppeniveau og diskuterer samtidig hvilke problemer, der kan være forbundet hermed. Baseret på vores egne erfaringer, diskuterer vi, hvordan mindfulness kan påvirke en gruppes...

  9. GIS-facilitated spatial narratives

    DEFF Research Database (Denmark)

    Møller-Jensen, Lasse; Jeppesen, Henrik; Kofie, Richard Y.

    2008-01-01

    -based' exploration of sites related to the narrative and as a tool that facilitates the design of spatial narratives before implementation within portable GIS devices. The Google Earth-based visualization of the spatial narrative is created by a Python script that outputs a web-accessible KML format file. The KML...

  10. Facilitating Creativity in Adult Learners

    Science.gov (United States)

    Tsai, Kuan Chen

    2013-01-01

    Creativity in education research has received increasing attention, although the major focus of this research has been on children. Despite pleas by several adult educators for promoting creativity, very few studies have focused on adult learners, leaving to it to be explored what approaches are useful for adult educators to facilitate creativity…

  11. Learning to Facilitate (Online) Meetings

    DEFF Research Database (Denmark)

    Reimann, Peter; Bull, Susan; Vatrapu, Ravi

    2013-01-01

    , etc.. We argue that facilitating meetings is a competence worth developing in students and describe the main knowledge and skill components that pertain to this competence. We then describe some implemented software tools that can be used in schools and colleges to provide opportunities for practicing...

  12. Facilitating Conditions for School Motivation.

    Science.gov (United States)

    Yeung, Alexander Seeshing; McInerney, Dennis M.

    Primary and high school students (277 in grades 5-6; 615 in grades 7-12) in the United States (47 percent boys) responded to 26 items of the Facilitating Conditions Questionnaire (FCQ). Results indicate 7 distinct FCQ factors: perceived value of schooling; affect toward schooling; peer positive academic climate (Peer Positive); encouragement from…

  13. Phosphoryl functionalized mesoporous silica for uranium adsorption

    Science.gov (United States)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun; Hongyu, Gong; Yujun, Zhang

    2017-04-01

    Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N2 adsorption-desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG0, ΔH0 and ΔS0) confirmed that the adsorption process was endothermic and spontaneous.

  14. The neural correlates of picture naming facilitated by auditory repetition

    Directory of Open Access Journals (Sweden)

    Heath Shiree

    2012-02-01

    Full Text Available Abstract Background Overt repetition of auditorily presented words can facilitate picture naming performance in both unimpaired speakers and individuals with word retrieval difficulties, but the underlying neurocognitive mechanisms and longevity of such effects remain unclear. This study used functional magnetic resonance imaging to examine whether different neurological mechanisms underlie short-term (within minutes and long-term (within days facilitation effects from an auditory repetition task in healthy older adults. Results The behavioral results showed that both short- and long-term facilitated items were named significantly faster than unfacilitated items, with short-term items significantly faster than long-term items. Neuroimaging analyses identified a repetition suppression effect for long-term facilitated items, relative to short-term facilitated and unfacilitated items, in regions known to be associated with both semantic and phonological processing. A repetition suppression effect was also observed for short-term facilitated items when compared to unfacilitated items in a region of the inferior temporal lobe linked to semantic processing and object recognition, and a repetition enhancement effect when compared to long-term facilitated items in a posterior superior temporal region associated with phonological processing. Conclusions These findings suggest that different neurocognitive mechanisms underlie short- and long-term facilitation of picture naming by an auditory repetition task, reflecting both phonological and semantic processing. More specifically, the brain areas engaged were consistent with the view that long-term facilitation may be driven by a strengthening of semantic-phonological connections. Short-term facilitation, however, appears to result in more efficient semantic processing and/or object recognition, possibly in conjunction with active recognition of the phonological form.

  15. Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains.

    Science.gov (United States)

    Xu, Lijun; Deng, Xingming

    2006-02-17

    Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

  16. Biohydrogen facilitated denitrification at biocathode in bioelectrochemical system (BES).

    Science.gov (United States)

    Liu, Hao; Yan, Qun; Shen, Wei

    2014-11-01

    Reductive removal of nitrate in bioelectrochemical system (BES) at abiotic cathode, biocathode and biohydrogen facilitated biocathode were investigated. It was found that nitrate removal efficiency reached 95% and 59% at the biohydrogen facilitated biocathode and biocathode respectively, while which was only 13% at the abiotic cathode. Meanwhile, activity of nitrate reductase reached 0.701 g-N/Lh for the biohydrogen facilitated group, which was about 9.3 times of the biocathode group. Moreover, electrochemical performances as power density, ohmic resistance, and polarization resistance of the biohydrogen facilitated group reached 76.96 mW/m(3), 8.63 ohm and 383 ohm, respectively, which were better than two other groups. Finally, an obvious shift of bacterial community responsible for the enhanced nitrate reduction between the two biocathode groups was observed. Therefore, nitrate reduction in BES could be enhanced at the biocathode than that of the abiotic cathode, and then be further boosted with the combination of biohydrogen.

  17. Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization.

    Science.gov (United States)

    Deshpande, Atul Suresh; Fang, Ping-An; Zhang, Xiaoyuan; Jayaraman, Thottala; Sfeir, Charles; Beniash, Elia

    2011-08-08

    The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.

  18. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

    Science.gov (United States)

    Kijas, Amanda W; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F

    2015-09-30

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

  19. Patient advocacy: barriers and facilitators

    Directory of Open Access Journals (Sweden)

    Nikravesh Mansoure

    2006-03-01

    Full Text Available Abstract Background During the two recent decades, advocacy has been a topic of much debate in the nursing profession. Although advocacy has embraced a crucial role for nurses, its extent is often limited in practice. While a variety of studies have been generated all over the world, barriers and facilitators in the patient advocacy have not been completely identified. This article presents the findings of a study exploring the barriers and facilitators influencing the role of advocacy among Iranian nurses. Method This study was conducted by grounded theory method. Participants were 24 Iranian registered nurses working in a large university hospital in Tehran, Iran. Semi-structured interviews were used for data collection. All interviews were transcribed verbatim and simultaneously Constant comparative analysis was used according to the Strauss and Corbin method. Results Through data analysis, several main themes emerged to describe the factors that hindered or facilitated patient advocacy. Nurses in this study identified powerlessness, lack of support, law, code of ethics and motivation, limited communication, physicians leading, risk of advocacy, royalty to peers, and insufficient time to interact with patients and families as barriers to advocacy. As for factors that facilitated nurses to act as a patient advocate, it was found that the nature of nurse-patient relationship, recognizing patients' needs, nurses' responsibility, physician as a colleague, and nurses' knowledge and skills could be influential in adopting the advocacy role. Conclusion Participants believed that in this context taking an advocacy role is difficult for nurses due to the barriers mentioned. Therefore, they make decisions and act as a patient's advocate in any situation concerning patient needs and status of barriers and facilitators. In most cases, they can not act at an optimal level; instead they accept only what they can do, which we called 'limited advocacy' in

  20. Facilitating Facilitators to Facilitate, in Problem or Enquiry Based Learning Sessions

    Science.gov (United States)

    Coelho, Catherine

    2014-01-01

    Problem based learning (PBL) has been used in dental education over the past 20 years and uses a patient case scenario to stimulate learning in a small group setting, where a trained facilitator does not teach but guides the group to bring about deep contextualized learning, to be empathetic to each other and to encourage fair and equitable…

  1. Expression and phosphorylation of neurofilament protein in different neuronal tissues

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.

  2. 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...

  3. The interplay between p16 serine phosphorylation and arginine methylation determines its function in modulating cellular apoptosis and senescence.

    Science.gov (United States)

    Lu, Yang; Ma, Wenlong; Li, Zhongwei; Lu, Jun; Wang, Xiuli

    2017-01-25

    Cyclin-dependent kinase inhibitor p16(INK4a) (p16) primarily functions as a negative regulator of the retinoblastoma protein (Rb) -E2F pathway, thus plays critical role in cell cycle progression, cellular senescence and apoptosis. In this study, we showed that the methylation of Arg 138 and the phosphorylation of Ser 140 on p16 were critical for the control of cell proliferation and apoptosis. Compared to wild type p16, mutant p16R138K possessed improved function in preventing cell proliferation and inducing apoptosis, while the Ser 140 mutation (p16S140A) exhibited the opposite alteration. We also demonstrated that H2O2 was able to induce the phosphorylation of p16, which facilitated the interaction between CDK4 (Cyclin-dependent protein kinase) and p16, in 293T (human emborynic kidney) cells. Furthermore, the elevated arginine methylation in p16S140A mutant and increased serine phosphorylation in p16R138K mutant suggest that a antagonizing mechanism coordinating Arg 138 methylation and Ser 140 phosphorylation to regulates p16 function as well as cellular apoptosis and senescence. These findings will therefore contribute to therapeutic treatment for p16-related gene therapy by providing theoretical and experimental evidence.

  4. Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt.

    Science.gov (United States)

    Han, Chang Yeob; Koo, Ja Hyun; Kim, Sung Hoon; Gardenghi, Sara; Rivella, Stefano; Strnad, Pavel; Hwang, Se Jin; Kim, Sang Geon

    2016-12-22

    Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.

  5. Serine 62-Phosphorylated MYC Associates with Nuclear Lamins and Its Regulation by CIP2A Is Essential for Regenerative Proliferation.

    Science.gov (United States)

    Myant, Kevin; Qiao, Xi; Halonen, Tuuli; Come, Christophe; Laine, Anni; Janghorban, Mahnaz; Partanen, Johanna I; Cassidy, John; Ogg, Erinn-Lee; Cammareri, Patrizia; Laiterä, Tiina; Okkeri, Juha; Klefström, Juha; Sears, Rosalie C; Sansom, Owen J; Westermarck, Jukka

    2015-08-11

    An understanding of the mechanisms determining MYC's transcriptional and proliferation-promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here, we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and that the protein phosphatase 2A (PP2A) inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum-induced MYC phosphorylation and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage. However, targeting of CIP2A did not influence the normal function of intestinal crypt cells. These data underline the importance of nuclear organization in the regulation of MYC phosphorylation, leading to an in vivo demonstration of a strategy for inhibiting MYC activity without detrimental physiological effects.

  6. Serine 62-Phosphorylated MYC Associates with Nuclear Lamins and Its Regulation by CIP2A Is Essential for Regenerative Proliferation

    Directory of Open Access Journals (Sweden)

    Kevin Myant

    2015-08-01

    Full Text Available An understanding of the mechanisms determining MYC’s transcriptional and proliferation-promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here, we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and that the protein phosphatase 2A (PP2A inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum-induced MYC phosphorylation and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage. However, targeting of CIP2A did not influence the normal function of intestinal crypt cells. These data underline the importance of nuclear organization in the regulation of MYC phosphorylation, leading to an in vivo demonstration of a strategy for inhibiting MYC activity without detrimental physiological effects.

  7. MD-2 interacts with Lyn kinase and is tyrosine phosphorylated following LPS-induced activation of the Toll-like receptor 4 signaling pathway

    Science.gov (United States)

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S.; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R.; Arditi, Moshe

    2011-01-01

    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrate that MD-2 is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific, it is blocked by the tyrosine kinase inhibitor, Herbimycin A, and by an inhibitor of endocytosis, Cytochalsin-D, suggesting that MD-2 phosphorylation occurs during trafficking of MD2 and not on cell surface. Furthermore, we identify two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine have reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD2 co-precipitates and colocalizes with Lyn kinase, most likely in ER. A Lyn-binding peptide inhibitor abolished MD2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phophorylation. Our study demonstrates that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response. PMID:21918188

  8. Interaction of human immunodeficiency virus type 1 Vif with APOBEC3G is not dependent on serine/threonine phosphorylation status.

    Science.gov (United States)

    Kopietz, Ferdinand; Jaguva Vasudevan, Ananda Ayyappan; Krämer, Melanie; Muckenfuss, Heide; Sanzenbacher, Ralf; Cichutek, Klaus; Flory, Egbert; Münk, Carsten

    2012-11-01

    The human immunodeficiency virus type 1 accessory protein Vif is important for viral infectivity because it counteracts the antiviral protein APOBEC3G (A3G). ³²P metabolic labelling of stimulated cells revealed in vivo phosphorylation of the control protein, whereas no serine/threonine phosphorylation was detected for Vif or the A3G protein. These data were confirmed by in vitro kinase assays using active recombinant kinase. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 efficiently phosphorylated its target ELK, but failed to phosphorylate Vif. Putative serine/threonine phosphorylation point mutations in Vif (T96, S144, S165, T188) using single-round infection assays demonstrated that these mutations did not alter Vif activity, with the exception of Vif.T96E. Interestingly, T96E and not T96A was functionally impaired, indicating that this residue is critical for Vif-A3G physical interaction and activity. Our data suggest that Vif and A3G are not serine/threonine phosphorylated in human cells and phosphorylation is not linked to their functional activities.

  9. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  10. Acute exercise modifies titin phosphorylation and increases cardiac myofilament stiffness

    Directory of Open Access Journals (Sweden)

    Anna Eliane Müller

    2014-11-01

    Full Text Available Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT and PEVK (increases PT. Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively, and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively. Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length ranging from 1.9-2.4µm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity.

  11. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  12. On novice facilitators doing research

    DEFF Research Database (Denmark)

    Tavella, Elena

    2016-01-01

    Opportunities for novices to facilitate Problem Structuring Methods (PSMs) workshops are limited, especially because of a lack of access to real-world interventions and confidence in their capabilities. Novices are usually young academics building their careers through publishing. Publishing...... is challenging if facilitation and opportunities for data collection are limited. To address this challenge, this paper suggests autoethnography as a framework for addressing difficulties that novices face in conducting research and publishing on PSMs. This suggestion grows out of a literature study...... on autoethnography and PSMs combined with reflections on the author’s experience as a PSM novice and young academic. Autoethnography is presented as a means to enable access to real-world interventions, enhance novices’ confidence, and identify research and publishing opportunities. The author outlines strengths...

  13. Rosamines targeting the cancer oxidative phosphorylation pathway.

    Directory of Open Access Journals (Sweden)

    Siang Hui Lim

    Full Text Available Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM, inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6 exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

  14. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N

    2005-01-01

    Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation i...

  15. Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    Science.gov (United States)

    Ludgate, Laurie; Liu, Kuancheng; Luckenbaugh, Laurie; Streck, Nicholas; Eng, Stacey; Voitenleitner, Christian; Delaney, William E.

    2016-01-01

    ABSTRACT Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have

  16. Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Bahassi, El Mustapha [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Myer, David L. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); McKenney, Richard J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Hennigan, Robert F. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States); Stambrook, Peter J. [Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267 (United States)]. E-mail: peter.stambrook@uc.edu

    2006-04-11

    The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

  17. Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Graham, Mark E; Robinson, Phillip J

    2004-01-01

    , or peptides altered in hydrophilicity such as phosphopeptides. We used microcolumns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analyzed. MS revealed that some phosphopeptides...... did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve...... and doubly phosphorylated peptide in dynamin III, analogous to the dynamin I sequence. A pair of dynamin III phosphorylation sites were found at Ser-759 and Ser-763 by tandem MS. The results directly define the in vivo phosphorylation sites in dynamins I and III for the first time. The findings indicate...

  18. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum.

    Science.gov (United States)

    Du, Lei; Ma, Li; Qi, Feifei; Zheng, Xianliang; Jiang, Chengying; Li, Ailei; Wan, Xiaobo; Liu, Shuang-Jiang; Li, Shengying

    2016-03-18

    4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (Km and kcat) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route.

  19. Phosphorylation of eIF2 alpha in Sf9 cells: a stress, survival and suicidal signal.

    Science.gov (United States)

    Aarti, Iyer; Rajesh, Kamindla; Ramaiah, Kolluru V A

    2010-06-01

    An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2alpha) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2alpha phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2alpha phosphorylation. Infection of Sf9 cells by wt and a mutant Deltapk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2alpha phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Deltapk2 baculovirus that lacks pk2, an inhibitor of eIF2alpha kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2alpha phosphorylation, thereby suggesting that eIF2alpha phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2alpha phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2alpha phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2alpha, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2alpha phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.

  20. Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation In Vivo

    Science.gov (United States)

    2006-11-01

    analysis indicates that the screen is preferentially isolating proteins with a known role in gene transcription and we are currently assessing the phosphorylation- dependence of the putative AR interacting proteins .

  1. The Synthesis of a Series of Phosphoryl Coumarins

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite (DIPPH) by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of the coumarins in the Atherton-Todd reaction were studied.

  2. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  3. Phosphorylation of the viral coat protein regulates RNA virus infection

    Directory of Open Access Journals (Sweden)

    Hoover HS

    2016-11-01

    Full Text Available Haley S Hoover, C Cheng Kao Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA Abstract: Coat proteins (CPs are the most abundant protein produced during a viral infection. CPs have been shown to regulate the infection processes of RNA viruses, including RNA replication and gene expression. The numerous activities of the CP in infection are likely to require regulation, possibly through posttranslational modifications. Protein posttranslational modifications are involved in signal transduction, expanding and regulating protein function, and responding to changes in the environment. Accumulating evidence suggests that phosphorylation of viral CPs is involved in the regulation of the viral infection process from enabling virion disassembly to regulation of viral protein synthesis and replication. CP phosphorylation also affects viral trafficking and virion assembly. This review focuses on the regulatory roles that phosphorylation of CPs has in the life cycle of viruses with RNA genomes. Keywords: viral capsid protein, posttranslational modification, phosphorylation, protein–RNA interaction

  4. A Green Synthesis of Diisopropyl Phosphoryl Amino Acid

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In this paper, we report that diisopropyl phosphoryl amino acid could be prepared with reasonable yields under solvent-free condition by adding amino acid to the mixture of diisopropyl phosphite and N-chlorodiisopropylamine.

  5. The FGFR4-G388R Polymorphism Promotes Mitochondrial STAT3 Serine Phosphorylation to Facilitate Pituitary Growth Hormone Cell Tumorigenesis

    OpenAIRE

    Toru Tateno; Asa, Sylvia L.; Lei Zheng; Thomas Mayr; Axel Ullrich; Shereen Ezzat

    2011-01-01

    Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting eff...

  6. Tau Phosphorylation by GSK3 in Different Conditions

    Science.gov (United States)

    Avila, Jesús; León-Espinosa, Gonzalo; García, Esther; García-Escudero, Vega; Hernández, Félix; DeFelipe, Javier

    2012-01-01

    Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau. PMID:22675648

  7. Fission yeast Rad52 phosphorylation restrains error prone recombination pathways.

    Directory of Open Access Journals (Sweden)

    Angela Bellini

    Full Text Available Rad52 is a key protein in homologous recombination (HR, a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

  8. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking.

    Science.gov (United States)

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan

    2016-06-01

    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

  9. Protein phosphorylation and its role in archaeal signal transduction.

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C; Albers, Sonja-Verena; Siebers, Bettina

    2016-09-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies.

  10. PHOSPHORYLATED TAU: TOXIC, PROTECTIVE, OR NONE OF THE ABOVE

    Science.gov (United States)

    Castellani, Rudy J.; Nunomura, Akihiko; Lee, Hyoung-gon; Perry, George; Smith, Mark A.

    2009-01-01

    Identification of phosphorylated tau as the major protein component of neurofibrillary tangles (NFTs) led to the concept that phosphorylated tau was inherently toxic and, as such, intimately involved in Alzheimer’s disease (AD) pathogenesis. While superficially logical, this construct ignores a number of key findings in AD, including i) that NFTs are encountered in viable neurons until late stage disease; ii) that NFTs persist within the neuronal cytoplasm for decades; iii) that NFTs are encountered, sometimes in significant numbers, in cognitively intact elderly; and iv) that neurons with NFTs contain normal content and structure of microtubules. Experimental data in transgenic animal models has further demonstrated that NFTs accumulate in neurons in spite of tau suppression and behavior normalization. These data call into question the inherent toxicity of phosphorylated tau, seemingly leaving the only viable hypothesis of the ad hoc “toxic intermediate” phosphorylated tau concept. However, since we also know that phosphorylated tau sequesters redox active heavy metals and protects against oxidative stress, here we suggest that phosphorylated tau serves a protective role against cellular toxicity. PMID:18688087

  11. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    Science.gov (United States)

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-04-18

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

  12. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Deaciuc, I.V.; Spitzer, J.A. (Louisiana State Univ. Medical Center, New Orleans (USA))

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  13. Stochastic facilitation in the brain?

    Science.gov (United States)

    Ward, Lawrence M.; Greenwood, Priscilla E.

    2016-05-01

    We describe the context for three unsolved problems of noise in the brain as well as provide some new results relevant to one of them. The problems are: are neural oscillations better described as noisy limit cycles or as noise-driven quasicycles, does noise facilitate synchronization and information transmission in the brain, and do noise-driven spatial patterns (quasipatterns) coexist with noise-driven quasicycles in the brain? We provide a few new results indicating that, in models at least, spatial quasipatterns of quasicycles can occur, and resemble patterns observed in other areas, such as predator-prey systems and chemical reactions.

  14. GIS-facilitated spatial narratives

    DEFF Research Database (Denmark)

    Møller-Jensen, Lasse; Jeppesen, Henrik; Kofie, Richard Y.

    2008-01-01

    -based' exploration of sites related to the narrative and as a tool that facilitates the design of spatial narratives before implementation within portable GIS devices. The Google Earth-based visualization of the spatial narrative is created by a Python script that outputs a web-accessible KML format file. The KML...... on the thematically and narrative linking of a set of locations within an area. A spatial narrative that describes the - largely unsuccessful - history of Danish plantations on the Gold Coast (1788-1850) is implemented through the Google Earth client. This client is seen both as a type of media in itself for ‘home...

  15. Phosphorylation of mTOR Ser2481 is a key target limiting the efficacy of rapalogs for treating hepatocellular carcinoma.

    Science.gov (United States)

    Watari, Kosuke; Nishitani, Ayumi; Shibata, Tomohiro; Noda, Masaki; Kawahara, Akihiko; Akiba, Jun; Murakami, Yuichi; Yano, Hirohisa; Kuwano, Michihiko; Ono, Mayumi

    2016-07-26

    Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

  16. Hyper-O-GlcNAcylation of YB-1 affects Ser102 phosphorylation and promotes cell proliferation in hepatocellular carcinoma.

    Science.gov (United States)

    Liu, Qingqing; Tao, Tao; Liu, Fang; Ni, Runzhou; Lu, Cuihua; Shen, Aiguo

    2016-12-10

    As an essential post-translational modification, O-GlcNAcylation has been thought to be able to modulate various nuclear and cytoplasmic proteins and is emerging as a key regulator of multiple biological processes, such as transcription, cell growth, signal transduction, and cell motility. Recently, authoritative glycomics analyses have reported extensive crosstalk between O-GlcNAcylation and phosphorylation, which always dynamically interplay with each other and regulate signaling, transcription, and other cellular processes. Also, plentiful studies have shown close correlation between YB-1 phosphorylation and tumorigenesis. Therefore, our study aimed to determine whether YB-1 was O-GlcNAc modified and whether such modification could interact with its phosphorylation during the process of HCC development. Western blot and immunohistochemistry were firstly conducted to reveal obvious up-regulation of YB-1, OGT and O-GlcNAc modification in HCC tissues. What is more, not only YB-1 was identified to be O-GlcNAcylated but hyper-O-GlcNAcylation was demonstrated to facilitate HCC cell proliferation in a YB-1 dependent manner. Moreover, we detected four specific O-GlcNAc sites and confirmed T126A to be the most effective mutant in HCC cell proliferation via close O-GlcNAcylation-phosphorylation interaction. Even more interestingly, we discovered that T126A-induced HCC cell retardation and subdued transcriptional activity of YB-1 could be partially reversed by T126A/S102E mutant. From all above, it is not difficult to find that glycosylated-YB-1 mainly enhanced cell proliferation through congenerous actions with YB-1 phosphorylation and thus played indispensable roles in fine-tuning cell proliferation and procession of HCC.

  17. Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Jianjun; Chen, Fei; Fan, Daidi, E-mail: fandaidi@nwu.edu.cn; Zhu, Chenhui; Ma, Xiaoxuan; Xue, Wenjiao

    2013-10-01

    Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein–iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (n{sub b}) and apparent association constant (K{sub app}) between iron and phosphorylated HLC were measured at n{sub b} = 23.7 and log K{sub app} = 4.57, respectively. The amount of iron (Fe{sup 2+} sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates. - Highlights: • The iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared. • One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. • The binding properties could be modulated through alterations in pH and phosphate content presented in HLC. • A novel strategy for preparing iron-binding proteins was provided.

  18. Adenosine A₂A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation.

    Directory of Open Access Journals (Sweden)

    Hai-Ying Shen

    Full Text Available Adenosine A2A receptors (A2AR are located postsynaptically in striatopallidal GABAergic neurons, antagonizing dopamine D2 receptor functions, and are also located presynaptically at corticostriatal terminals, facilitating glutamate release. To address the hypothesis that these two A2AR populations differently control the action of psychostimulants, we characterized A2AR modulation of cocaine-induced effects at the level of DARPP-32 phosphorylation at Thr-34 and Thr-75, c-Fos expression, and psychomotor activity using two lines of cell-type selective A2AR knockout (KO mice with selective A2AR deletion in GABAergic neurons (striatum-A2AR-KO mice, or with A2AR deletion in both striatal GABAergic neurons and projecting cortical glutamatergic neurons (forebrain-A2AR-KO mice. We demonstrated that striatum-A2AR KO mice lacked A2ARs exclusively in striatal GABAergic terminals whereas forebrain-A2AR KO mice lacked A2ARs in both striatal GABAergic and glutamatergic terminals leading to a blunted A2AR-mediated facilitation of synaptosomal glutamate release. The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75. Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75. This distinct modulation of DARPP-32 phosphorylation was associated with opposite responses to cocaine-induced striatal c-Fos expression and psychomotor activity in striatum-A2AR KO (enhanced and forebrain-A2AR KO mice (reduced. Thus, A2ARs in glutamatergic corticostriatal terminals and in GABAergic striatal neurons modulate the action of psychostimulants and DARPP-32 phosphorylation in opposite ways. We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and

  19. Voice congruency facilitates word recognition.

    Directory of Open Access Journals (Sweden)

    Sandra Campeanu

    Full Text Available Behavioral studies of spoken word memory have shown that context congruency facilitates both word and source recognition, though the level at which context exerts its influence remains equivocal. We measured event-related potentials (ERPs while participants performed both types of recognition task with words spoken in four voices. Two voice parameters (i.e., gender and accent varied between speakers, with the possibility that none, one or two of these parameters was congruent between study and test. Results indicated that reinstating the study voice at test facilitated both word and source recognition, compared to similar or no context congruency at test. Behavioral effects were paralleled by two ERP modulations. First, in the word recognition test, the left parietal old/new effect showed a positive deflection reflective of context congruency between study and test words. Namely, the same speaker condition provided the most positive deflection of all correctly identified old words. In the source recognition test, a right frontal positivity was found for the same speaker condition compared to the different speaker conditions, regardless of response success. Taken together, the results of this study suggest that the benefit of context congruency is reflected behaviorally and in ERP modulations traditionally associated with recognition memory.

  20. Facilitation of learning: part 2.

    Science.gov (United States)

    Warburton, Tyler; Houghton, Trish; Barry, Debbie

    2016-04-27

    The previous article in this series of 11, Facilitation of learning: part 1, reviewed learning theories and how they relate to clinical practice. Developing an understanding of these theories is essential for mentors and practice teachers to enable them to deliver evidence-based learning support. This is important given that effective learning support is dependent on an educator who possesses knowledge of their specialist area as well as the relevent tools and methods to support learning. The second domain of the Nursing and Midwifery Council's Standards to Support Learning and Assessment in Practice relates to the facilitation of learning. To fulfil this domain, mentors and practice teachers are required to demonstrate their ability to recognise the needs of learners and provide appropriate support to meet those needs. This article expands on some of the discussions from part 1 of this article and considers these from a practical perspective, in addition to introducing some of the tools that can be used to support learning.

  1. H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis.

    Science.gov (United States)

    Wang, Qian; Wei, Haojie; Du, Juan; Cao, Yan; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Chen, Dandan; Ma, Wei

    2016-01-01

    Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.

  2. Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-mediated phosphorylation of Thr231 in vitro.

    Science.gov (United States)

    Moszczynski, Alexander J; Gohar, May; Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Wendy; Strong, Michael J

    2015-03-01

    We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr(175) (pThr(175)) in association with GSK3β activation. We have now examined whether pThr(175) induces GSK3β activation and whether this leads to pathologic fibril formation through Thr(231) phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr(175)Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3β) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3β inhibitors or small hairpin RNA knockdown of GSK3β abolished fibril formation and prevented cell death. Inhibition of Thr(231) phosphorylation (Thr(231)Ala) prevented pathologic tau fibril formation, regardless of Thr(175) state, whereas Thr(231)Asp (pseudophosphorylated at Thr(231)) developed pathologic tau fibrils. Ser(235) mutations did not affect fibril formation, indicating an unprimed mechanism of Thr(231) phosphorylation. These findings suggest a mechanism of tau pathology by which pThr(175) induces GSK3β phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T. (UTSMC)

    2009-09-11

    We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

  4. PP2A(Cdc55) Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation.

    Science.gov (United States)

    Godfrey, Molly; Touati, Sandra A; Kataria, Meghna; Jones, Andrew; Snijders, Ambrosius P; Uhlmann, Frank

    2017-02-02

    In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2A(Cdc55) phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2A(Cdc55) specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Facilitating facilitators to facilitate, in problem or enquiry based learning sessions

    Directory of Open Access Journals (Sweden)

    Catherine Coelho

    2014-06-01

    Full Text Available Problem based learning (PBL has been used in dental education over the past 20 years and uses a patient case scenario to stimulate learning in a small group setting, where a trained facilitator does not teach but guides the group to bring about deep contextualized learning, to be empathetic to each other and to encourage fair and equitable contribution from individual learners. Learners are encouraged to appreciate that they individually perform better when they actively participate in the group and share resources, than when they learn in isolation (Bandura, 1977, Freire, 1972, Lave and Wenger, 1991, Kolb, 1984 and Vygotsky, 1978.

  6. The Phosphorylation-Dependent Regulation of Mitochondrial Proteins in Stress Responses

    Directory of Open Access Journals (Sweden)

    Yusuke Kanamaru

    2012-01-01

    Full Text Available To maintain cellular homeostasis, cells are equipped with precise systems that trigger the appropriate stress responses. Mitochondria not only provide cellular energy but also integrate stress response signaling pathways, including those regulating cell death. Several lines of evidence suggest that the mitochondrial proteins that function in this process, such as Bcl-2 family proteins in apoptosis and phosphoglycerate mutase family member 5 (PGAM5 in necroptosis, are regulated by several kinases. It has also been suggested that the phosphorylation-dependent regulation of mitochondrial fission machinery, dynamin-related protein 1 (Drp1, facilitates appropriate cellular stress responses. However, mitochondria themselves are also damaged by various stresses. To avoid the deleterious effects exerted by damaged mitochondria, cells remove these mitochondria in a selective autophagic degradation process called mitophagy. Interestingly, several kinases, such as PTEN-induced putative kinase 1 (PINK1 in mammals and stress-responsive mitogen-activated protein (MAP kinases in yeast, have recently been shown to be involved in mitophagy. In this paper, we focus on the phosphorylation-dependent regulation of mitochondrial proteins and discuss the roles of this regulation in the mitochondrial and cellular stress responses.

  7. A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination.

    Science.gov (United States)

    Luo, Kuntian; Li, Lei; Li, Yunhui; Wu, Chenming; Yin, Yujiao; Chen, Yuping; Deng, Min; Nowsheen, Somaira; Yuan, Jian; Lou, Zhenkun

    2016-12-01

    Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway. © 2016 Luo et al.; Published by Cold Spring Harbor Laboratory Press.

  8. Detecting KaiC phosphorylation rhythms of the cyanobacterial circadian oscillator in vitro and in vivo

    Science.gov (United States)

    Kim, Yong-Ick; Boyd, Joseph S.; Espinosa, Javier; Golden, Susan S.

    2016-01-01

    The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins, KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphoryation states of KaiC and other phosphorylated proteins from in vivo samples. PMID:25662456

  9. Phosphorylation of FOXP3 by LCK downregulates MMP9 expression and represses cell invasion.

    Directory of Open Access Journals (Sweden)

    Kumiko Nakahira

    Full Text Available Forkhead Box P3 (FOXP3 is a member of the forkhead/winged helix family of the transcription factors and plays an important role not only as a master gene in T-regulatory cells, but also as a tumor suppressor. In this study, we identified lymphocyte-specific protein tyrosine kinase (LCK, which correlates with cancer malignancy, as a binding partner of FOXP3. FOXP3 downregulated LCK-induced MMP9, SKP2, and VEGF-A expression. We observed that LCK phosphorylated Tyr-342 of FOXP3 by immunoprecipitation and in vitro kinase assay, and the replacement of Tyr-342 with phenylalanine (Y342F abolished the ability to suppress MMP9 expression. Although FOXP3 decreased the invasive ability induced by LCK in MCF-7 cells, Y342F mutation in FOXP3 diminished this suppressive effect. Thus we demonstrate for the first time that LCK upregulates FOXP3 by tyrosine phosphorylation, resulting in decreased MMP9, SKP2, and VEGF-A expression, and suppressed cellular invasion. We consider that further clarification of transcriptional mechanism of FOXP3 may facilitate the development of novel therapeutic approaches to suppress cancer malignancy.

  10. Aurora A phosphorylates MCAK to control ran-dependent spindle bipolarity.

    Science.gov (United States)

    Zhang, Xin; Ems-McClung, Stephanie C; Walczak, Claire E

    2008-07-01

    During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

  11. Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.

    Science.gov (United States)

    Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela

    2014-10-07

    Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.

  12. Phosphorylation of ATPase subunits of the 26S proteasome.

    Science.gov (United States)

    Mason, G G; Murray, R Z; Pappin, D; Rivett, A J

    1998-07-01

    The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

  13. Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity.

    Science.gov (United States)

    Bertling, Enni; Englund, Jonas; Minkeviciene, Rimante; Koskinen, Mikko; Segerstråle, Mikael; Castrén, Eero; Taira, Tomi; Hotulainen, Pirta

    2016-05-11

    Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance. Dendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for

  14. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro.

    Directory of Open Access Journals (Sweden)

    Rakesh Verma

    Full Text Available Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.

  15. Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells.

    Science.gov (United States)

    Nili, U; de Wit, H; Gulyas-Kovacs, A; Toonen, R F; Sørensen, J B; Verhage, M; Ashery, U

    2006-12-01

    Activation of protein kinase C (PKC) after robust stimulation is necessary for vesicle pool replenishment in secretory cells. Here we studied the contribution of a prominent downstream PKC target, Munc18-1, to this process in bovine chromaffin cells. In these cells, both activation of endogenous PKC and overexpressing of Munc18-1 promote vesicle pool replenishment after an extensive stimulation. In order to study the physiological relevance of PKC-dependent Munc18-1 phosphorylation, we generated two Munc18-1 phospho-mutants; one that mimics a constitutively PKC-phosphorylated Munc18-1 (i.e. a phosphomimetic mutant; Munc18-1(S313D)) and a second that cannot be PKC-phosphorylated (Munc18-1(3A)). Overexpression of Munc18-1(3A) caused a significant decrease in vesicle pool replenishment following a depleting stimulation, while Munc18-1(S313D) caused a significant increase in vesicle pool replenishment. These findings suggested that the phosphorylation of Munc18-1 by PKC potentiates vesicle pool replenishment. This hypothesis was further strengthened by the finding that overexpression of wild type Munc18-1 in the presence of a PKC inhibitor caused a significant reduction in vesicle pool replenishment, similar to that observed with Munc18-1(3A). Moreover, overexpression of Munc18-1(S313D) in the presence of the PKC inhibitor partly alleviated this attenuation, elucidating Munc18-1's unique contribution to vesicle pool replenishment. Finally, we demonstrate that Munc18-1 promotes vesicle docking in a phosphorylation-independent manner. This is deduced from the findings that both the wild type and the two Munc18-1 phospho-mutants enhanced docking to the same extent in bovine chromaffin cells. We conclude that Munc18-1 facilitates docking in a PKC phosphorylation-independent manner, and that its phosphorylation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage.

  16. Facilitating Value Co-Creation

    DEFF Research Database (Denmark)

    Veith, Anne; Assaf, Albert; Josiassen, Alexander

    2013-01-01

    ) introduced a new dominant logic in the marketing literature, the Service-Dominant Logic (S-D Logic), in which service, interactions, and enhanced experiences help create value, and this potential for value is what attracts consumers. Therefore organizations must be customer-centric in order to facilitate...... unique, positive experiences. As the name indicates, both organizations and consumers (should) obtain value when co-creating, which is why both parties are willing to increase their degree of involvement, e.g. spending more resources, sharing tacit knowledge, etc., because a high degree of involvement......). Therefore, for instance, being part of the process is a key incentive for consumers. Postmodern consumers' search for unique experiences calls for individualization, personalization, etc. Although Prahalad & Ramaswamy (2004), Karpen et al. (2008), and Karpen et al. (2011) have presented S-D Logic...

  17. Facilitating Collaboration through Design Games

    DEFF Research Database (Denmark)

    Brandt, Eva; Messeter, Jørn

    2004-01-01

    In recent years both companies and research communities call for collaborative work practices and user-centered approaches in various design fields. There are several challenges and issues to take into consideration. For instance there is a need to find ways of collaborating across various...... in collaboration with industrial partners and potential users, and use of the games in three educational settings.The overall aim of the design games is to help facilitate a user-centered design process for cross-disciplinary design groups early in the design process. Framing collaborative design activities...... understanding of the development task. This paper presents a set of four design games, which offers solutions to the challenges mentioned. The design games have been developed in the Space Studio during several projects and years. Here experiences are discussed on the basis of two research projects carried out...

  18. Facilitating Conversations about Managerial Identities

    DEFF Research Database (Denmark)

    Madsen, Mona Toft

    for identity work was introduced. The empirical starting point was progressive performativity and constructionistic process consultation with the intention to engage with a practical context, a company. The empirical study was based on a) individual interviews with three middle managers in a project......-based organization in the engineering consulting sector b) a reflection meeting, where the same three managers were gathered, and conversations were facilitated based on identity work in the context of earlier interviews. More specifically, three themes were discussed; flat organizational structure, tensions between...... project work and professional development, and the role of Department Heads. Theoretically, the study contributes to discussions on the need for legitimizing different mixtures of bureaucratic and post bureaucratic ideals. Methodological reflections are made in the discussion as well....

  19. Facilitating submetering implementation. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Bowers, M.A.

    1996-05-01

    Residential submetering is the measurement and billing of electric use in individual apartments in master-metered buildings. In master-metered building situations, residents do not bear electricity costs in proportion to consumption levels. As a result, studies have confirmed that residents in master-metered buildings tend to consume more electricity than residents with individual apartment metering, and have established electrical submetering as an effective energy conservation measure. The New York State Energy Research & Development Authority (NYSERDA) has commissioned a project called Facilitating Submetering Implementation to identify and analyze barriers to the implementation of residential electrical submetering in New York and to formulate recommendations that would facilitate the removal of these barriers, streamlining the process. Experienced professionals in the technical, legal, regulatory, analytical, financial, and other aspects of submetering were retained to interview key interested parties and conduct public forums. This and other data were then analyzed to ascertain the barriers to submetering and develop recommendations designed to reduce or eliminate these barriers. The key barriers to submetering implementation were found to be the Public Service Commission (PSC) requirement for a vote of a majority of shareholders (for coops and condos) and the high initial cost that cannot easily be recouped by owners of both rental and shareholder-owned buildings. The key recommendations are to repeal the voting requirement, maintain the utility incentives, adopt a uniform dispute resolution mechanism, and increase awareness through an Ad-hoc Submetering Committee and supporting educational materials. Other funding sources not fully available can also be made available with regulatory agency support.

  20. Expression and phosphorylation of the Na+-Cl- cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1.

    Science.gov (United States)

    Vallon, Volker; Schroth, Jana; Lang, Florian; Kuhl, Dietmar; Uchida, Shinichi

    2009-09-01

    The Na-Cl cotransporter NCC is expressed in the distal convoluted tubule, activated by phosphorylation, and has been implicated in renal NaCl and K(+) homeostasis. The serum and glucocorticoid inducible kinase 1 (SGK1) contributes to renal NaCl retention and K(+) excretion, at least in part, by stimulating the epithelial Na(+) channel and Na(+)-K(+)-ATPase in the downstream segments of aldosterone-sensitive Na(+)/K(+) exchange. In this study we confirmed in wild-type mice (WT) that dietary NaCl restriction increases renal NCC expression and its phosphorylation at Thr(53), Thr(58), and Ser(71), respectively. This response, however, was attenuated in mice lacking SGK1 (Sgk1(-/-)), which may contribute to impaired NaCl retention in those mice. Total renal NCC expression and phosphorylation at Thr(53), Thr(58), and Ser(71) in WT were greater under low- compared with high-K(+) diet. This finding is consistent with a regulation of NCC to modulate Na(+) delivery to downstream segments of Na(+)/K(+) exchange, thereby modulating K(+) excretion. Dietary K(+)-dependent variation in renal expression of total NCC and phosphorylated NCC were not attenuated in Sgk1(-/-) mice. In fact, high-K(+) diet-induced NCC suppression was enhanced in Sgk1(-/-) mice. The hyperkalemia induced in Sgk1(-/-) mice by a high-K(+) diet may have augmented NCC suppression, thereby increasing Na(+) delivery and facilitating K(+) excretion in downstream segments of impaired Na(+)/K(+) exchange. In summary, changes in NaCl and K(+) intake altered NCC expression and phosphorylation, an observation consistent with a role of NCC in NaCl and K(+) homeostasis. The two maneuvers dissociated plasma aldosterone levels from NCC expression and phosphorylation, implicating additional regulators. Regulation of NCC expression and phosphorylation by dietary NaCl restriction appears to involve SGK1.

  1. Cellular Functions Regulated by Phosphorylation of EGFR on Tyr845

    Directory of Open Access Journals (Sweden)

    Ken-ichi Sato

    2013-05-01

    Full Text Available The Src gene product (Src and the epidermal growth factor receptor (EGFR are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845 in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase. A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation.

  2. Determining in vivo phosphorylation sites using mass spectrometry.

    Science.gov (United States)

    Breitkopf, Susanne B; Asara, John M

    2012-04-01

    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High-resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole-cell and tissue extracts. For the first, endogenous or epitope-tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO(2)) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole-cell lysate, followed by peptide fractionation by strong cation-exchange chromatography, phosphopeptide enrichment by IMAC or TiO(2), and LC-MS/MS. Alternatively, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment, and LC-MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC-MS/MS.

  3. Tau phosphorylation affects its axonal transport and degradation

    Science.gov (United States)

    Rodríguez-Martín, Teresa; Cuchillo-Ibáñez, Inmaculada; Noble, Wendy; Nyenya, Fanon; Anderton, Brian H.; Hanger, Diane P.

    2013-01-01

    Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases. PMID:23601672

  4. Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

    LENUS (Irish Health Repository)

    Yates, Darran M

    2009-04-01

    Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

  5. LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

    Science.gov (United States)

    Jaleel, Mahaboobi; Nichols, R. Jeremy; Deak, Maria; Campbell, David G.; Gillardon, Frank; Knebel, Axel; Alessi, Dario R.

    2007-01-01

    Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop

  6. Evaluation of the Facilitated Communication Pilot

    Science.gov (United States)

    Cooper-Martin, Elizabeth

    2014-01-01

    The Office of Special Education and Student Services asked the Office of Shared Accountability to evaluate the "Facilitated Communication Pilot." In facilitated communication (FC), people with communication impairments express themselves by typing with the aid of a communication partner, called a facilitator, who provides physical (and…

  7. Reduced coupling of oxidative phosphorylation in vivo precedes electron transport chain defects due to mild oxidative stress in mice.

    Directory of Open Access Journals (Sweden)

    Michael P Siegel

    Full Text Available Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ treatment of wild type mice and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1(-/- models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.

  8. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    DEFF Research Database (Denmark)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco

    2014-01-01

    In S. cerevisiae, the 5'-3' exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood....... Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription...... termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII...

  9. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  10. Exploring the intramolecular phosphorylation sites in human Chk2

    DEFF Research Database (Denmark)

    Olsen, Birgitte B; Larsen, Martin R; Boldyreff, Brigitte;

    2008-01-01

    A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time....... Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except...... for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria....

  11. Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1

    Directory of Open Access Journals (Sweden)

    Morderer D. Ye.

    2014-01-01

    Full Text Available ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography, in vitro kinase reaction, Western blotting, gel staining with fluorescent stains. Results. We show that the fraction of calmodulin-binding proteins is able to phosphorylate the recombinant fragments encoding the coiled-coil region and the SH3 domain-containing region of ITSN1 in the presence of Ca ions and calmodulin. Conclusions. The coiled-coil region and the SH3 domain-containing region of ITSN1 undergo Ca/calmodulin-dependent phosphorylation in vitro, suggesting a possible regulation of ITSN1 by Ca signaling.

  12. Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

    Science.gov (United States)

    Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

    2013-01-01

    Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

  13. Regulatory phosphorylation of Ikaros by Bruton's tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Hong Ma

    Full Text Available Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4 within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros.

  14. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    Energy Technology Data Exchange (ETDEWEB)

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.

    2011-12-31

    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  15. Surface-phosphorylated copolymer promotes direct bone bonding.

    Science.gov (United States)

    Gopalakrishnanchettiyar, Sailaja S; Mohanty, Mira; Kumary, Thrikkovil V; Valappil, Mohanan P; Parameshwaran, Ramesh; Varma, Harikrishna K

    2009-10-01

    The bone bonding potential of surface-phosphorylated poly (2-hydroxyethyl methacrylate-co-methyl methacrylate) [poly (HEMA-co-MMA)] has been investigated and compared with commercially available poly (methyl methacrylate) bone cement (CMW1 radiopaque, Depuy; Johnson & Johnson, Blackpool, Lancashire, England, United Kingdom) as control. Poly (HEMA-co-MMA) is synthesized by free radical-initiated copolymerization and surface functionalized by phosphorylation. The X-ray photoelectron spectroscopy confirms the presence of surface-bound phosphate groups on poly (HEMA-co-MMA). The surface-phosphorylated poly (HEMA-co-MMA) promotes in vitro biomineralization, cell viability, cell adhesion, and expression of bone-specific markers such as osteocalcin and alkaline phosphatase. The bone implantation study performed in rabbits as per ISO 10993-6; 1994 (E) shows that surface-phosphorylated poly (HEMA-co-MMA) elicits bone bonding and new bone formation. New woven bone trabeculae are formed at the defect site of surface-phosphorylated poly (HEMA-co-MMA) within 1 week, while for control sample, inflammatory cells--predominantly, macrophages, fibroblasts, and fibrocytes--are present at the cortical margins around the defect. The 4 and 12 weeks postimplantation results show that the major part of the defects around the surface-phosphorylated poly (HEMA-co-MMA) implant is bridged with new woven bone, with significant remodeling (evident from resorption bays) along both the margins of the defect, but for control implants, the defects are only partially closed, with slight remodeling along the margins, but most of them are separated by fibrous tissue.

  16. Facilitating peer learning in study groups

    DEFF Research Database (Denmark)

    Adriansen, Hanne Kirstine; Madsen, Lene Møller

    2009-01-01

    the preliminary results from the facilitated study groups. After one term (February-May), student satisfaction with both the social and the disciplinary environment had increased. The project shows how academic and social integration can be achieved with minimum faculty member involvement. This is done by relying...... 'Facilitating study environment' at one of DPU's educations in spring 2009. The pilot project consisted of three elements: Facilitated study groups, a student bar with facilitated activities, and academic identity events. Subsequently, we have studied students' experiences with the project. This paper outlines...... on the students' own resources, using peer-learning and facilitating these activities....

  17. Social facilitation of wound healing.

    Science.gov (United States)

    Detillion, Courtney E; Craft, Tara K S; Glasper, Erica R; Prendergast, Brian J; DeVries, A Courtney

    2004-09-01

    It is well documented that psychological stress impairs wound healing in humans and rodents. However, most research effort into influences on wound healing has focused on factors that compromise, rather than promote, healing. In the present study, we determined if positive social interaction, which influences hypothalamic-pituitary-adrenal (HPA) axis activity in social rodents, promotes wound healing. Siberian hamsters received a cutaneous wound and then were exposed to immobilization stress. Stress increased cortisol concentrations and impaired wound healing in isolated, but not socially housed, hamsters. Removal of endogenous cortisol via adrenalectomy eliminated the effects of stress on wound healing in isolated hamsters. Treatment of isolated hamsters with oxytocin (OT), a hormone released during social contact and associated with social bonding, also blocked stress-induced increases in cortisol concentrations and facilitated wound healing. In contrast, treating socially housed hamsters with an OT antagonist delayed wound healing. Taken together, these data suggest that social interactions buffer against stress and promote wound healing through a mechanism that involves OT-induced suppression of the HPA axis. The data imply that social isolation impairs wound healing, whereas OT treatment may ameliorate some effects of social isolation on health.

  18. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...

  19. The phosphorylation pattern of bovine heart complex I subunits

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Sardanelli, Anna Maria; Signorile, Anna;

    2007-01-01

    The phosphoproteome of bovine heart complex I of the respiratory chain has been analysed with a procedure based on nondenaturing gel electrophoretic separation of complex I from small quantities of mitochondria samples, in-gel digestion, in combination with phosphopeptide enrichment by titanium...... dioxide and MS. The results, complemented by analyses of purified samples of complex I, showed phosphorylation of five subunits of the complex, 42 kDa (human gene NDUFA10), ESSS, B14.5a (human gene NDUFA7), B14.5b (human gene NDUFC2) and B16.6 (GRIM-19). MS also revealed the presence of phosphorylated...

  20. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj

    2009-01-01

    -substrate specificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms....... Furthermore, a protocol for the use of the artificial neural network based predictors, NetPhos and NetPhosK, is provided. Finally, we point to possible developments with the intention of providing the community with improved and additional phosphorylation predictors for large-scale modeling of cellular...... signaling networks....

  1. Chemical phosphorylation of deoxyribonucleosides and thermolytic DNA oligonucleotides.

    Science.gov (United States)

    Ausín, Cristina; Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L

    2006-10-01

    The phosphorylating reagent bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid-phase synthesis of deoxyribonucleoside 5'-/3'-phosphate or -thiophosphate monoesters and oligonucleotide 5'-phosphate/-thiophosphate monoesters. Bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions ( approximately 25 degrees C) for potential therapeutic applications.

  2. Laboratory reengineering facilitates cost management.

    Science.gov (United States)

    Ellis, J E; Moser, L H

    1998-08-01

    In 1993, The Medical University of South Carolina (MUSC) in Charleston undertook a change management initiative to achieve a more cost-competitive position in its market and become a more attractive partner for a possible future affiliation with another provider organization. A key element of this change process was a reorganization of the medical center's laboratory department. Through consolidation of MUSC's separate laboratories and the introduction of a new, more efficient chemistry analyzer system, the medical center realized annual laboratory savings of approximately $1.3 million.

  3. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.

    Directory of Open Access Journals (Sweden)

    Lori K Van Ness

    Full Text Available Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

  4. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.

    Science.gov (United States)

    Van Ness, Lori K; Jayaraman, Dhileepkumar; Maeda, Junko; Barrett-Wilt, Gregory A; Sussman, Michael R; Ané, Jean-Michel

    2016-01-01

    Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

  5. D6 PROTEIN KINASE activates auxin transport-dependent growth and PIN-FORMED phosphorylation at the plasma membrane.

    Science.gov (United States)

    Barbosa, Inês C R; Zourelidou, Melina; Willige, Björn C; Weller, Benjamin; Schwechheimer, Claus

    2014-06-23

    The directed cell-to-cell transport of the phytohormone auxin by efflux and influx transporters is essential for proper plant growth and development. Like auxin efflux facilitators of the PIN-FORMED (PIN) family, D6 PROTEIN KINASE (D6PK) from Arabidopsis thaliana localizes to the basal plasma membrane of many cells, and evidence exists that D6PK may directly phosphorylate PINs. We find that D6PK is a membrane-bound protein that is associated with either the basal domain of the plasma membrane or endomembranes. Inhibition of the trafficking regulator GNOM leads to a rapid internalization of D6PK to endomembranes. Interestingly, the dissociation of D6PK from the plasma membrane is also promoted by auxin. Surprisingly, we find that auxin transport-dependent tropic responses are critically and reversibly controlled by D6PK and D6PK-dependent PIN phosphorylation at the plasma membrane. We conclude that D6PK abundance at the plasma membrane and likely D6PK-dependent PIN phosphorylation are prerequisites for PIN-mediated auxin transport.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  7. Evaluation of novel supports for selective and efficient enrichment of phosphorylated peptides

    CSIR Research Space (South Africa)

    Bensadek, D

    2011-06-01

    Full Text Available A series of novel emulsion based supports MagReSynTM TiO 2 (A-D) were evaluated using 32P labelled phosphopeptides and nano LC-MS/MS. The performance of MagReSynTM TiO 2 (D) was found to be significantly better in terms of selectivity and specificity...

  8. Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    ZHANG,Jian-Chen(张建臣); CAO,Shu-Xiaa(曹书霞); XU,Juna(徐军); LIAO,Xin-Cheng(廖新成); ZHAO,Yu-Fen(赵玉芬)

    2004-01-01

    Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M+H]+, [M+Na]+ and [M-H]- ions of phosphoryl amino acids were summarized. In addition to several similar patterns,each of them showed its characteristic fragmention.

  9. Juggling Efficiency

    DEFF Research Database (Denmark)

    Andersen, Rikke Sand; Vedsted, Peter

    2015-01-01

    efficiency in order to deal with uncertainties and meet more complex or unpredictable needs. Lastly, building on the empirical case of cancer diagnostics, we discuss the implications of the pervasiveness of the logic of efficiency in the clinical setting and argue that provision of medical care in today......'s primary care settings requires careful balancing of increasing demands of efficiency, greater complexity of biomedical knowledge and consideration for individual patient needs....

  10. Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

    Science.gov (United States)

    Marlow, Victoria L.; Porter, Michael; Hobley, Laura; Kiley, Taryn B.; Swedlow, Jason R.; Davidson, Fordyce A.

    2014-01-01

    Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation. PMID:24123822

  11. Phosphorylation of the SQ H2A.X motif is required for proper meiosis and mitosis in Tetrahymena thermophila.

    Science.gov (United States)

    Song, Xiaoyuan; Gjoneska, Elizabeta; Ren, Qinghu; Taverna, Sean D; Allis, C David; Gorovsky, Martin A

    2007-04-01

    Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro- and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.

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

    Science.gov (United States)

    Frades, Itziar; Andreasson, Erik

    2016-04-01

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

  13. Construction and Analysis of N-phosphoryl Peptide Libraries

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Ming Yu NIU; Kui LU; Xin Cheng LIAO; Yu Fen ZHAO

    2004-01-01

    N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

  14. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

    2016-04-06

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  15. Genetic defects in the oxidative phosphorylation (OXPHOS) system.

    NARCIS (Netherlands)

    Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.

    2004-01-01

    The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual gene

  16. Plk phosphorylation regulates the microtubule-stabilizing protein TCTP.

    Science.gov (United States)

    Yarm, Frederic R

    2002-09-01

    The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

  17. Spatial separation of Plk1 phosphorylation and activity

    Directory of Open Access Journals (Sweden)

    Wytse eBruinsma

    2015-06-01

    Full Text Available Polo-like kinase 1 (Plk1 is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2 and at the end of mitosis it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.

  18. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.

    2003-01-01

    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...

  19. Histone 3 s10 phosphorylation: "caught in the R loop!".

    Science.gov (United States)

    Skourti-Stathaki, Konstantina; Proudfoot, Nicholas J

    2013-11-21

    In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.

  20. Annealing properties of potato starches with different degrees of phosphorylation

    DEFF Research Database (Denmark)

    Muhrbeck, Per; Svensson, E

    1996-01-01

    Changes in the gelatinization temperature interval and gelatinization enthalpy with annealing time at 50 degrees C were followed for a number of potato starch samples, with different degrees of phosphorylation, using differential scanning calorimetry. The gelatinization temperature increased with...... and crystalline structure of amylopectin helices. (C) 1997 Elsevier Science Ltd....

  1. Genetic defects in the oxidative phosphorylation (OXPHOS) system.

    NARCIS (Netherlands)

    Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.

    2004-01-01

    The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual

  2. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  3. Enteric GFAP expression and phosphorylation in Parkinson's disease

    NARCIS (Netherlands)

    Clairembault, Thomas; Kamphuis, W.; Leclair-Visonneau, Laurène; Rolli-Derkinderen, Malvyne; Coron, Emmanuel; Neunlist, Michel; Hol, Elly M; Derkinderen, Pascal

    2014-01-01

    Enteric glial cells (EGCs) are in many respects similar to astrocytes of the central nervous system and express similar proteins including glial fibrillary acidic protein (GFAP). Changes in GFAP expression and/or phosphorylation have been reported during brain damage or central nervous system degene

  4. Phosphorylation of as1-casein is regulated by different genes

    NARCIS (Netherlands)

    Bijl, E.; Valenberg, van H.J.F.; Huppertz, T.; Hooijdonk, van A.C.M.; Bovenhuis, H.

    2014-01-01

    Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the i

  5. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission

    Science.gov (United States)

    Jungas, Thomas; Perchey, Renaud T.; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud

    2016-01-01

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  6. Circadian KaiC phosphorylation: a multi-layer network.

    Directory of Open Access Journals (Sweden)

    Congxin Li

    2009-11-01

    Full Text Available Circadian KaiC phosphorylation in cyanobacteria reconstituted in vitro recently initiates a series of studies experimentally and theoretically to explore its mechanism. In this paper, we report a dynamic diversity in hexameric KaiC phosphoforms using a multi-layer reaction network based on the nonequivalence of the dual phosphorylation sites (S431 and T432 in each KaiC subunit. These diverse oscillatory profiles can generate a kaleidoscopic phase modulation pattern probably responsible for the genome-wide transcription rhythms directly and/or indirectly in cyanobacteria. Particularly, our model reveals that a single KaiC hexamer is an energy-based, phosphorylation-dependent and self-regulated circadian oscillator modulated by KaiA and KaiB. We suggest that T432 is the main regulator for the oscillation amplitude, while S431 is the major phase regulator. S431 and T432 coordinately control the phosphorylation period. Robustness of the Kai network was examined by mixing samples in different phases, and varying protein concentrations and temperature. Similar results were obtained regardless of the deterministic or stochastic method employed. Therefore, the dynamic diversities and robustness of Kai oscillator make it a qualified core pacemaker that controls the cellular processes in cyanobacteria pervasively and accurately.

  7. ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

    NARCIS (Netherlands)

    Zwiers, H; Veldhuis, H D; Schotman, P; Gispen, W H

    1976-01-01

    Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated(32)P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide

  8. The phosphatase calcineurin regulates pathological TDP-43 phosphorylation.

    Science.gov (United States)

    Liachko, Nicole F; Saxton, Aleen D; McMillan, Pamela J; Strovas, Timothy J; Currey, Heather N; Taylor, Laura M; Wheeler, Jeanna M; Oblak, Adrian L; Ghetti, Bernardino; Montine, Thomas J; Keene, C Dirk; Raskind, Murray A; Bird, Thomas D; Kraemer, Brian C

    2016-10-01

    Detergent insoluble inclusions of TDP-43 protein are hallmarks of the neuropathology in over 90 % of amyotrophic lateral sclerosis (ALS) cases and approximately half of frontotemporal dementia (FTLD-TDP) cases. In TDP-43 proteinopathy disorders, lesions containing aggregated TDP-43 protein are extensively post-translationally modified, with phosphorylated TDP-43 (pTDP) being the most consistent and robust marker of pathological TDP-43 deposition. Abnormally phosphorylated TDP-43 has been hypothesized to mediate TDP-43 toxicity in many neurodegenerative disease models. To date, several different kinases have been implicated in the genesis of pTDP, but no phosphatases have been shown to reverse pathological TDP-43 phosphorylation. We have identified the phosphatase calcineurin as an enzyme binding to and catalyzing the removal of pathological C-terminal phosphorylation of TDP-43 in vitro. In C. elegans models of TDP-43 proteinopathy, genetic elimination of calcineurin results in accumulation of excess pTDP, exacerbated motor dysfunction, and accelerated neurodegenerative changes. In cultured human cells, treatment with FK506 (tacrolimus), a calcineurin inhibitor, results in accumulation of pTDP species. Lastly, calcineurin co-localizes with pTDP in degenerating areas of the central nervous system in subjects with FTLD-TDP and ALS. Taken together, these findings suggest calcineurin acts on pTDP as a phosphatase in neurons. Furthermore, patient treatment with calcineurin inhibitors may have unappreciated adverse neuropathological consequences.

  9. Studies on the synthesis of phosphorylated and alanylated cytokinins.

    NARCIS (Netherlands)

    Shadid, B.

    1990-01-01

    New approaches are described in this thesis towards the syntheses of phosphorylated and alanylated cytokinins.In chapter 1 a general picture of the stucture of cytokinins, their occurence in nature, their biological synthesis, their effects on plants and their chemical synthesis is described.A liter

  10. Stem rust spores elicit rapid RPG1 phosphorylation

    Science.gov (United States)

    Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutant...

  11. One-Pot Synthesis of N-Phosphoryl Amino Acids

    Institute of Scientific and Technical Information of China (English)

    GUO Xin; FU Hua; LIN Chang-Xue; ZHAO Yu-Fen

    2003-01-01

    @@ Phosphoramidates have been considered as an important class of rationally designed therapeutics especially asoligonucleotide analogs employed as antisene and antigene agents. [1] N-Phosphoryl amino acids are of biological andpharmaceutical interest, [2] and can be used as the building blocks in synthesis of polypeptides. [3

  12. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Science.gov (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  13. Decoding the phosphorylation code in Hedgehog signal transduction

    Institute of Scientific and Technical Information of China (English)

    Yongbin Chen; Jin Jiang

    2013-01-01

    Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis,and its deregulation leads to numerous human disorders including cancer.Binding of Hh to Patched (Ptc),a twelve-transmembrane protein,alleviates its inhibition of Smoothened (Smo),a seven-transmembrane protein related to G-proteincoupled receptors (GPCRs),leading to Smo phosphorylation and activation.Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a fulllength activator,leading to derepression/activation of Hh target genes.Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli,and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities.In this review,we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction,and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms.

  14. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A

    1997-01-01

    Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus seque...

  15. The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

    Science.gov (United States)

    Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

    2017-08-22

    SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

  16. Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity.

    Science.gov (United States)

    Wu, Qian; Sun, Miao; Bernard, Laura P; Zhang, Huaye

    2017-09-29

    Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Bioinformatics Study of Cancer-Related Mutations within p53 Phosphorylation Site Motifs

    Directory of Open Access Journals (Sweden)

    Xiaona Ji

    2014-07-01

    Full Text Available p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.

  18. The crystal structure of phosphorylated MAPK13 reveals common structural features and differences in p38 MAPK family activation.

    Science.gov (United States)

    Yurtsever, Zeynep; Scheaffer, Suzanne M; Romero, Arthur G; Holtzman, Michael J; Brett, Tom J

    2015-04-01

    The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.

  19. PR65A phosphorylation regulates PP2A complex signaling.

    Directory of Open Access Journals (Sweden)

    Kumar Kotlo

    Full Text Available Serine-threonine Protein phosphatase 2 A (PP2A, a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac; a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa; and one of at least 18 associated variable regulatory proteins (B subunits classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314. Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A and non-phosphorylated (N-PR65A amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A, elongation factor 2, heat shock protein 60 (HSP60, NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.

  20. A redox-regulated tyrosine phosphorylation cascade in rat spermatozoa.

    Science.gov (United States)

    Lewis, B; Aitken, R J

    2001-01-01

    Rat spermatozoa from both the caput and cauda epididymidis were shown to generate superoxide anion (O2-.) both spontaneously and following stimulation with NAD(P)H. Caput spermatozoa gave a significantly greater O2- response to NADPH stimulation than caudal cells, whereas in both cell types the responses to exogenous NADPH and NADH were approximately equivalent. Analysis of H2O2 production revealed that this oxidant was generated only by caudal epididymal cells and only in these cells did the stimulation of reactive oxygen species (ROS) production with NADPH lead to an increase in tyrosine phosphorylation. Stimulation of ROS production with NADPH increased intracellular cyclic adenosine monophosphate (cAMP) levels in both caput and caudal epididymal cells, but only in caudal cells did cAMP stimulate tyrosine phosphorylation, in keeping with the NADPH results. On the basis of these findings we propose that tyrosine phosphorylation in rat spermatozoa is driven by ROS acting via 2 different but complementary mechanisms; O2-. stimulates tyrosine kinase activity indirectly through the elevation of intracellular cAMP while H2O2 acts directly on the kinase/phosphatase system, stimulating the former and inhibiting the latter. Zinc was examined as a potential regulator of this signal transduction cascade and was shown to suppress tyrosine phosphorylation in caput cells but to promote this activity in caudal spermatozoa, possibly through an inhibitory effect on tyrosine phosphatase activity. These results reveal the maturation of a redox-regulated, cAMP-mediated, signal transduction cascade during epididymal transit in the rat that is sensitive to zinc and plays a key role in the control of tyrosine phosphorylation events associated with capacitation.

  1. Disruption of GluA2 phosphorylation potentiates stress responsivity.

    Science.gov (United States)

    Ellis, Alexandra S; Fosnocht, Anne Q; Lucerne, Kelsey E; Briand, Lisa A

    2017-08-30

    Cocaine addiction is characterized by persistent craving and addicts frequently relapse even after long periods of abstinence. Exposure to stress can precipitate relapse in humans and rodents. Stress and drug use can lead to common alterations in synaptic plasticity and these commonalities may contribute to the ability of stress to elicit relapse. These common changes in synaptic plasticity are mediated, in part, by alterations in the trafficking and stabilization of AMPA receptors. Exposure to both cocaine and stress can lead to alterations in protein kinase C-mediated phosphorylation of GluA2 AMPA subunits and thus alter the trafficking of GluA2-containing AMPARs. However, it is not clear what role AMPAR trafficking plays in the interactions between stress and cocaine. The current study utilized a mouse with a point mutation within the GluA2 subunit c-terminus resulting in a disruption of PKC-mediated GluA2 phosphorylation to examine stress responsivity. Although no differences were seen in the response to a forced swim stress in naïve mice, GluA2 K882A knock-in mice exhibited an increased stress response following cocaine self-administration. Furthermore, we demonstrated that disrupting GluA2 phosphorylation increases vulnerability to stress-induced reinstatement of both cocaine seeking and cocaine-conditioned reward. Finally, GluA2 K882A knock-in mice exhibit an increased vulnerability to social defeat as indicated by increased social avoidance. Taken together these results indicate that disrupting GluA2 phosphorylation leads to increased responsivity to acute stress following cocaine exposure and increased vulnerability to chronic stress. These results highlight the GluA2 phosphorylation site as a novel target for the stress-related disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Phosphorylation of the chromatin binding domain of KSHV LANA.

    Directory of Open Access Journals (Sweden)

    Crystal Woodard

    Full Text Available The Kaposi sarcoma associated herpesvirus (KSHV latency associated nuclear antigen (LANA is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329 that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21. Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

  3. Phosphorylation regulates NCC stability and transporter activity in vivo.

    Science.gov (United States)

    Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang; Lin, Shih-Hua

    2013-10-01

    A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

  4. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  5. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  6. 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...

  7. Structure-activity approach in the reactivation of tabun-phosphorylated human acetylcholinesterase with bispyridinium para-aldoximes.

    Science.gov (United States)

    Kovarik, Zrinka; Calić, Maja; Sinko, Goran; Bosak, Anita

    2007-06-01

    We investigated interactions of bispyridinium para-aldoximes N,N'-(propano)bis(4-hydroxyiminomethyl) pyridinium bromide (TMB-4), N,N'-(ethano)bis(4-hydroxyiminomethyl)pyridinium methanosulphonate (DMB-4), and N,N'-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride (MMB-4) with human erythrocyte acetylcholinesterase phosphorylated by tabun. We analysed aldoxime conformations to determine the flexibility of aldoxime as an important feature for binding to the acetylcholinesterase active site. Tabun-inhibited human erythrocyte acetylcholinesterase was completely reactivated only by the most flexible bispyridinium aldoxime - TMB-4 with a propylene chain between two rings. Shorter linkers than propylene (methylene or ethylene) as in MMB-4 and DMB-4 did not allow appropriate orientation in the active site, and MMB-4 and DMB-4 were not efficient reactivators of tabun-phosphorylated acetylcholinesterase. Since aldoximes are also reversible inhibitors of native acetylcholinesterase, we determined dissociation constants and their protective index against acetylcholinesterase inactivation by tabun.

  8. Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

    Science.gov (United States)

    Novak, Zsofia A; Wainman, Alan; Gartenmann, Lisa; Raff, Jordan W

    2016-06-20

    Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome.

  9. The social facilitation of eating. A review.

    Science.gov (United States)

    Herman, C Peter

    2015-03-01

    The social facilitation of eating (i.e., people eating more in groups than when alone) has been studied for about three decades now. In this paper, we review the empirical research (diary studies, observational studies, and experimental studies) of social facilitation, attending to factors that increase or decrease socially facilitated eating. We also review the various explanations (e.g., "time extension") that have been offered for the effect and offer our own speculations as to the underlying mechanisms. Further, we discuss promising directions for future research on the social facilitation of eating. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Ghana - Land Tenure Facilitation Impact Evaluation

    Data.gov (United States)

    Millennium Challenge Corporation — The MCC-supported Land Title Facilitation Activity (LTF) in Ghana was designed to increase investment and productivity by strengthening property rights. In Ghana,...

  11. Organisational Socialisation Facilitates Overseas Assignment

    Directory of Open Access Journals (Sweden)

    Pei-Chen (Chiu-Yi/Joy Lee

    2014-09-01

    Full Text Available Cross-cultural adjustment is the biggest challenge that derails expatriate success. In responding to the importance of Taiwanese expatriates’ adjustment in Mainland China, this study tests, as a critical factor, Socialisation affecting the adjustment of Taiwanese expatriates in Mainland China. As a result, this study critically reviews previous studies on expatriate success, shedding light on key factors in the area of expatriate adjustment and providing multiple guidelines for human resource supervisors and senior executives hoping to ensure expatriate success. The definitions of success and methodological approaches are discussed, and suggestions for improving empirical research in this area are presented. Academic researchers and international enterprises can perhaps clarify whether all international expatriates are affected by the factor as organisational socialisation, and also whether the proposed model can be utilised in studying expatriates in different professional areas from those of this study. Finally, the results and their implications for research and the business world are discussed. This may assist not only Taiwanese MNCs but also global enterprises to perform their international business more efficiently in Mainland China.

  12. Computational identification of interplay between phosphorylation and O-β-glycosylation of human occludin as potential mechanism to impair hepatitis C virus entry.

    Science.gov (United States)

    Butt, Azeem Mehmood; Feng, Dandan; Nasrullah, Izza; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang; Lu, Jun

    2012-08-01

    Hepatitis C virus (HCV) is one of the leading causes of liver diseases. Several host factors that facilitate the attachment and entry of HCV have been discovered, of which human occludin seems to be the most promising. Studies have shown that activity of occludin is dependent upon its phosphorylation status, and that during HCV infection deregulation of phosphorylated occludin collectively leads to a reduction in tight junction (TJ) integrity of hepatocytes and favors HCV entry. However, detailed information of the posttranslational modifications (PTMs) of occludin still remains largely unknown. In addition to phosphorylation, serine/threonine residues of several proteins are also regulated by a unique type of modification known as O-β-glycosylation and this crosstalk serves as a functional switch. To identify the O-β-glycosylation potential and how interplay between phosphorylation and O-β-glycosylation can be exploited for the inhibition of HCV entry, here we report a computational analysis of PTMs of human occludin. Several conserved phosphorylation residues and kinases that can alter the ability of occludin to regulate the integrity of TJs were identified. In addition to previously reported Tyr residues, two additional Tyr residues (Tyr29 and Tyr287) were identified as target sites of Src kinase. To our knowledge, this is the first study to report the O-β-GlcNAc potential of occludin and target sites of ERK (Ser8, Ser310, and Thr345), GSK-3 (Ser8, Ser341) and Cdk5 (Thr376). Furthermore, based on findings from this study, a potential novel interplay between phosphorylation and O-β-glycosylation at the two Yin Yang sites (Ser408 and Ser490) is also proposed.

  13. (S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.

    Science.gov (United States)

    Moutal, Aubin; François-Moutal, Liberty; Perez-Miller, Samantha; Cottier, Karissa; Chew, Lindsey Anne; Yeon, Seul Ki; Dai, Jixun; Park, Ki Duk; Khanna, May; Khanna, Rajesh

    2016-04-01

    The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions.

  14. Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-cyclin B kinases to regulate lipid synthesis in yeast.

    Science.gov (United States)

    Su, Wen-Min; Han, Gil-Soo; Casciano, Jessica; Carman, George M

    2012-09-28

    Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.

  15. Protein tyrosine phosphatase PTPRB regulates Src phosphorylation and tumour progression in NSCLC.

    Science.gov (United States)

    Qi, Yinliang; Dai, Yuanchang; Gui, Shuyu

    2016-10-01

    Protein tyrosine-phosphatases (PTPs) play important roles in various biological processes. Deregulation in PTP function has been implicated in carcinogenesis and tumour progression in many cancer types. However, the role of protein tyrosine phosphatase receptor type B (PTPRB) in non-small-cell lung cancer (NSCLC) tumorigenesis has not been investigated. Lentiviral vector expressing PTPRB cDNA or shRNA was infected into A549 and H1299 cell lines, followed by cell proliferation, colony formation, soft agar and invasion assays. A549 xenograft mouse model was used to evaluate in vivo function of PTPRB. Quantitative polymerase chain reaction (PCR) was used to measure PTPRB expression in NSCLC patient samples. Kaplan Meier analysis was performed to assess association between PTPRB expression and patient overall survival (OS). Multivariate analysis was performed to evaluate prognostic significance of PTPRB. Overexpression of PTPRB reduced cell proliferation rate, colony formation efficiency, soft agar growth and cell invasion in A549 and H1299 cells, as well as tumour growth rate in A549 xenograft. Knockdown of PTPRB increased Src phosphorylation and cell invasion, which was reversed by Src inhibitor PP2. Additionally, PTPRB was down-regulated in NSCLC patient and was associated with patient OS. PTPRB regulates Src phosphorylation and tumorigenesis in NSCLC. PTPRB may serve as an independent prognostic biomarker for NSCLC patients.

  16. H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Monika Podhorecka

    2010-01-01

    Full Text Available Double-strand breaks (DSBs are the most deleterious DNA lesions, which, if left unrepaired, may have severe consequences for cell survival, as they lead to chromosome aberrations, genomic instability, or cell death. Various physical, chemical, and biological factors are involved in DSB induction. Cells respond to DNA damage by activating the so-called DNA damage response (DDR, a complex molecular mechanism developed to detect and repair DNA damage. The formation of DSBs triggers activation of many factors, including phosphorylation of the histone variant H2AX, producing γH2AX. Phosphorylation of H2AX plays a key role in DDR and is required for the assembly of DNA repair proteins at the sites containing damaged chromatin as well as for activation of checkpoints proteins which arrest the cell cycle progression. In general, analysis of γH2AX expression can be used to detect the genotoxic effect of different toxic substances. When applied to clinical samples from cancer patients, evaluation of γH2AX levels may allow not only to monitor the efficiency of anticancer treatment but also to predict of tumor cell sensitivity to DNA damaging anticancer agents and toxicity of anticancer treatment toward normal cells.

  17. Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis.

    Science.gov (United States)

    Qiao, Liang; Roussel, Christophe; Wan, Jingjing; Yang, Pengyuan; Girault, Hubert H; Liu, Baohong

    2007-12-01

    An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

  18. Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2006-08-01

    Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.

  19. Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein.

    Science.gov (United States)

    Kurat, Christoph F; Lambert, Jean-Philippe; van Dyk, Dewald; Tsui, Kyle; van Bakel, Harm; Kaluarachchi, Supipi; Friesen, Helena; Kainth, Pinay; Nislow, Corey; Figeys, Daniel; Fillingham, Jeffrey; Andrews, Brenda J

    2011-12-01

    The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.

  20. Novel antibodies to phosphorylated α-synuclein serine 129 and NFL serine 473 demonstrate the close molecular homology of these epitopes.

    Science.gov (United States)

    Rutherford, Nicola J; Brooks, Mieu; Giasson, Benoit I

    2016-08-08

    Pathological inclusions containing aggregated, highly phosphorylated (at serine129) α-synuclein (αS pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting αS inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of αS inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 αS and pSer473 NFL epitopes. We determined the relative abilities of the known αS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and αS, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating αS at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 αS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological αS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 αS antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 αS antibodies, but highlights

  1. Efficiency and Logistics

    CERN Document Server

    Hompel, Michael; Klumpp, Matthias

    2013-01-01

    The „EffizienzCluster LogistikRuhr“ was a winner in the Leading Edge Science Cluster competition run by the German federal Ministry of Education and Research. The mission and aim of the „EffizienzCluster LogistikRuhr“ is to facilitate tomorrow’s individuality – in the sense of individual goods supply, mobility, and production – using 75 percent of today’s resources. Efficiency – both in economical and ecological terms – is enabled by state-of-the-art and innovative logistical solutions including transportation, production and intralogistics. These proceedings “Efficiency and Logistics” give first answers from 27 research projects as an insight into the current state of research of Europe’s leading research and development cluster in logistics and as a contribution to the discussion on how logistics as a science can help to cope with foreseeable resource shortage and sustainability as global challenges.

  2. Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells.

    Directory of Open Access Journals (Sweden)

    Avigail Dreazen Wittenberg

    Full Text Available Constitutive expression of active Akt (Akttg drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6, an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-. rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.

  3. Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells.

    Science.gov (United States)

    Wittenberg, Avigail Dreazen; Azar, Shahar; Klochendler, Agnes; Stolovich-Rain, Miri; Avraham, Shlomit; Birnbaum, Lea; Binder Gallimidi, Adi; Katz, Maximiliano; Dor, Yuval; Meyuhas, Oded

    2016-01-01

    Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.

  4. Facilitation of calcium-dependent potassium current.

    Science.gov (United States)

    Thompson, S H

    1994-12-01

    The activation of Ca-dependent K+ current, Ic, was studied in macropatches on the cell bodies of molluscan neurons. When a depolarizing voltage-clamp pulse was applied repeatedly, Ic facilitated in a manner that resembled the facilitation of synaptic transmitter release. Facilitation was characterized by an increase in Ic amplitude, a progressive increase in instantaneous outward current, and a decrease in utilization time. Experiments were done to investigate the mechanism responsible for Ic facilitation. Facilitation was reduced by microinjection of an exogenous Ca2+ buffer into the cytoplasm, indicating that facilitation is a Ca(2+)-dependent process. It was also reduced at elevated temperatures. Conversely, facilitation was greatly potentiated by blocking the Na/Ca exchange mechanism. It is concluded that the facilitation of Ca-dependent K+ current results from the accumulation of Ca2+ at the inner face of the membrane during the repeated activation of Ca2+ channels by depolarization. The Ca2+ indicator fluo-3 was used in fluorescence imaging experiments to measure changes in [Ca]i near the cell membrane during repeated depolarizing pulses and the interpretation of these results was aided by numerical simulations of Ca2+ accumulation, diffusion, and buffering in the peripheral cytoplasm. These experiments showed that the time course of Ic facilitation matches the time course of Ca2+ accumulation at the membrane. It was found that the strength of Ic facilitation varies among patches on the same neuron, suggesting that the accumulation of Ca2+ is not uniform along the inner surface of the membrane and that gradients in [Ca]i develop and are maintained during trains of depolarizing pulses. Potential mechanisms that may lead to local differences in Ca2+ accumulation and Ic facilitation are discussed.

  5. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba

    2009-06-01

    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  6. Modifiers for quality assurance in group facilitation

    NARCIS (Netherlands)

    Kolfschoten, G.L.; Grünbacher, P.; Briggs, R.O.

    2011-01-01

    A key task of a professional facilitator is to assure the quality of the knowledge products created through collaborative effort. To manage the quality of the knowledge a group generates, facilitators attend to, judge, and question the quality of the contributions a group makes, the decisions it mak

  7. Teachers as Friendship Facilitators: Respeto and Personalismo.

    Science.gov (United States)

    Turnbull, Ann P.; Pereira, Lourdes; Blue-Banning, Martha

    2000-01-01

    This article highlights three teachers who were actively involved as friendship facilitators by illuminating their use of a friendship-facilitator framework with three students with moderate/severe disabilities. The framework includes three strategies: finding opportunities to bring children and youth together, acknowledging individual strengths…

  8. Reconceptualizing the Pedagogical Value of Student Facilitation

    Science.gov (United States)

    Oztok, Murat

    2016-01-01

    Sustained discourse is critical to the learning potential of online courses. And, while research has surfaced many factors that mediate interaction, it further suggests that sustained interaction remains elusive. In this paper, I propose that student facilitation may have an impact on the quality of facilitators' interactions following a week of…

  9. "Stepping Up": A Focus on Facilitator Development

    Science.gov (United States)

    Kostouros, Patricia; Warthe, D. Gaye; Carter-Snell, Catherine; Burnett, Che

    2016-01-01

    This article examines the impact on peer facilitators in "Stepping Up," a dating violence prevention program at a Canadian university. A focus group held eight months following the delivery of the program determined the personal impact of involvement in the program. Results indicate that peer facilitators experienced personal growth as…

  10. Interaction Patterns and Facilitation of Peer Learning.

    Science.gov (United States)

    Shaw, Marvin E.; And Others

    1979-01-01

    Data show that giving information to members of a group is more important in determining the perception by others that the person is facilitating group performance. Asking for information and opinions is more important in actual facilitation of group learning. Social-emotional support becomes important after initial phases of group interaction.…

  11. The Limited Facilitative Effect of Typographical Signals.

    Science.gov (United States)

    Golding, Jonathan M.; Fowler, Susan B.

    1992-01-01

    Two experiments with 188 college students investigated the facilitative effect of typographical signals such as underlining, headings, or other devices to help readers identify specific points. Results do not support a general facilitative effect of typographical signals but suggest that use of signals depends on the reader's strategic processing.…

  12. The Role of Touch in Facilitated Communication.

    Science.gov (United States)

    Kezuka, Emiko

    1997-01-01

    A study investigated the role of touch in the use of facilitated communication with Japanese individuals with autism. Five experiments were conducted involving a "telepathy game" using a rod with an attached strain gauge. Results found the facilitator's contact controlled the motor responses of the subjects. (Author/CR)

  13. Inflammatory stress increases hepatic CD36 translational efficiency via activation of the mTOR signalling pathway.

    Directory of Open Access Journals (Sweden)

    Chuan Wang

    Full Text Available Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD. Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α and interleukin-6 (IL-6 stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.

  14. Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating

    DEFF Research Database (Denmark)

    Assentoft, Mette; Kaptan, Shreyas; Fenton, Robert A

    2013-01-01

    of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation...... is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic...... cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range...

  15. Facilitated diffusion on mobile DNA: configurational traps and sequence heterogeneity

    CERN Document Server

    Brackley, C A; Marenduzzo, D; 10.1103/PhysRevLett.109.168103

    2012-01-01

    We present Brownian dynamics simulations of the facilitated diffusion of a protein, modelled as a sphere with a binding site on its surface, along DNA, modelled as a semi-flexible polymer. We consider both the effect of DNA organisation in 3D, and of sequence heterogeneity. We find that in a network of DNA loops, as are thought to be present in bacterial DNA, the search process is very sensitive to the spatial location of the target within such loops. Therefore, specific genes might be repressed or promoted by changing the local topology of the genome. On the other hand, sequence heterogeneity creates traps which normally slow down facilitated diffusion. When suitably positioned, though, these traps can, surprisingly, render the search process much more efficient.

  16. Using facilitative skills in project management

    DEFF Research Database (Denmark)

    Rasmussen, Lauge Baungaard; Hansen, Mette Sanne; Jacobsen, Peter

    2013-01-01

    managers are frequently finding themselves in situations where using facilitating skills is not an option, but a requirement. Facilitation is to be viewed as a process of ‘obstetric’ aid to meet the challenges of coping with the changing conditions for project management described briefly above...... facilitating skills are identified and discussed in relation to the changing circumstances for project management. The approach used to achieve this paper’s objective includes a literature review, model building and reflection on facilitation skills based on the author’s experiences from facilitating workshops......Project management can be seen as a profession, discipline and conceptual framework. It has been developed from different fields, including military engineering, mechanical engineering, social sciences and construction. During recent decades, there has been a number of challenges as to its efficacy...

  17. Practical-theological facilitation as skilled helping

    Directory of Open Access Journals (Sweden)

    Elmo Pienaar

    2013-11-01

    Full Text Available The article discussed the idea of skilled helping in relation to what has been put forward as practical theological facilitation. It has been argued that various helping relationships, amongst which the author refers to coaching, facilitation, and therapy has more in common than what differentiates them if epistemology is viewed as a unifying concept. As such the scope of practical theology in terms of the contexts and themes in which it might be involved is said to widen. The public dimension of the organisational context, more so than the congregational context, has been put forward as an important habitus of practical-theological facilitation. The organisational involvement of the practical-theological facilitator in terms of professional-vocational skilled helping takes on an actual role through facilitation and other helping modalities.

  18. The Self-catalytic Esterification Reaction of O-Phosphoryl Serine Derivative

    Institute of Scientific and Technical Information of China (English)

    Jin Tang DU; Yan Mei LI; Zhong Zhou CHEN; Shi Zhong LUO; Yu Fen ZHAO

    2005-01-01

    O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. This type of reactions were proposed through an intermediate of mixed phosphoric-carboxylic anhydride that might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.

  19. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins

    DEFF Research Database (Denmark)

    Diella, F.; Cameron, S.; Gemund, C.

    2004-01-01

    need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins. Description: Phospho. ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed...... to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho. ELM version 2.0 contains 1703 phosphorylation site...

  20. Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription.

    Science.gov (United States)

    Heidemann, Martin; Hintermair, Corinna; Voß, Kirsten; Eick, Dirk

    2013-01-01

    The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique domain composed of heptapeptide repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the "CTD code". In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  1. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation.

    Science.gov (United States)

    Brunelle, Joslyn K; Bell, Eric L; Quesada, Nancy M; Vercauteren, Kristel; Tiranti, Valeria; Zeviani, Massimo; Scarpulla, Richard C; Chandel, Navdeep S

    2005-06-01

    Mammalian cells detect decreases in oxygen concentrations to activate a variety of responses that help cells adapt to low oxygen levels (hypoxia). One such response is stabilization of the protein HIF-1 alpha, a component of the transcription factor HIF-1. Here we show that a small interfering RNA (siRNA) against the Rieske iron-sulfur protein of mitochondrial complex III prevents the hypoxic stabilization of HIF-1 alpha protein. Fibroblasts from a patient with Leigh's syndrome, which display residual levels of electron transport activity and are incompetent in oxidative phosphorylation, stabilize HIF-1 alpha during hypoxia. The expression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevents the hypoxic stabilization of HIF-1 alpha. These findings provide genetic evidence that oxygen sensing is dependent on mitochondrial-generated reactive oxygen species (ROS) but independent of oxidative phosphorylation.

  2. Random-coil chemical shifts of phosphorylated amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Bienkiewicz, Ewa A.; Lumb, Kevin J. [Colorado State University, Department of Biochemistry and Molecular Biology (United States)

    1999-11-15

    The {sup 1}H, {sup 13}C, {sup 15}N and {sup 31} P random-coil chemical shifts and phosphate pK{sub a} values of the phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH{sub 2} have been obtained in water at 25 deg. C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

  3. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K

    2014-01-01

    released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

  4. ERK phosphorylation regulates sleep and plasticity in Drosophila.

    Directory of Open Access Journals (Sweden)

    William M Vanderheyden

    Full Text Available Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK(SEM pan-neuronally in the adult fly using GeneSwitch (Gsw Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

  5. Reactions of. cap alpha. -phosphorylated carbonyl compounds with amino alcohols

    Energy Technology Data Exchange (ETDEWEB)

    Moskva, V.V.; Sitdikova, T.Sh.; Zykova, T.V.; Alparova, M.V.; Shagvaleev, F.Sh.

    1986-10-20

    2-Aminoethanol reacts with carbonyl compounds with the formation, depending on the structure of the latter, either of a mixture of azomethines and oxazolidines, or of only azomethines. In the development of investigations on the reactivity of ..cap alpha..-phosphorylated carbonyl compounds the authors studied the reactions of a number of amino alcohols with phosphorylated acetaldehyde and acetone. In both cases they observed the formation of compounds of enamine structure, oxazolidines and azomethines were not observed. By means of NMR spectroscopy they established clearly the formation of the E-isomeric products. The /sup 1/H, /sup 31/P, and /sup 13/C NMR spectra were recorded on a WP-80 spectrometer. Chemical shifts of protons and /sup 13/C nuclei are given relative to TMS, and phosphorus nuclei relative to orthophosphoric acid.

  6. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.

    OpenAIRE

    1989-01-01

    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylma...

  7. Snapshot of a phosphorylated substrate intermediate by kinetic crystallography

    OpenAIRE

    Käck, Helena; Gibson, Katharine J.; Lindqvist, Ylva; Schneider, Gunter

    1998-01-01

    The ATP-dependent enzyme dethiobiotin synthetase from Escherichia coli catalyses the formation of dethiobiotin from CO2 and 7,8-diaminopelargonic acid. The reaction is initiated by the formation of a carbamate and proceeds through a phosphorylated intermediate, a mixed carbamic phosphoric anhydride. Here, we report the crystal structures at 1.9- and 1.6-Å resolution, respectively, of the enzyme–MgATP–diaminopelargonic acid and enzyme–MgADP–carbamic–phosphoric acid anhydride complexes, observe...

  8. Regulation of PCNA Function by Tyrosine Phosphorylation in Prostate Cancer

    Science.gov (United States)

    2012-10-01

    prostate cancer. 15. SUBJECT TERMS PCNA, Tyrosine Phosphorylation, Prostate Cancer 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...directed peptide is a rational strategy to target proliferation-competent PCNA, limitations associated with peptides as a therapeutic agent, particularly...radical prostatectomy: Where have we been? Where are we going? Urol Oncol 2007;25:11–8. 2. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the

  9. FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4.

    Science.gov (United States)

    Zhang, Qiang; Karnak, David; Tan, Mingjia; Lawrence, Theodore S; Morgan, Meredith A; Sun, Yi

    2016-02-04

    FBXW7 is a haploinsufficient tumor suppressor with loss-of-function mutations occurring in human cancers. FBXW7 inactivation causes genomic instability, but the mechanism remains elusive. Here we show that FBXW7 facilitates nonhomologous end-joining (NHEJ) repair and that FBXW7 depletion causes radiosensitization. In response to ionizing radiation, ATM phosphorylates FBXW7 at serine 26 to recruit it to DNA double-strand break (DSB) sites, whereas activated DNA-PKcs phosphorylates XRCC4 at serines 325/326, which promotes binding of XRCC4 to FBXW7. SCF(FBXW7) E3 ligase then promotes polyubiquitylation of XRCC4 at lysine 296 via lysine 63 linkage for enhanced association with the Ku70/80 complex to facilitate NHEJ repair. Consistent with these findings, a small-molecule inhibitor that abrogates XRCC4 polyubiquitylation reduces NHEJ repair. Our study demonstrates one mechanism by which FBXW7 contributes to genome integrity and implies that inactivated FBXW7 in human cancers could be a strategy for increasing the efficacy of radiotherapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Supraspinal brain-derived neurotrophic factor signaling: a novel mechanism for descending pain facilitation.

    Science.gov (United States)

    Guo, Wei; Robbins, Meredith T; Wei, Feng; Zou, Shiping; Dubner, Ronald; Ren, Ke

    2006-01-04

    In the adult mammalian brain, brain-derived neurotrophic factor (BDNF) is critically involved in long-term synaptic plasticity. Here, we show that supraspinal BDNF-tyrosine kinase receptor B (TrkB) signaling contributes to pain facilitation. We show that BDNF-containing neurons in the periaqueductal gray (PAG), the central structure for pain modulation, project to and release BDNF in the rostral ventromedial medulla (RVM), a relay between the PAG and spinal cord. BDNF in PAG and TrkB phosphorylation in RVM neurons are upregulated after inflammation. Intra-RVM sequestration of BDNF and knockdown of TrkB by RNA interference attenuate inflammatory pain. Microinjection of BDNF (10-100 fmol) into the RVM facilitates nociception, which is dependent on NMDA receptors (NMDARs). In vitro studies with RVM slices show that BDNF induces tyrosine phosphorylation of the NMDAR NR2A subunit in RVM via a signal transduction cascade involving IP(3), PKC, and Src. The supraspinal BDNF-TrkB signaling represents a previously unknown mechanism underlying the development of persistent pain. Our findings also caution that application of BDNF for recovery from CNS disorders could lead to undesirable central pain.

  11. Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max

    2012-01-01

    maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins....

  12. Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2013-01-01

    The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences...... on oxidative phosphorylation in mitochondria should therefore include the use of relevant concentrations of the organic solvent in order to validate the contribution....

  13. Absolute Phosphorylation Stoichiometry Analysis by Motif-Targeting Quantitative Mass Spectrometry.

    Science.gov (United States)

    Tsai, Chia-Feng; Ku, Wei-Chi; Chen, Yu-Ju; Ishihama, Yasushi

    2017-01-01

    Direct measurement of site-specific phosphorylation stoichiometry can unambiguously distinguish whether the degree of phosphorylation is regulated by upstream kinase/phosphatase activity or by transcriptional regulation to alter protein expression level. Here, we describe a motif-targeting quantitative proteomic approach that integrates dephosphorylation, isotope tag labeling, and enzymatic kinase reaction for large-scale phosphorylation stoichiometry measurement of the human proteome.

  14. Roles of “junk phosphorylation” in modulating biomolecular association of phosphorylated proteins?

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Jørgensen, Claus; Linding, Rune

    2010-01-01

    Protein phosphorylation dynamically regulates cellular activities in response to environmental cues. Sequence conservation analysis of recent proteome-wide phosphorylation data revealed that many previously unidentified phosphorylation sites are not well-conserved leading to the proposal that man...... evolutionary approaches to interpret physiological important sites....

  15. Detection of tyrosine phosphorylated proteins in trichinella spiralis L1 larvae

    Directory of Open Access Journals (Sweden)

    Allegretti S.

    2001-06-01

    Full Text Available Western-blotting analysis showed the presence of tyrosine phosphorylated proteins in crude extracts of T. spiralis larvae and these phosphorylated proteins were located by immunofluorescence on the striations of the larval cuticle. The patterns of phosphorylated proteins were modified when larvae were incubated with bile.

  16. NetPhosYeast: prediction of protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.

    2007-01-01

    We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast-an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation s...

  17. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    Science.gov (United States)

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

  18. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg;

    2010-01-01

    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...... as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations....

  19. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    Science.gov (United States)

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  20. Coilin phosphorylation mediates interaction with SMN and SmB'.

    Science.gov (United States)

    Toyota, Cory G; Davis, Misty D; Cosman, Angela M; Hebert, Michael D

    2010-04-01

    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

  1. Coilin phosphorylation mediates interaction with SMN and SmB′

    Science.gov (United States)

    Toyota, Cory G.; Davis, Misty D.; Cosman, Angela M.; Hebert, Michael D.

    2010-01-01

    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB. PMID:19997741

  2. TTBK2: A Tau Protein Kinase beyond Tau Phosphorylation

    Directory of Open Access Journals (Sweden)

    Jung-Chi Liao

    2015-01-01

    Full Text Available Tau tubulin kinase 2 (TTBK2 is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1 kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

  3. Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Fan Xia

    2008-05-01

    Full Text Available The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf, which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.

  4. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    Science.gov (United States)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco; Haaning, Line Lindegaard; Jensen, Torben Heick

    2014-01-01

    In S. cerevisiae, the 5′-3′ exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood. Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII transcription kinetics, a feature that may contribute to the cellular phenotypes of the mutant. Consistently, the rat1-1 allele is synthetic lethal with the rpb1-E1103G mutation, causing increased RNAPII speed, and is suppressed by the rpb2-10 mutation, causing slowed transcription. Thus, Rat1p plays more complex roles in controlling transcription than previously thought. PMID:24501251

  5. Catalytic constants enable the emergence of bistability in dual phosphorylation.

    Science.gov (United States)

    Conradi, Carsten; Mincheva, Maya

    2014-06-06

    Dual phosphorylation of proteins is a principal component of intracellular signalling. Bistability is considered an important property of such systems and its origin is not yet completely understood. Theoretical studies have established parameter values for multistationarity and bistability for many types of proteins. However, up to now no formal criterion linking multistationarity and bistability to the parameter values characterizing dual phosphorylation has been established. Deciding whether an unclassified protein has the capacity for bistability, therefore requires careful numerical studies. Here, we present two general algebraic conditions in the form of inequalities. The first employs the catalytic constants, and if satisfied guarantees multistationarity (and hence the potential for bistability). The second involves the catalytic and Michaelis constants, and if satisfied guarantees uniqueness of steady states (and hence absence of bistability). Our method also allows for the direct computation of the total concentration values such that multistationarity occurs. Applying our results yields insights into the emergence of bistability in the ERK-MEK-MKP system that previously required a delicate numerical effort. Our algebraic conditions present a practical way to determine the capacity for bistability and hence will be a useful tool for examining the origin of bistability in many models containing dual phosphorylation.

  6. Novice facilitators and the use of scripts for managing facilitated modelling workshops

    DEFF Research Database (Denmark)

    Tavella, Elena; Papadopoulos, Thanos

    2015-01-01

    There is limited research on the use of scripts by novice facilitators (novices) in Facilitated Modelling (FM) workshops. To address this gap, this paper illustrates how novices—supported by scripts—switch between and combine facilitation skills and competencies to successfully manage FM workshops...

  7. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    Science.gov (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  8. The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

    Directory of Open Access Journals (Sweden)

    Simon Reiss

    2013-05-01

    Full Text Available The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα is an essential host factor of hepatitis C virus (HCV replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A. This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58, indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56. PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis

  9. Batch efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Schwickerath, Ulrich; Silva, Ricardo; Uria, Christian, E-mail: Ulrich.Schwickerath@cern.c, E-mail: Ricardo.Silva@cern.c [CERN IT, 1211 Geneve 23 (Switzerland)

    2010-04-01

    A frequent source of concern for resource providers is the efficient use of computing resources in their centers. This has a direct impact on requests for new resources. There are two different but strongly correlated aspects to be considered: while users are mostly interested in a good turn-around time for their jobs, resource providers are mostly interested in a high and efficient usage of their available resources. Both things, the box usage and the efficiency of individual user jobs, need to be closely monitored so that the sources of the inefficiencies can be identified. At CERN, the Lemon monitoring system is used for both purposes. Examples of such sources are poorly written user code, inefficient access to mass storage systems, and dedication of resources to specific user groups. As a first step for improvements CERN has launched a project to develop a scheduler add-on that allows careful overloading of worker nodes that run idle jobs.

  10. Batch efficiency

    CERN Document Server

    Schwickerath, U; Uria, C; CERN. Geneva. IT Department

    2010-01-01

    A frequent source of concern for resource providers is the efficient use of computing resources in their centers. This has a direct impact on requests for new resources. There are two different but strongly correlated aspects to be considered: while users are mostly interested in a good turn-around time for their jobs, resource providers are mostly interested in a high and efficient usage of their available resources. Both things, the box usage and the efficiency of individual user jobs, need to be closely monitored so that the sources of the inefficiencies can be identified. At CERN, the Lemon monitoring system is used for both purposes. Examples of such sources are poorly written user code, inefficient access to mass storage systems, and dedication of resources to specific user groups. As a first step for improvements CERN has launched a project to develop a scheduler add-on that allows careful overloading of worker nodes that run idle jobs.

  11. Mcm2 phosphorylation and the response to replicative stress

    Directory of Open Access Journals (Sweden)

    Stead Brent E

    2012-05-01

    Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

  12. Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome.

    Science.gov (United States)

    Valenti, Daniela; De Rasmo, Domenico; Signorile, Anna; Rossi, Leonardo; de Bari, Lidia; Scala, Iris; Granese, Barbara; Papa, Sergio; Vacca, Rosa Anna

    2013-04-01

    A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.

  13. Quantitative encoding of a partial agonist effect on individual opioid receptors by multi-site phosphorylation and threshold detection

    Science.gov (United States)

    Lau, Elaine K.; Trester-Zedlitz, Michelle; Trinidad, Jonathan C.; Kotowski, Sarah J.; Krutchinsky, Andrew N.; Burlingame, Alma L.; von Zastrow, Mark

    2013-01-01

    Many drugs act as partial agonists of seven-transmembrane signaling receptors when compared to endogenous ligands. Partial agonism is well described as a 'macroscopic' property manifest at the level of physiological systems or cell populations, but it is not known whether partial agonists encode discrete regulatory information at the 'microscopic' level of individual receptors. We addressed this question by focusing on morphine, a partial agonist drug for µ-type opioid peptide receptors, and combining quantitative mass spectrometry with cell biological analysis to investigate morphine's reduced efficacy for promoting receptor endocytosis when compared to a peptide full agonist. We show that these chemically distinct ligands produce a complex, and qualitatively similar mixture of phosphorylated opioid receptor forms in intact cells. Quantitatively, however, the agonists promote markedly disproportional production of multi-site phosphorylation involving a specific Ser/Thr motif, whose modification at more than one residue is essential for efficient recruitment of the adaptor protein β-arrestin to clathrin-coated pits that mediate subsequent endocytosis of MORs. These results reveal quantitative encoding of agonist-selective endocytosis at the level of individual opioid receptors, based on the conserved biochemical principles of multi-site phosphorylation and threshold detection. PMID:21868358

  14. The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation.

    Science.gov (United States)

    Beavis, A D; Lehninger, A L

    1986-07-15

    Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

  15. FACT facilitates chromatin transcription by RNA polymerases I and III

    DEFF Research Database (Denmark)

    Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

    2009-01-01

    Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...

  16. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  17. Spatial memory deficits and motor coordination facilitation in cGMP-dependent protein kinase type II-deficient mice.

    Science.gov (United States)

    Wincott, Charlotte M; Kim, Seonil; Titcombe, Roseann F; Tukey, David S; Girma, Hiwot K; Pick, Joseph E; Devito, Loren M; Hofmann, Franz; Hoeffer, Charles; Ziff, Edward B

    2013-01-01

    Activity-dependent trafficking of AMPA receptors to synapses regulates synaptic strength. Activation of the NMDA receptor induces several second messenger pathways that contribute to receptor trafficking-dependent plasticity, including the NO pathway, which elevates cGMP. In turn, cGMP activates the cGMP-dependent protein kinase type II (cGKII), which phosphorylates the AMPA receptor subunit GluA1 at serine 845, a critical step facilitating synaptic delivery in the mechanism of activity-dependent synaptic potentiation. Since cGKII is expressed in the striatum, amygdala, cerebral cortex, and hippocampus, it has been proposed that mice lacking cGKII may present phenotypic differences compared to their wild-type littermates in emotion-dependent tasks, learning and memory, and drug reward salience. Previous studies have shown that cGKII KO mice ingest higher amounts of ethanol as well as exhibit elevated anxiety levels compared to wild-type (WT) littermates. Here, we show that cGKII KO mice are significantly deficient in spatial learning while exhibiting facilitated motor coordination, demonstrating a clear dependence of memory-based tasks on cGKII. We also show diminished GluA1 phosphorylation in the postsynaptic density (PSD) of cGKII KO prefrontal cortex while in hippocampal PSD fractions, phosphorylation was not significantly altered. These data suggest that the role of cGKII may be more robust in particular brain regions, thereby impacting complex behaviors dependent on these regions differently.

  18. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene

    2008-01-01

    we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation...... sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site...

  19. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

    DEFF Research Database (Denmark)

    Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek

    2015-01-01

    neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

  20. Facilitating lifelong learning with OpenU

    NARCIS (Netherlands)

    Rubens, Wilfred; Counotte, Anda

    2012-01-01

    Rubens, W., & Counotte, A. (2012). Facilitating lifelong learning with OpenU. In R. Jacobi, & N. van der Woert (Eds.), Trendreport Open Educational Resources 2012 (pp. 22-26). Utrecht: SURF Foundation - Special Interest Group Open Educational Resources SURF.

  1. Digital Cadastres Facilitating Land Information Management ...

    African Journals Online (AJOL)

    Digital Cadastres Facilitating Land Information Management. ... Journal Home > Vol 3, No 1 (2014) >. Log in or Register ... Raw materials required for the manufacturing industry which drives the economy are extracted from the land resource.

  2. Facilitating LOS Debriefings: A Training Manual

    Science.gov (United States)

    McDonnell, Lori K.; Jobe, Kimberly K.; Dismukes, R. Key

    1997-01-01

    This manual is a practical guide to help airline instructors effectively facilitate debriefings of Line Oriented Simulations (LOS). It is based on a recently completed study of Line Oriented Flight Training (LOFT) debriefings at several U.S. airlines. This manual presents specific facilitation tools instructors can use to achieve debriefing objectives. The approach of the manual is to be flexible so it can be tailored to the individual needs of each airline. Part One clarifies the purpose and objectives of facilitation in the LOS setting. Part Two provides recommendations for clarifying roles and expectations and presents a model for organizing discussion. Part Tree suggests techniques for eliciting active crew participation and in-depth analysis and evaluation. Finally, in Part Four, these techniques are organized according to the facilitation model. Examples of how to effectively use the techniques are provided throughout, including strategies to try when the debriefing objectives are not being fully achieved.

  3. Facilitating Creativity in Science Students' through Teacher ...

    African Journals Online (AJOL)

    Facilitating Creativity in Science Students' through Teacher Professional Development. ... and the creativity checklist (CCh) was the instrument used to collect data. ... The research questions were answered using mean while the hypotheses ...

  4. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    Science.gov (United States)

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-04

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  5. Role of the Phosphorylation of mTOR in the Differentiation of AML Cells Triggered with CD44 Antigen

    KAUST Repository

    Darwish, Manar M

    2013-05-01

    Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

  6. Facilitated inter-firm collaboration in Ghana

    DEFF Research Database (Denmark)

    Kuada, John Ernest; Sørensen, Olav Jull

    2005-01-01

    Since the mid-1980s, aid agencies have endorsed the need to support private enterprise development in developing countries as an instrument for overall economic development and poverty reduction. Facilitation of collaboration between developed and developing country firms has become one of the mo...... the mechanisms of third-party inter-firm facilitation and assesses how effective the catalyst institutions are in nurturing collaborations between companies in developed and developing countries. The discussions are illustrated with case examples drawn from Ghana....

  7. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  8. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-01

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  9. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    Science.gov (United States)

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. dbPPT: a comprehensive database of protein phosphorylation in plants.

    Science.gov (United States)

    Cheng, Han; Deng, Wankun; Wang, Yongbo; Ren, Jian; Liu, Zexian; Xue, Yu

    2014-01-01

    As one of the most important protein post-translational modifications, the reversible phosphorylation is critical for plants in regulating a variety of biological processes such as cellular metabolism, signal transduction and responses to environmental stress. Numerous efforts especially large-scale phosphoproteome profiling studies have been contributed to dissect the phosphorylation signaling in various plants, while a large number of phosphorylation events were identified. To provide an integrated data resource for further investigations, here we present a comprehensive database of dbPPT (database of Phosphorylation site in PlanTs, at http://dbppt.biocuckoo.org), which contains experimentally identified phosphorylation sites in proteins from plants. The phosphorylation sites in dbPPT were manually curated from the literatures, whereas datasets in other public databases were also integrated. In total, there were 82,175 phosphorylation sites in 31,012 proteins from 20 plant organisms in dbPPT, presenting a larger quantity of phosphorylation sites and a higher coverage of plant species in comparison with other databases. The proportions of residue types including serine, threonine and tyrosine were 77.99, 17.81 and 4.20%, respectively. All the phosphoproteins and phosphorylation sites in the database were critically annotated. Since the phosphorylation signaling in plants attracted great attention recently, such a comprehensive resource of plant protein phosphorylation can be useful for the research community. Database URL: http://dbppt.biocuckoo.or

  11. The Impact of Phosphorylation on Electron Capture Dissociation of Proteins: A Top-Down Perspective

    Science.gov (United States)

    Chen, Bifan; Guo, Xiao; Tucholski, Trisha; Lin, Ziqing; McIlwain, Sean; Ge, Ying

    2017-09-01

    Electron capture dissociation (ECD) is well suited for the characterization of phosphoproteins, with which labile phosphate groups are generally preserved during the fragmentation process. However, the impact of phosphorylation on ECD fragmentation of intact proteins remains unclear. Here, we have performed a systematic investigation of the phosphorylation effect on ECD of intact proteins by comparing the ECD cleavages of mono-phosphorylated α-casein, multi-phosphorylated β-casein, and immunoaffinity-purified phosphorylated cardiac troponin I with those of their unphosphorylated counterparts, respectively. In contrast to phosphopeptides, phosphorylation has significantly reduced deleterious effects on the fragmentation of intact proteins during ECD. On a global scale, the fragmentation patterns are highly comparable between unphosphorylated and phosphorylated precursors under the same ECD conditions, despite a slight decrease in the number of fragment ions observed for the phosphorylated forms. On a local scale, single phosphorylation of intact proteins imposes minimal effects on fragmentation near the phosphorylation sites, but multiple phosphorylations in close proximity result in a significant reduction of ECD bond cleavages. [Figure not available: see fulltext.

  12. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    Science.gov (United States)

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

  13. Association between intrinsic disorder and serine/threonine phosphorylation in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Gajinder Pal Singh

    2015-01-01

    Full Text Available Serine/threonine phosphorylation is an important mechanism that is involved in the regulation of protein function. In eukaryotes, phosphorylation occurs predominantly in intrinsically disordered regions of proteins. Though serine/threonine phosphorylation and protein disorder are much less prevalent in prokaryotes, some bacteria have high levels of serine/threonine phosphorylation and disorder, including the medically important M. tuberculosis. Here I show that serine/threonine phosphorylation sites in M. tuberculosis are highly enriched in intrinsically disordered regions, indicating similarity in the substrate recognition mechanisms of eukaryotic and M. tuberculosis kinases. Serine/threonine phosphorylation has been linked to the pathogenicity and survival of M. tuberculosis. Thus, a better understanding of how its kinases recognize their substrates could have important implications in understanding and controlling the biology of this deadly pathogen. These results also indicate that the association between serine/threonine phosphorylation and disorder is not a feature restricted to eukaryotes.

  14. Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

    Science.gov (United States)

    Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

    2015-11-01

    Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system.

  15. Combined epiretinal and internal limiting membrane peeling facilitated by high dilution indocyanine green negative staining

    Directory of Open Access Journals (Sweden)

    Mark M Kaehr

    2015-01-01

    Full Text Available We describe the utilization of indocyanine green (ICG dye to facilitate combined/en bloc removal of epiretinal membranes (ERM along with internal limiting membranes (ILM. The method utilizes a highly diluted preparation of ICG in dextrose water solvent (D5W. Elimination of fluid air exchange step facilitating staining in the fluid phase and low intensity lighting help minimize potential ICG toxicity. The technique demonstrates how ICG facilitates negative staining of ERMs and how ILM peeling concomitantly can allow complete and efficient ERM removal minimizing surgical time and the necessity for dual or sequential staining.

  16. Happy creativity: Listening to happy music facilitates divergent thinking.

    Science.gov (United States)

    Ritter, Simone M; Ferguson, Sam

    2017-01-01

    Creativity can be considered one of the key competencies for the twenty-first century. It provides us with the capacity to deal with the opportunities and challenges that are part of our complex and fast-changing world. The question as to what facilitates creative cognition-the ability to come up with creative ideas, problem solutions and products-is as old as the human sciences, and various means to enhance creative cognition have been studied. Despite earlier scientific studies demonstrating a beneficial effect of music on cognition, the effect of music listening on creative cognition has remained largely unexplored. The current study experimentally tests whether listening to specific types of music (four classical music excerpts systematically varying on valance and arousal), as compared to a silence control condition, facilitates divergent and convergent creativity. Creativity was higher for participants who listened to 'happy music' (i.e., classical music high on arousal and positive mood) while performing the divergent creativity task, than for participants who performed the task in silence. No effect of music was found for convergent creativity. In addition to the scientific contribution, the current findings may have important practical implications. Music listening can be easily integrated into daily life and may provide an innovative means to facilitate creative cognition in an efficient way in various scientific, educational and organizational settings when creative thinking is needed.

  17. Happy creativity: Listening to happy music facilitates divergent thinking

    Science.gov (United States)

    Ferguson, Sam

    2017-01-01

    Creativity can be considered one of the key competencies for the twenty-first century. It provides us with the capacity to deal with the opportunities and challenges that are part of our complex and fast-changing world. The question as to what facilitates creative cognition—the ability to come up with creative ideas, problem solutions and products—is as old as the human sciences, and various means to enhance creative cognition have been studied. Despite earlier scientific studies demonstrating a beneficial effect of music on cognition, the effect of music listening on creative cognition has remained largely unexplored. The current study experimentally tests whether listening to specific types of music (four classical music excerpts systematically varying on valance and arousal), as compared to a silence control condition, facilitates divergent and convergent creativity. Creativity was higher for participants who listened to ‘happy music’ (i.e., classical music high on arousal and positive mood) while performing the divergent creativity task, than for participants who performed the task in silence. No effect of music was found for convergent creativity. In addition to the scientific contribution, the current findings may have important practical implications. Music listening can be easily integrated into daily life and may provide an innovative means to facilitate creative cognition in an efficient way in various scientific, educational and organizational settings when creative thinking is needed. PMID:28877176

  18. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity.

    Science.gov (United States)

    Lewandowski, Katherine T; Piwnica-Worms, Helen

    2014-01-15

    The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

  19. HDAC2 phosphorylation-dependent KIf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

    Institute of Scientific and Technical Information of China (English)

    Bin Zheng; Mei Han; Ya-nan Shu; Yimg-jie Li; Sui-bing Miao; Xin-hua Zhang; Hui-jing Shi; Tian zhang; Jin-kun Wen

    2011-01-01

    Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension,atherosclerosis and restenosis after angioplasty,leading to pathophysiological vascular remodeling.As an important growth arrest gene,p21 plays critical roles in vascular remodeling.Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling.Nevertheless,the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood.Here,we show that,under basal conditions,RARa forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (KIf5) at the p21 promoter to inhibit its expression.Upon RARα agonist stimulation,HDAC2 is phosphorylated by CK2α.Phosphorylation of HDAC2,on the one hand,promotes its dissociation from RARα,thus allowing the liganded-RARα to interact with co-activators; on the other hand,it increases its interaction with KIf5,thus leading to deacetylation of Klf5.Deacetylation of KIf5 facilitates its dissociation from thep21 promoter,relieving its repressive effect on thep21 promoter.Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of KIf5 from the p21 promoter and impairs RAR agonist-induced p21 activation.Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonlst treatment,allowing for optimum agonistinduced p21 expression.

  20. Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase.

    Science.gov (United States)

    Kinumi, Tomoya; Niki, Etsuo; Shigeri, Yasushi; Matsumoto, Hiroyuki

    2005-12-01

    A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.