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Sample records for phosphorylation site database

  1. The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update

    PubMed Central

    Sadowski, Ivan; Breitkreutz, Bobby-Joe; Stark, Chris; Su, Ting-Cheng; Dahabieh, Matthew; Raithatha, Sheetal; Bernhard, Wendy; Oughtred, Rose; Dolinski, Kara; Barreto, Kris; Tyers, Mike

    2013-01-01

    PhosphoGRID is an online database that curates and houses experimentally verified in vivo phosphorylation sites in the Saccharomyces cerevisiae proteome (www.phosphogrid.org). Phosphosites are annotated with specific protein kinases and/or phosphatases, along with the condition(s) under which the phosphorylation occurs and/or the effects on protein function. We report here an updated data set, including nine additional high-throughput (HTP) mass spectrometry studies. The version 2.0 data set contains information on 20 177 unique phosphorylated residues, representing a 4-fold increase from version 1.0, and includes 1614 unique phosphosites derived from focused low-throughput (LTP) studies. The overlap between HTP and LTP studies represents only ∼3% of the total unique sites, but importantly 45% of sites from LTP studies with defined function were discovered in at least two independent HTP studies. The majority of new phosphosites in this update occur on previously documented proteins, suggesting that coverage of phosphoproteins in the yeast proteome is approaching saturation. We will continue to update the PhosphoGRID data set, with the expectation that the integration of information from LTP and HTP studies will enable the development of predictive models of phosphorylation-based signaling networks. Database URL: http://www.phosphogrid.org/ PMID:23674503

  2. Charge environments around phosphorylation sites in proteins

    PubMed Central

    Kitchen, James; Saunders, Rebecca E; Warwicker, Jim

    2008-01-01

    Background Phosphorylation is a central feature in many biological processes. Structural analyses have identified the importance of charge-charge interactions, for example mediating phosphorylation-driven allosteric change and protein binding to phosphopeptides. Here, we examine computationally the prevalence of charge stabilisation around phosphorylated sites in the structural database, through comparison with locations that are not phosphorylated in the same structures. Results A significant fraction of phosphorylated sites appear to be electrostatically stabilised, largely through interaction with sidechains. Some examples of stabilisation across a subunit interface are evident from calculations with biological units. When considering the immediately surrounding environment, in many cases favourable interactions are only apparent after conformational change that accompanies phosphorylation. A simple calculation of potential interactions at longer-range, applied to non-phosphorylated structures, recovers the separation exhibited by phosphorylated structures. In a study of sites in the Phospho.ELM dataset, for which structural annotation is provided by non-phosphorylated proteins, there is little separation of the known phospho-acceptor sites relative to background, even using the wider interaction radius. However, there are differences in the distributions of patch polarity for acceptor and background sites in the Phospho.ELM dataset. Conclusion In this study, an easy to implement procedure is developed that could contribute to the identification of phospho-acceptor sites associated with charge-charge interactions and conformational change. Since the method gives information about potential anchoring interactions subsequent to phosphorylation, it could be combined with simulations that probe conformational change. Our analysis of the Phospho.ELM dataset also shows evidence for mediation of phosphorylation effects through (i) conformational change associated with

  3. P(3)DB: An Integrated Database for Plant Protein Phosphorylation.

    PubMed

    Yao, Qiuming; Bollinger, Curtis; Gao, Jianjiong; Xu, Dong; Thelen, Jay J

    2012-01-01

    Protein phosphorylation is widely recognized as the most widespread, enzyme-catalyzed post-translational modification in eukaryotes. In particular, plants have appropriated this signaling mechanism as evidenced by the twofold higher frequency of protein kinases within the genome compared to other eukaryotes. While all aspects of plant protein phosphorylation research have grown in the past 10 years; phosphorylation site mapping using high-resolution mass spectrometry has grown exponentially. In Arabidopsis alone there are thousands of experimentally determined phosphorylation sites. To archive these events in a user-intuitive format we have developed P(3)DB, the Plant Protein Phosphorylation Database (p3db.org). This database is a repository for plant protein phosphorylation site data, currently hosting information on 32,963 non-redundant sites collated from 23 experimental studies from six plant species. These data can be queried for a protein-of-interest using an integrated BLAST module to query similar sequences with known phosphorylation sites among the multiple plants currently investigated. The paper demonstrates how this resource can help identify functionally conserved phosphorylation sites in plants using a multi-system approach.

  4. Prediction of functional phosphorylation sites by incorporating evolutionary information.

    PubMed

    Niu, Shen; Wang, Zhen; Ge, Dongya; Zhang, Guoqing; Li, Yixue

    2012-09-01

    Protein phosphorylation is a ubiquitous protein post-translational modification, which plays an important role in cellular signaling systems underlying various physiological and pathological processes. Current in silico methods mainly focused on the prediction of phosphorylation sites, but rare methods considered whether a phosphorylation site is functional or not. Since functional phosphorylation sites are more valuable for further experimental research and a proportion of phosphorylation sites have no direct functional effects, the prediction of functional phosphorylation sites is quite necessary for this research area. Previous studies have shown that functional phosphorylation sites are more conserved than non-functional phosphorylation sites in evolution. Thus, in our method, we developed a web server by integrating existing phosphorylation site prediction methods, as well as both absolute and relative evolutionary conservation scores to predict the most likely functional phosphorylation sites. Using our method, we predicted the most likely functional sites of the human, rat and mouse proteomes and built a database for the predicted sites. By the analysis of overall prediction results, we demonstrated that protein phosphorylation plays an important role in all the enriched KEGG pathways. By the analysis of protein-specific prediction results, we demonstrated the usefulness of our method for individual protein studies. Our method would help to characterize the most likely functional phosphorylation sites for further studies in this research area.

  5. Prioritizing functional phosphorylation sites based on multiple feature integration

    PubMed Central

    Xiao, Qingyu; Miao, Benpeng; Bi, Jie; Wang, Zhen; Li, Yixue

    2016-01-01

    Protein phosphorylation is an important type of post-translational modification that is involved in a variety of biological activities. Most phosphorylation events occur on serine, threonine and tyrosine residues in eukaryotes. In recent years, many phosphorylation sites have been identified as a result of advances in mass-spectrometric techniques. However, a large percentage of phosphorylation sites may be non-functional. Systematically prioritizing functional sites from a large number of phosphorylation sites will be increasingly important for the study of their biological roles. This study focused on exploring the intrinsic features of functional phosphorylation sites to predict whether a phosphosite is likely to be functional. We found significant differences in the distribution of evolutionary conservation, kinase association, disorder score, and secondary structure between known functional and background phosphorylation datasets. We built four different types of classifiers based on the most representative features and found that their performances were similar. We also prioritized 213,837 human phosphorylation sites from a variety of phosphorylation databases, which will be helpful for subsequent functional studies. All predicted results are available for query and download on our website (Predict Functional Phosphosites, PFP, http://pfp.biosino.org/). PMID:27090940

  6. Mixed mechanisms of multi-site phosphorylation.

    PubMed

    Suwanmajo, Thapanar; Krishnan, J

    2015-06-01

    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose-response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction. PMID:25972433

  7. [Identifying phosphopeptide by searching a site annotated protein database].

    PubMed

    Cheng, Kai; Wang, Fangjun; Bian, Yangyang; Ye, Mingling; Zou, Hanfa

    2015-01-01

    Phosphoproteome analysis is one of the important research fields in proteomics. In shotgun proteomics, phosphopeptides could be identified directly by setting phosphorylation as variable modifications in database search. However, search space increases significantly when variable modifications are set in post-translation modifications (PTMs) analysis, which will decrease the identification sensitivity. Because setting a variable modification on a specific type of amino acid residue means all of this amino acid residues in the database might be modified, which is not consistent with actual conditions. Phosphorylation and dephosphorylation are regulated by protein kinases and phosphatases, which can only occur on particular substrates. Therefore only residues within specific sequence are potential sites which may be modified. To address this issue, we extracted the characteristic sequence from the identified phosphorylation sites and created an annotated database containing phosphorylation site information, which allowed the searching engine to set variable modifications only on the serine, threonine and tyrosine residues that were identified to be phosphorylated previously. In this database only annotated serine, threonine and tyrosine can be modified. This strategy significantly reduced the search space. The performance of this new database searching strategy was evaluated by searching different types of data with Mascot, and higher sensitivity for phosphopeptide identification was achieved with high reliability.

  8. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

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

    SciTech Connect

    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

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

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

    PubMed

    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.

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

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    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. Predicting and analyzing protein phosphorylation sites in plants using musite.

    PubMed

    Yao, Qiuming; Gao, Jianjiong; Bollinger, Curtis; Thelen, Jay J; Xu, Dong

    2012-01-01

    Although protein phosphorylation sites can be reliably identified with high-resolution mass spectrometry, the experimental approach is time-consuming and resource-dependent. Furthermore, it is unlikely that an experimental approach could catalog an entire phosphoproteome. Computational prediction of phosphorylation sites provides an efficient and flexible way to reveal potential phosphorylation sites and provide hypotheses in experimental design. Musite is a tool that we previously developed to predict phosphorylation sites based solely on protein sequence. However, it was not comprehensively applied to plants. In this study, the phosphorylation data from Arabidopsis thaliana, B. napus, G. max, M. truncatula, O. sativa, and Z. mays were collected for cross-species testing and the overall plant-specific prediction as well. The results show that the model for A. thaliana can be extended to other organisms, and the overall plant model from Musite outperforms the current plant-specific prediction tools, Plantphos, and PhosphAt, in prediction accuracy. Furthermore, a comparative study of predicted phosphorylation sites across orthologs among different plants was conducted to reveal potential evolutionary features. A bipolar distribution of isolated, non-conserved phosphorylation sites, and highly conserved ones in terms of the amino acid type was observed. It also shows that predicted phosphorylation sites conserved within orthologs do not necessarily share more sequence similarity in the flanking regions than the background, but they often inherit protein disorder, a property that does not necessitate high sequence conservation. Our analysis also suggests that the phosphorylation frequencies among serine, threonine, and tyrosine correlate with their relative proportion in disordered regions. Musite can be used as a web server (http://musite.net) or downloaded as an open-source standalone tool (http://musite.sourceforge.net/).

  13. Determining in vivo Phosphorylation Sites using Mass Spectrometry

    PubMed Central

    Breitkopf, Susanne B.; Asara, John M.

    2012-01-01

    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems since it controls cell growth, proliferation, survival, etc. High resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity and throughput. The protocol described here focuses 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 (IP) 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 (TiO2) 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 (SCX), phosphopeptide enrichment by IMAC or TiO2 and LC-MS/MS. Alternatively, one can fractionate the protein lysate by SDS-PAGE, followed by digestion, phosphopeptide enrichment and LC-MS/MS. One can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS. PMID:22470061

  14. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  15. Methods for generating phosphorylation site-specific immunological reagents

    DOEpatents

    Anderson, Carl W.; Appella, Ettore; Sakaguchi, Kazuyasu

    2001-01-01

    The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid residue to a phosphate group. A preferred linkage is a CF.sub.2 group. Such a linkage is used to generate the phosphoserine mimetic F.sub.2 Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF.sub.2 group linkage is also used to produce the phosphothreonine mimetic F.sub.2 Pmb, and to produce the phosphotyrosine mimetic, F.sub.2 Pmp.

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

    PubMed

    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.

  17. General database for ground water site information.

    PubMed

    de Dreuzy, Jean-Raynald; Bodin, Jacques; Le Grand, Hervé; Davy, Philippe; Boulanger, Damien; Battais, Annick; Bour, Olivier; Gouze, Philippe; Porel, Gilles

    2006-01-01

    In most cases, analysis and modeling of flow and transport dynamics in ground water systems require long-term, high-quality, and multisource data sets. This paper discusses the structure of a multisite database (the H+ database) developed within the scope of the ERO program (French Environmental Research Observatory, http://www.ore.fr). The database provides an interface between field experimentalists and modelers, which can be used on a daily basis. The database structure enables the storage of a large number of data and data types collected from a given site or multiple-site network. The database is well suited to the integration, backup, and retrieval of data for flow and transport modeling in heterogeneous aquifers. It relies on the definition of standards and uses a templated structure, such that any type of geolocalized data obtained from wells, hydrological stations, and meteorological stations can be handled. New types of platforms other than wells, hydrological stations, and meteorological stations, and new types of experiments and/or parameters could easily be added without modifying the database structure. Thus, we propose that the database structure could be used as a template for designing databases for complex sites. An example application is the H+ database, which gathers data collected from a network of hydrogeological sites associated with the French Environmental Research Observatory.

  18. Phosphorylation Site Dynamics of Early T-cell Receptor Signaling

    PubMed Central

    Rigbolt, Kristoffer T. G.; Hu, Bin; Hlavacek, William S.; Blagoev, Blagoy

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein–protein interactions and phosphorylation events have been studied extensively, we lack 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 with central roles in TCR signaling. The model was used to generate predictions suggesting unexpected roles for the phosphatase PTPN6 (SHP-1) and shortcut recruitment of the actin regulator WAS. Predictions were validated experimentally. This integration of proteomics and modeling illustrates a novel, generalizable framework for solidifying quantitative understanding of a signaling network and for elucidating missing links. PMID:25147952

  19. Characterization of the phosphorylation sites of the squid (Loligo pealei) high-molecular-weight neurofilament protein from giant axon axoplasm.

    PubMed

    Jaffe, H; Sharma, P; Grant, P; Pant, H

    2001-02-01

    Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C-terminal tail domain KSP repeats by proline-directed kinases. The squid giant axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid-specific neurofilaments, particularly the highly phosphorylated NF-220. Of the 228 serine-threonine candidate phosphate acceptor sites in the NF-220 tail domain (residues 401-1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C-terminal repeat segment (residues 840-1160). Characterization of the in vivo NF-220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF-220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C-terminal repeat segment (residues 840-1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline-directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline-directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho-sites requires sequential phosphorylation by several kinases.

  20. Early Site Permit Demonstration Program: Nuclear Power Plant Siting Database

    1994-01-28

    This database is a repository of comprehensive licensing and technical reviews of siting regulatory processes and acceptance criteria for advanced light water reactor (ALWR) nuclear power plants. The program is designed to be used by applicants for an early site permit or combined construction permit/operating license (10CFRR522, Subparts A and C) as input for the development of the application. The database is a complete, menu-driven, self-contained package that can search and sort the supplied datamore » by topic, keyword, or other input. The software is designed for operation on IBM compatible computers with DOS.« less

  1. Computational Analysis of the Predicted Evolutionary Conservation of Human Phosphorylation Sites

    PubMed Central

    Trost, Brett; Kusalik, Anthony; Napper, Scott

    2016-01-01

    Protein kinase-mediated phosphorylation is among the most important post-translational modifications. However, few phosphorylation sites have been experimentally identified for most species, making it difficult to determine the degree to which phosphorylation sites are conserved. The goal of this study was to use computational methods to characterize the conservation of human phosphorylation sites in a wide variety of eukaryotes. Using experimentally-determined human sites as input, homologous phosphorylation sites were predicted in all 432 eukaryotes for which complete proteomes were available. For each pair of species, we calculated phosphorylation site conservation as the number of phosphorylation sites found in both species divided by the number found in at least one of the two species. A clustering of the species based on this conservation measure was concordant with phylogenies based on traditional genomic measures. For a subset of the 432 species, phosphorylation site conservation was compared to conservation of both protein kinases and proteins in general. Protein kinases exhibited the highest degree of conservation, while general proteins were less conserved and phosphorylation sites were least conserved. Although preliminary, these data tentatively suggest that variation in phosphorylation sites may play a larger role in explaining phenotypic differences among organisms than differences in the complements of protein kinases or general proteins. PMID:27046079

  2. Sequence- and Structure-Based Analysis of Tissue-Specific Phosphorylation Sites

    PubMed Central

    Karabulut, Nermin Pinar; Frishman, Dmitrij

    2016-01-01

    Phosphorylation is the most widespread and well studied reversible posttranslational modification. Discovering tissue-specific preferences of phosphorylation sites is important as phosphorylation plays a role in regulating almost every cellular activity and disease state. Here we present a comprehensive analysis of global and tissue-specific sequence and structure properties of phosphorylation sites utilizing recent proteomics data. We identified tissue-specific motifs in both sequence and spatial environments of phosphorylation sites. Target site preferences of kinases across tissues indicate that, while many kinases mediate phosphorylation in all tissues, there are also kinases that exhibit more tissue-specific preferences which, notably, are not caused by tissue-specific kinase expression. We also demonstrate that many metabolic pathways are differentially regulated by phosphorylation in different tissues. PMID:27332813

  3. Biochemical and biological analysis of Mek1 phosphorylation site mutants.

    PubMed Central

    Huang, W; Kessler, D S; Erikson, R L

    1995-01-01

    Recently, we described the constitutive activation of Mek1 by mutation of its two serine phosphorylation sites. We have now characterized the biochemical properties of these Mek1 mutants and performed microinjection experiments to investigate the effect of an activated Mek on oocyte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp218, Asp222] and [Asp218, Glu222], activated Mek1 over 6000-fold. The specific activity of the [Asp218, Asp222] and [Asp218, Glu222] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Although the mutants with double acidic substitutions could not be further activated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Mek1. Injection of the [Asp218, Asp222] Mek1 mutant into Xenopus oocytes activated both MAP kinase and histone H1 kinase and induced germinal vesicle breakdown, an effect that was only partially blocked by inhibition of protein synthesis. These data provide a measure of Mek's potential to influence cell functions and a quantitative basis to assess the biological effects of Mek1 mutants in a variety of circumstances. Images PMID:7612960

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

    PubMed

    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.

  5. Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites.

    PubMed

    Nishi, Hafumi; Demir, Emek; Panchenko, Anna R

    2015-01-30

    Cellular fate depends on the spatiotemporal separation and integration of signaling processes that can be provided by phosphorylation events. In this study, we identify the crucial points in signaling crosstalk that can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences of phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative and redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition, we found that in silico phosphorylation of sites with similar functional consequences has comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation.

  6. Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites

    PubMed Central

    Nishi, Hafumi; Demir, Emek; Panchenko, Anna R.

    2014-01-01

    Cellular fate depends on the spatio-temporal separation and integration of signaling processes which can be provided by phosphorylation events. In this study we identify the crucial points in signaling crosstalk which can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences on phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative or redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition we found that in silico phosphorylation of sites with similar functional consequences have comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation. PMID:25451034

  7. Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site.

    PubMed

    Chen, Tiane; Kocinsky, Hetal S; Cha, Boyoung; Murtazina, Rakhilya; Yang, Jianbo; Tse, C Ming; Singh, Varsha; Cole, Robert; Aronson, Peter S; de Jonge, Hugo; Sarker, Rafiquel; Donowitz, Mark

    2015-01-23

    The epithelial brush-border Na(+)/H(+) exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser(554), Ser(607), and Ser(663), equivalent to mouse Ser(552), Ser(605), and Ser(659)), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser(663) phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser(554), Ser(607), and Ser(663), all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser(663). The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.

  8. An ensemble method approach to investigate kinase-specific phosphorylation sites.

    PubMed

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2014-01-01

    Protein phosphorylation is one of the most significant and well-studied post-translational modifications, and it plays an important role in various cellular processes. It has made a considerable impact in understanding the protein functions which are involved in revealing signal transductions and various diseases. The identification of kinase-specific phosphorylation sites has an important role in elucidating the mechanism of phosphorylation; however, experimental techniques for identifying phosphorylation sites are labor intensive and expensive. An exponentially increasing number of protein sequences generated by various laboratories across the globe require computer-aided procedures for reliably and quickly identifying the phosphorylation sites, opening a new horizon for in silico analysis. In this regard, we have introduced a novel ensemble method where we have selected three classifiers (least square support vector machine, multilayer perceptron, and k-Nearest Neighbor) and three different feature encoding parameters (dipeptide composition, physicochemical properties of amino acids, and protein-protein similarity score). Each of these classifiers is trained on each of the three different parameter systems. The final results of the ensemble method are obtained by fusing the results of all the classifiers by a weighted voting algorithm. Extensive experiments reveal that our proposed method can successfully predict phosphorylation sites in a kinase-specific manner and performs significantly better when compared with other existing phosphorylation site prediction methods.

  9. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    NASA Technical Reports Server (NTRS)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

  10. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Δ ura8Δ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  11. Why does troponin I have so many phosphorylation sites? Fact and fancy.

    PubMed

    Solaro, R John; van der Velden, Jolanda

    2010-05-01

    We discuss a current controversy regarding the relative role of phosphorylation sites on cardiac troponin I (cTnI) (Fig. 1) in physiological and patho-physiological cardiac function. Studies with mouse models and in vitro studies indicate that multi-site phosphorylations are involved in both control of maximum tension and sarcomeric responsiveness to Ca(2+). Thus one hypothesis is that cardiac function reflects a balance of cTnI phosphorylations and a tilt in this balance may be maladaptive in acquired and genetic disorders of the heart. Studies on human heart samples taken mainly at end-stage heart failure, and in depth proteomic analysis of human and rat heart samples demonstrate that Ser23/Ser24 are the major and perhaps the only sites likely to be relevant to control cardiac function. Thus functional significance of Ser23/Ser24 phosphorylation is taken as fact, whereas the function of some other sites is treated as fancy. Maybe the extremes will meet: in any case we both agree that further work needs to be carried out with relatively large mammals and with determination of the time course of changes in phosphorylation to identify transient modifications that may be relevant at a beat-to-beat basis. Moreover, we agree that the changes and effects of cTnI phosphorylation need to be fully integrated into the effects of other phosphorylations in the cardiac myocyte.

  12. Recognition of protein phosphorylation site based on amino acids sequence features

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Ding, Changjiang; Lu, Jun

    2012-09-01

    Protein phosphorylation is one of the most important reversible post-translational modifications (PTMs), and the theoretical recognition of the phosphorylation site is one of the important content of the computational biology. In the paper, we use the amino acid component, the position-dependent residue statistics and the non-adjacent residue pair frequency as the recognition parameters, and use Jensen-Shannon Divergence with Quadratic Discriminant analysis(JSDQD) as the method for predicting the phosphorylation sites. The 7-fold cross-validation test accuracies for the CK2, PKA and PKC kinase families are 90%, 90% and 86%, respectively.

  13. Comparison of Savannah River Site`s meteorological databases

    SciTech Connect

    Weber, A.H.

    1993-07-01

    A five-year meteorological database from the 61-meter, H-Area tower for the period 1987--1991 was compared to an earlier database for the period 1982--1986. The amount of invalid data for the newer 87--91 database was one third that for the earlier database. The data recovery percentage for the last four years of the 87-91 database was well above 90%. Considerable effort was necessary to fill in for missing data periods for the newer database for the H-Area tower. Therefore, additional databases that have been prepared for the remaining SRS meteorological towers have had missing and erroneous data flagged, but not replaced. The F-Area tower`s database was used for cross-comparison purposes because of its proximity to H Area. The primary purpose of this report is to compare the H-Tower databases for 82-86 and 87-91. Statistical methods enable the use of probability statements to be made concerning the hypothesis of no differences between the distributions of the two time periods, assuming each database is a random sample from its respective distribution. This assumption is required for the statistical tests to be valid. A number of statistical comparisons can be made between the two data sets, even though the 82-86 database exist only as distributions of frequency and mean speed.

  14. Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation.

    PubMed

    Deziel, M R; Lippes, H A; Rampal, A L; Jung, C Y

    1989-01-01

    1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.

  15. Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44

    SciTech Connect

    Silva, Laurie A.; Strang, Blair L.; Lin, Eric W.; Kamil, Jeremy P.; Coen, Donald M.

    2011-09-01

    The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides. Viruses containing alanine substitutions of most UL44 residues that are phosphorylated in infected cells exhibited at most modest effects on viral DNA synthesis and yield. However, substitution of highly phosphorylated sites adjacent to the nuclear localization signal abolished viral replication. The results taken together are consistent with UL44 being phosphorylated directly by UL97 during infection, and a crucial role for phosphorylation-mediated nuclear localization of UL44 for viral replication, but lend little support to the widely held hypothesis that UL97-mediated phosphorylation of UL44 is crucial for viral DNA synthesis.

  16. Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor*

    PubMed Central

    Bray, Lauriane; Froment, Carine; Pardo, Pierre; Candotto, Cédric; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine; Moulédous, Lionel

    2014-01-01

    The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, 412TNST415 at the end of the C terminus of the receptor, and additional sites involved in desensitization (372TS373) and internalization (Ser395). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns. PMID:25326382

  17. Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

    PubMed Central

    Huang, Haomin; Hittle, James; Zappacosta, Francesca; Annan, Roland S.; Hershko, Avram; Yen, Timothy J.

    2008-01-01

    BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007. Genes Dev. 21:2205–2219; Matsumura, S., F. Toyoshima, and E. Nishida. 2007. J. Biol. Chem. 282:15217–15227). S670, the most conserved residue, is phosphorylated at kinetochores at the onset of mitosis and dephosphorylated before anaphase onset. Unlike the Plk1-dependent S676 phosphorylation, S670 phosphorylation is sensitive to microtubule attachments but not to kinetochore tension. Functionally, phosphorylation of S670 is essential for error correction and for kinetochores with end-on attachments to establish tension. Furthermore, in vitro data suggest that the phosphorylation status of BubR1 is important for checkpoint inhibition of the anaphase-promoting complex/cyclosome. Finally, RNA interference experiments show that Mps1 is a major but not the exclusive kinase that specifies BubR1 phosphorylation in vivo. The combined data suggest that BubR1 may be an effector of multiple kinases that are involved in discrete aspects of kinetochore attachments and checkpoint regulation. PMID:19015317

  18. CaMKII Phosphorylation of Na(V)1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure.

    PubMed

    Herren, Anthony W; Weber, Darren M; Rigor, Robert R; Margulies, Kenneth B; Phinney, Brett S; Bers, Donald M

    2015-05-01

    The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.

  19. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

    PubMed Central

    Lasalde, Clarivel; Rivera, Andrea V.; León, Alfredo J.; González-Feliciano, José A.; Estrella, Luis A.; Rodríguez-Cruz, Eva N.; Correa, María E.; Cajigas, Iván J.; Bracho, Dina P.; Vega, Irving E.; Wilkinson, Miles F.; González, Carlos I.

    2014-01-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity. PMID:24198248

  20. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

    PubMed

    Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

    2014-02-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

  1. A Hepatitis C Virus NS5A Phosphorylation Site That Regulates RNA Replication

    PubMed Central

    LeMay, K. L.; Treadaway, J.; Angulo, I.

    2013-01-01

    The hepatitis C virus NS5A protein is essential for RNA replication and virion assembly. NS5A is phosphorylated on multiple residues during infections, but these sites remain uncharacterized. Here we identify serine 222 of genotype 2a NS5A as a phosphorylation site that functions as a negative regulator of RNA replication. This site is a component of the hyperphosphorylated form of NS5A, which is in good agreement with previous observations that hyperphosphorylation negatively affects replication. PMID:23115292

  2. Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.

    PubMed

    Kakade, Poonam S; Budnar, Srikanth; Kalraiya, Rajiv D; Vaidya, Milind M

    2016-06-01

    Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

  3. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

    PubMed Central

    Camurdanoglu, B. Z.; Hrovat, C.; Dürnberger, G.; Madalinski, M.; Mechtler, K.; Herbst, R.

    2016-01-01

    The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance. PMID:27666825

  4. The Mouse SAGE Site: database of public mouse SAGE libraries.

    PubMed

    Divina, Petr; Forejt, Jirí

    2004-01-01

    The Mouse SAGE Site is a web-based database of all available public libraries generated by the Serial Analysis of Gene Expression (SAGE) from various mouse tissues and cell lines. The database contains mouse SAGE libraries organized in a uniform way and provides web-based tools for browsing, comparing and searching SAGE data with reliable tag-to-gene identification. A modified approach based on the SAGEmap database is used for reliable tag identification. The Mouse SAGE Site is maintained on an ongoing basis at the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and is accessible at the internet address http://mouse.biomed.cas.cz/sage/.

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

    PubMed Central

    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

  6. Application of phosphorylation site-specific antibodies to measure nuclear receptor signaling: characterization of novel phosphoantibodies for estrogen receptor α

    PubMed Central

    Al-Dhaheri, Mariam H.; Rowan, Brian G.

    2006-01-01

    An understanding of posttranslational events in nuclear receptor signaling is crucial for drug design and clinical therapeutic strategies. Phosphorylation is a well-characterized posttranslational modification that regulates subcellular localization and function of nuclear receptors and coregulators. Although the role of single phosphorylation sites in nuclear receptor function has been described, the contribution of combinations of multiple phosphorylation sites to receptor function remains unclear. The development of phosphoantibodies to each phosphorylation site in a nuclear receptor is a powerful tool to address the role of phosphorylation in multiply phosphorylated receptors. However, phosphoantibodies must be rigorously validated prior to use. This review describes the general methodology for design, characterization and validation of phosphoantibodies using the example of eight phosphoantibodies raised against phosphorylation sites in estrogen receptor α (ERα). PMID:16741565

  7. Creation of geographic information database of subsatellite calibration test site

    NASA Astrophysics Data System (ADS)

    Zyelyk, Ya. I.; Semeniv, O. V.

    2014-12-01

    The prototype of geographic information database (DB) of the sub-satellite calibration test site has been created, to which user can be accessed from the free open-source geographic information system Quantum GIS (QGIS) environment. QGIS is used as an integrator of all data and applications and visualizer of the satellite imagery and vector layers of test sites in the cartographic interface. Conversion of the database from the local representation in the MS Access to the server representation in the PostgreSQL environment has been performed. Dynamic application to QGIS for user interaction from QGIS environment with the object-relational database and to display information from the database has been created. Functional-algorithmic part of these application and the interface for user interaction with the database has been developed.

  8. Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

    PubMed Central

    2009-01-01

    Background Estrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. PMID:20043841

  9. Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

    PubMed

    Gagné, Jean-Philippe; Moreel, Xavier; Gagné, Pierre; Labelle, Yves; Droit, Arnaud; Chevalier-Paré, Mélissa; Bourassa, Sylvie; McDonald, Darin; Hendzel, Michael J; Prigent, Claude; Poirier, Guy G

    2009-02-01

    Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

  10. The identification and analysis of phosphorylation sites on the Atg1 protein kinase

    PubMed Central

    Yeh, Yuh-Ying; Shah, Khyati H; Chou, Chi-Chi; Hsiao, He-Hsuan; Wrasman, Kristie M; Stephan, Joseph S; Stamatakos, Demetra; Khoo, Kay-Hooi

    2011-01-01

    Autophagy is a conserved, degradative process that has been implicated in a number of human diseases and is a potential target for therapeutic intervention. It is therefore important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. The Atg1 protein kinase is a key element of this control as a number of signaling pathways target this enzyme and its associated protein partners. These studies have established that Atg1 activities are controlled, at least in part, by protein phosphorylation. To further this understanding, we used a combined mass spectrometry and molecular biology approach to identify and characterize additional sites of phosphorylation in the Saccharomyces cerevisiae Atg1. Fifteen candidate sites of phosphorylation were identified, including nine that had not been noted previously. Interestingly, our data suggest that the phosphorylation at one of these sites, Ser-34, is inhibitory for both Atg1 kinase activity and autophagy. This site is located within a glycine-rich loop that is highly conserved in protein kinases. Phosphorylation at this position in several cyclin-dependent kinases has also been shown to result in diminished enzymatic activity. In addition, these studies identified Ser-390 as the site of autophosphorylation responsible for the anomalous migration exhibited by Atg1 on SDS-polyacrylamide gels. Finally, a mutational analysis suggested that a number of the sites identified here are important for full autophagy activity in vivo. In all, these studies identified a number of potential sites of regulation within Atg1 and will serve as a framework for future work with this enzyme. PMID:21460632

  11. Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit.

    PubMed

    Rogers, Samuel; McCloy, Rachael; Watkins, D Neil; Burgess, Andrew

    2016-07-01

    Entry into mitosis is driven by the activity of kinases, which phosphorylate over 7000 proteins on multiple sites. For cells to exit mitosis and segregate their genome correctly, these phosphorylations must be removed in a specific temporal order. This raises a critical and important question: how are specific phosphorylation sites on an individual protein removed? Traditionally, the temporal order of dephosphorylation was attributed to decreasing kinase activity. However, recent evidence in human cells has identified unique patterns of dephosphorylation during mammalian mitotic exit that cannot be fully explained by the loss of kinase activity. This suggests that specificity is determined in part by phosphatases. In this review, we explore how the physicochemical properties of an individual phosphosite and its surrounding amino acids can affect interactions with a phosphatase. These positive and negative interactions in turn help determine the specific pattern of dephosphorylation required for correct mitotic exit. PMID:27417119

  12. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

    SciTech Connect

    Waldron, Richard T. . E-mail: rwaldron@mednet.ucla.edu; Whitelegge, Julian P.; Faull, Kym F.; Rozengurt, Enrique

    2007-05-04

    Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.

  13. Basal and hydrogen peroxide stimulated sites of phosphorylation in heterogeneous nuclear ribonucleoprotein C1/C2.

    PubMed

    Stone, James R; Maki, Jenny L; Collins, Tucker

    2003-02-11

    Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.

  14. Site-Specific Connexin Phosphorylation Is Associated with Reduced Heterocellular Communication between Smooth Muscle and Endothelium

    PubMed Central

    Straub, Adam C.; Johnstone, Scott R.; Heberlein, Katherine R.; Rizzo, Michael J.; Best, Angela K.; Boitano, Scott; Isakson, Brant E.

    2010-01-01

    Background/Aims Myoendothelial junctions (MEJs) represent a specialized signaling domain between vascular smooth muscle cells (VSMC) and endothelial cells (EC). The functional consequences of phosphorylation state of the connexins (Cx) at the MEJ have not been explored. Methods/Results: Application of adenosine 3′,5′-cyclic monophosphate sodium (pCPT) to mouse cremasteric arterioles reduces the detection of connexin 43 (Cx43) phosphorylated at its carboxyl terminal serine 368 site (S368) at the MEJ in vivo. After single-cell microinjection of a VSMC in mouse cremaster arterioles, only in the presence of pCPT was dye transfer to EC observed. We used a vascular cell co-culture (VCCC) and applied the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA) or fibroblast growth factor-2 (FGF-2) to induce phosphorylation of Cx43 S368. This phosphorylation event was associated with a significant reduction in dye transfer and calcium communication. Using a novel method to monitor increases in intracellular calcium across the in vitro MEJ, we noted that PMA and FGF-2 both inhibited movement of inositol 1,4,5-triphosphate (IP3), but to a lesser extent Ca2+. Conclusion These data indicate that site-specific connexin phosphorylation at the MEJ can potentially regulate the movement of solutes between EC and VSMC in the vessel wall. PMID:20016202

  15. Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.

    PubMed

    Wiese, Heike; Kuhlmann, Katja; Wiese, Sebastian; Stoepel, Nadine S; Pawlas, Magdalena; Meyer, Helmut E; Stephan, Christian; Eisenacher, Martin; Drepper, Friedel; Warscheid, Bettina

    2014-02-01

    Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site

  16. Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting

    PubMed Central

    Kim, Kyung Lock; Kim, Daehyung; Lee, Seongsil; Kim, Su-Jeong; Noh, Jung Eun; Kim, Joung-Hun; Chae, Young Chan; Lee, Jong-Bong; Ryu, Sung Ho

    2016-01-01

    Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling. PMID:27009355

  17. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation.

    PubMed

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-08-16

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1-activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  18. Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2

    PubMed Central

    Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin; Kaoud, Tamer S.; Arshava, Boris; Dutta, Kaushik; Dalby, Kevin N.; Ghose, Ranajeet

    2015-01-01

    The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS. PMID:26054059

  19. Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization

    PubMed Central

    Chen, Chin-Pei; Lin, Meng-Han; Chan, Ya-Ting; Chen, Li-Chyong; Ma, Che; Fischer, Wolfgang B.

    2016-01-01

    Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization. PMID:27353136

  20. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    PubMed

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.

  1. Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response.

    PubMed

    Fu, Ying; Westenbroek, Ruth E; Scheuer, Todd; Catterall, William A

    2013-11-26

    L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by β-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The β-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by β-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.

  2. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    PubMed

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. PMID:27017623

  3. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

    PubMed

    Zeymer, Cathleen; Werbeck, Nicolas D; Zimmermann, Sabine; Reinstein, Jochen; Hansen, D Flemming

    2016-09-12

    States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  4. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

    PubMed Central

    Zeymer, Cathleen; Werbeck, Nicolas D.; Zimmermann, Sabine

    2016-01-01

    Abstract States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  5. CPhos: a program to calculate and visualize evolutionarily conserved functional phosphorylation sites.

    PubMed

    Zhao, Boyang; Pisitkun, Trairak; Hoffert, Jason D; Knepper, Mark A; Saeed, Fahad

    2012-11-01

    Profiling using high-throughput MS has discovered an overwhelming number of novel protein phosphorylation sites ("phosphosites"). However, the functional relevance of these sites is not always clear. In light of recent studies on the evolutionary mechanism of phosphorylation, we have developed CPhos, a Java program that can assess the conservation of phosphosites among species using an information theory-based approach. The degree of conservation established using CPhos can be used to assess the functional significance of phosphosites. CPhos has a user friendly graphical user interface and is available both as a web service and as a standalone Java application to assist phosphoproteomic researchers in analyzing and prioritizing lists of phosphosites for further experimental validation. CPhos can be accessed or downloaded at http://helixweb.nih.gov/CPhos/.

  6. Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics.

    PubMed

    Barton, Byron; Ayer, Gretchen; Maughan, David W; Vigoreaux, Jim O

    2007-01-01

    Flightin is a myosin rod binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of myofibril assembly and precedes the emergence of flight competency in young adults. To investigate the role of flightin phosphorylation in vivo we generated three flightin null (fln(0)) Drosophila strains that express a mutant flightin transgene with two (Thr158, Ser 162), three (Ser139, Ser141, Ser145) or all five potential phosphorylation sites mutated to alanines. These amino acid substitutions result in lower than normal levels of flightin accumulation and transgenic strains that are unable to beat their wings. On two dimensional gels of IFM proteins, the transgenic strain with five mutant sites (fln(5STA)) is devoid of all phosphovariants, the transgenic strain with two mutant sites (fln(2TSA)) expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites (fln(3SA)) expresses all nine phosphovariants, as the wild-type strain. These results suggest that phosphorylation of Thr158 and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show normal, albeit long, IFM sarcomeres in newly eclosed adults. In contrast, sarcomeres in fully mature fln(5STA) and fln(2TSA) adults show extensive breakdown while those in fln(3SA) are not as disordered. The fiber hypercontraction phenotype that characterizes fln(0) is fully evident in fln(5STA) and fln(2TSA) but partially rescued in fln(3SA). Mechanics on skinned fibers from newly eclosed flies show alterations in viscous modulus for fln(5STA) and fln(2TSA) that result in a significant reduction in oscillatory power output. Expression of fln(5STA) and fln(2TSA), but not fln(3SA), in a wild-type (fln(+)/fln(+)) background resulted in a dominant negative effect manifested as flight impairments and hypercontracted IFM

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

    PubMed

    Koland, John G

    2014-01-01

    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 molecules such as EGFR

  8. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  9. CEOS database of worldwide calibration facilities and validation test sites

    NASA Astrophysics Data System (ADS)

    Butler, James J.; Wanchoo, Lalit; Le, Truong

    2001-02-01

    12 Since 1995, the CEOS Calibration/Validation (Cal/Val) Database has provided the international Earth remote sensing science community with a) a central repository for information on current and planned Calibration/Validation activities and b) a means to foster collaboration on common Cal/Val issues. The Cal/Val Database uses an ORACLE relation database management system to store the data and is accessed via the World Wide Web (WWW) using PERL scripts to search and query the database. The search queries are structured such that users can define any combination of fields, either through selection of valids, or by directly typing the information. All query results are displayed in the text form. The text displays are interactive allowing the user to point and click to access more detailed information. System functionality provides an on-line form of all of the three questionnaires for submitting new information and allows a user with the assigned password to edit archived information for their facility. This functionality allows users to update information, as it becomes available. In 2000, the Cal/Val database was updated through a process of additional surveying of existing and planned Cal/Val capabilities to support the NASA's Earth Science Enterprise (ESE) and other international Earth observing missions. A set of three updated questionnaires was prepared: one for calibration laboratories, one for test sites, and one for field instruments. The information requested included: a description of the facility, instruments available, instrument characteristics, types of measurements performed, programs/projects that have used the facility, etc. These questionnaires with cover letter were mailed to over 250 research groups that included CEOS members and facilities within the USA. The information collected from worldwide facilities was used to construct and update this on-line database for use not only by the CEOS members, but also the broader international Earth

  10. The Hanford Site generic component failure-rate database compared with other generic failure-rate databases

    SciTech Connect

    Reardon, M.F.; Zentner, M.D.

    1992-11-01

    The Risk Assessment Technology Group, Westinghouse Hanford Company (WHC), has compiled a component failure rate database to be used during risk and reliability analysis of nonreactor facilities. Because site-specific data for the Hanford Site are generally not kept or not compiled in a usable form, the database was assembled using information from a variety of other established sources. Generally, the most conservative failure rates were chosen from the databases reviewed. The Hanford Site database has since been used extensively in fault tree modeling of many Hanford Site facilities and systems. The purpose of this study was to evaluate the reasonableness of the data chosen for the Hanford Site database by comparing the values chosen with the values from the other databases.

  11. PeptiSite: a structural database of peptide binding sites in 4D.

    PubMed

    Acharya, Chayan; Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2014-03-21

    We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include: (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a multimeric protein assembly with cofactors and metal ions and (3) analysis of consensus interaction motifs within the ensembles and identification of conserved determinants of these interactions. Currently the database contains 585 proteins with 650 peptide-binding sites. http://peptisite.ucsd.edu/ link allows searching for the sites of interest and interactive visualization of the ensembles using the ActiveICM web-browser plugin. This structural database for protein-peptide interactions enables understanding of structural principles of these interactions and may assist the development of an efficient peptide docking benchmark. PMID:24406170

  12. Newly Identified Phosphorylation Site in the Vesicular Stomatitis Virus P Protein Is Required for Viral RNA Synthesis

    PubMed Central

    Mondal, Arindam; Victor, Ken G.; Pudupakam, R. S.; Lyons, Charles E.

    2014-01-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis. PMID:24257610

  13. In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: implications for biological function.

    PubMed

    Ahmad, Ishtiaq; Hoessli, Daniel C; Gupta, Ramneek; Walker-Nasir, Evelyne; Rafik, Saleem M; Choudhary, M Iqbal; Shakoori, Abdul Rauf

    2007-04-15

    Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence of other interacting proteins. Bioinformatics tools are therefore helpful to predict protein multifunctionality through the identification of serine and threonine residues wherein the hydroxyl group is likely to become modified by phosphorylation or glycosylation. Moreover, serines and threonines where both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should be helpful to assess the contribution of dynamic protein modifications in selectin-mediated inflammatory responses and cell-cell adhesion processes that are difficult to determine experimentally.

  14. The hinge region of chicken annexin I contains no site for tyrosine phosphorylation.

    PubMed

    Sidis, Y; Horseman, N D

    1993-08-30

    Annexin I (AnxI) is a calcium-dependent membrane binding protein which has been implicated in various physiological activities. The region of the chicken anxI cDNA encoding the first 130 amino terminal residues was cloned by reverse transcription PCR in order to determine the relationship of its variable amino-terminal regulatory region with other known annexins. This nucleotide sequence shows 86% identity with pigeon AnxI isoforms, and 57% with its human homolog. The protein encoded by the chicken anxI cDNA lacks the canonical epidermal growth factor receptor/kinase phosphorylation site, which is present in AnxI of other species. In contrast, the putative protein kinase C phosphorylation site of the amino-terminus is present in the chicken AnxI. Whereas the pigeon genome contains two anxI genes, genomic Southern analysis shows that in the chicken AnxI is encoded by only a single gene. These data suggest that AnxI has undergone significant sequence variation in the avians, and clarifies the relationships of the avian anxI genes with their ancestral homologs.

  15. IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.

    PubMed

    Fillebeen, Carine; Caltagirone, Annie; Martelli, Alain; Moulis, Jean-Marc; Pantopoulos, Kostas

    2005-05-15

    In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

  16. Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

    PubMed Central

    Prakash, Aishwarya; Cao, Vy Bao; Doublié, Sylvie

    2016-01-01

    The NEIL1 DNA glycosylase is one of eleven mammalian DNA glycosylases that partake in the first step of the base excision repair (BER) pathway. NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating (alanine) mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1 in vitro, and was able to phosphorylate the enzyme at residues S207, S306, and S61. PMID:27518429

  17. Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation.

    PubMed

    Cuozzo, J W; Tao, K; Wu, Q L; Young, W; Sahagian, G G

    1995-06-30

    The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins. PMID:7797559

  18. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

    PubMed Central

    Weng, Q P; Andrabi, K; Klippel, A; Kozlowski, M T; Williams, L T; Avruch, J

    1995-01-01

    The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:7777579

  19. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    SciTech Connect

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  20. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-01

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity. PMID:26583259

  1. Prediction of protein phosphorylation sites using classification trees and SVM classifier

    NASA Astrophysics Data System (ADS)

    Betkier, Piotr; Szymański, Zbigniew

    2011-10-01

    The paper presents a method of solving the problem of protein phosphorylation sites recognition. Six classifiers were created for prediction whether specified amino acid sequences represented as a 9-character strings react with given types of the kinase-enzymes. The method consists of three steps. Positions in the amino acid sequences significant for classification are found with the use of classification trees in the first step. Afterwards, the symbols composing the sequences are mapped to the real numbers domain using the Gini index method. The last step consists of creating the SVM classifiers as the final prediction models. The paper contains evaluation of the obtained results and the description of the methods applied to evaluate the quality of the classifiers.

  2. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad. PMID:11717309

  3. Akt Phosphorylates Connexin43 on Ser373, a “Mode-1” Binding Site for 14-3-3

    PubMed Central

    PARK, DARREN J.; WALLICK, CHRISTOPHER J.; MARTYN, KENDRA D.; LAU, ALAN F.; JIN, CHENGSHI; WARN-CRAMER, BONNIE J.

    2009-01-01

    Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques. PMID:18163231

  4. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival.

    PubMed

    Virdee, K; Parone, P A; Tolkovsky, A M

    2000-09-21

    Phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD.

  5. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    PubMed

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. PMID:25887881

  6. Identification and characterization of columbid annexin Icp37. Insights into the evolution of annexin I phosphorylation sites.

    PubMed

    Haigler, H T; Mangili, J A; Gao, Y; Jones, J; Horseman, N D

    1992-09-25

    Annexin I (AnxI) contains phosphorylation sites in its "hinge region" that have been implicated in the regulation of cell growth and/or differentiation. A pigeon (Columba livia) isoform of this protein, annexin Icp35 (cp35), has a very similar amino acid sequence overall but an unrelated sequence that lacks phosphorylation sites in the hinge region. We now report the identification and characterization of annexin Icp37 (cp37) from pigeon. Genomic cloning and Southern blot analysis demonstrated that cp37 and cp35 were encoded by separated genes. Prolactin induced the expression of cp35 mRNA but not cp37. The amino acid sequence of cp37 was deduced from a cDNA clone and found to share 93 and 75% sequence identity with cp35 and human AnxI, respectively. The amino acid sequence of cp37 bore similarities to both AnxI and cp35 in the critical hinge region. Like AnxI, cp37 contained consensus phosphorylation sites in its amino acid sequence and was phosphorylated on tyrosine by the EGF receptor/kinase and on serine by protein kinase C in vitro. Despite the functional similarities between cp37 and AnxI, the nucleotide sequence that encoded the hinge region of cp37 was very similar to the analogous region of cp35, but different from that of AnxI. We propose that certain features shared by cp37 and AnxI are the products of convergent evolution. The fact that evolution independently selected for two annexin I-like genes (cp37 and anxI) encoding analogous phosphorylation sites is strong evidence that phosphorylation is important for the regulation of the biological activity of these proteins.

  7. Uncertainty in site inspection and tracking database estimates of savings

    SciTech Connect

    Sonnenblick, R.; Eto, J.

    1988-12-31

    The authors systematically analyze impact evaluation results of three commercial lighting rebate DSM programs. The research includes (1) analysis of ex ante and ex post estimates of program performance, broken down into critical program parameters: hours of operation, watts saved per measure, and measures installed per site; (2) construction of probability distributions of program performance, both in the aggregate and for these critical program parameters; and (3) use of these analyses and distributions to draw conclusions about the accuracy of savings estimates from a variety of evaluation methods. The analysis suggests that realization rates (a ratio of metered savings estimates to tracking database savings estimates) for the sample of participants they examine are subject to tremendous variability, calling into question the usefulness of a point estimate of the realization rate. Discrepancies in estimates of hours of operation are responsible for most of the uncertainty in the realization rate. Finally, the impact of shorter measure lifetimes on savings estimates suggest that persistence studies should be an evaluation priority.

  8. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH)

    SciTech Connect

    Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

    1987-05-01

    BCKDH is phosphorylated by a copurifying kinase at two serine residues on the El..cap alpha.. subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, ..cap alpha..-chloroisocaproate (CIC) and branched chain ..cap alpha..-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37/sup 0/C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions.

  9. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  10. Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

    PubMed Central

    Kobayashi, Ryosuke; Kato, Akihisa; Oda, Shinya; Koyanagi, Naoto; Oyama, Masaaki; Kozuka-Hata, Hiroko; Arii, Jun

    2015-01-01

    Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo. PMID:25810545

  11. Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

    PubMed Central

    Lutterbach, B; Hann, S R

    1994-01-01

    The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site. Images PMID:8035827

  12. Adapting the rangeland database for managing ecological site description data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field data collection for writing Ecological Data Descriptions (ESD) creates a paperwork burden that reduces efficiency of ESD preparation. The recently developed Rangeland Database and Field Data Entry System is well suited to managing ESD data. This database was developed to automate data entry an...

  13. Systematic Mapping of Posttranslational Modifications in Human Estrogen Receptor-α with Emphasis on Novel Phosphorylation Sites*S⃞

    PubMed Central

    Atsriku, Christian; Britton, David J.; Held, Jason M.; Schilling, Birgit; Scott, Gary K.; Gibson, Bradford W.; Benz, Christopher C.; Baldwin, Michael A.

    2009-01-01

    A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer. PMID:18984578

  14. Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization.

    PubMed

    Nguyen, Chan; Hosokawa, Tomohisa; Kuroiwa, Mahomi; Ip, Nancy Y; Nishi, Akinori; Hisanaga, Shin-Ichi; Bibb, James A

    2007-11-01

    While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca(2+) channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M(r) 32 000 required protein phosphatase 1/2A activity and extracellular Ca(2+). In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na(+) or Ca(2+) sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.

  15. High LET - induced H2AX phosphorylation at sites of DNA double strand breaks

    NASA Astrophysics Data System (ADS)

    Desai, N.; Cucinotta, F.; Wu, H.

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  16. Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

    PubMed

    Nakrieko, Kerry-Ann; Mould, Ruth M; Smith, Alison G

    2004-02-01

    Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.

  17. Design and implementation of the site and engineering properties database; Yucca Mountain Site Characterzation Project

    SciTech Connect

    Krebs-Jespersen, M.L.

    1992-02-01

    The Yucca Mountain Site Characterization Project (YMP) is conducting studies to determine whether the Yucca Mountain site in southern Nevada will meet regulatory criteria for a potential mined geologic disposal system for high-level radioactive waste. Data gathered as part of these studies must be compiled and tabulated in a controlled manner for use in design and performance analyses. An integrated data management system has been developed to facilitate this process; this system relies on YMP participants to share in the development of the database and to ensure the integrity of the data. The site and Engineering Properties Database (SEPDB) is unique in that, unlike most databases where one data set is stored for use by one defined user, the SEPDB stores different sets of data which must be structured so that a variety of users can be given access to the information. All individuals responsible for activities supporting the license application should, to the extent possible,work with the same data and the same assumptions. For this reason, it is important that these data sets are readily accessible, comprehensive, and current. The SEPDB contains scientific and engineering data for use in performance assessment and design activities. These data sets currently consist of geologic, hydrologic, and rock properties information from drill holes and field measurements. The users of the SEPDB include engineers and scientists from several government research laboratories (Lawrence Livermore National Laboratory, Los Alamos National Laboratory, and Sandia National Laboratories), the US Geological Survey, and several government contractors. This manuscript describes the detailed requirements, contents, design, and status of the SEPDB, the procedures for submitting data to and/or requesting data from the SEPDB, and a SEPDB data dictionary (Appendix A) for defining the present contents.

  18. Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.

    PubMed

    Xie, Xitao; Langlais, Paul; Zhang, Xiaodong; Heckmann, Bradlee L; Saarinen, Alicia M; Mandarino, Lawrence J; Liu, Jun

    2014-06-15

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.

  19. Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites

    PubMed Central

    Wu, Qian; Paul, Atanu; Su, Dan; Mehmood, Shahid; Foo, Tzeh Keong; Ochi, Takashi; Bunting, Emma L.; Xia, Bing; Robinson, Carol V.; Wang, Bin; Blundell, Tom L.

    2016-01-01

    Summary BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR. PMID:26778126

  20. Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.

    PubMed

    Xie, Xitao; Langlais, Paul; Zhang, Xiaodong; Heckmann, Bradlee L; Saarinen, Alicia M; Mandarino, Lawrence J; Liu, Jun

    2014-06-15

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL. PMID:24801391

  1. Nerve Agent Exposure Elicits Site-Specific Changes in Protein Phosphorylation in Mouse Brain

    PubMed Central

    Zhu, Hongwen; O’Brien, Jennifer J.; O’Callaghan, James P.; Miller, Diane B.; Zhang, Qiang; Rana, Minal; Tsui, Tiffany; Peng, Youyi; Tomesch, John; Hendrick, Joseph P.; Wennogle, Lawrence P; Snyder, Gretchen L.

    2010-01-01

    Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies. PMID:20423708

  2. Novel HSAN1 mutation in serine palmitoyltransferase resides at a putative phosphorylation site that is involved in regulating substrate specificity.

    PubMed

    Ernst, Daniela; Murphy, Sinéad M; Sathiyanadan, Karthik; Wei, Yu; Othman, Alaa; Laurá, Matilde; Liu, Yo-Tsen; Penno, Anke; Blake, Julian; Donaghy, Michael; Houlden, Henry; Reilly, Mary M; Hornemann, Thorsten

    2015-03-01

    1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position.

  3. Novel HSAN1 mutation in serine palmitoyltransferase resides at a putative phosphorylation site that is involved in regulating substrate specificity.

    PubMed

    Ernst, Daniela; Murphy, Sinéad M; Sathiyanadan, Karthik; Wei, Yu; Othman, Alaa; Laurá, Matilde; Liu, Yo-Tsen; Penno, Anke; Blake, Julian; Donaghy, Michael; Houlden, Henry; Reilly, Mary M; Hornemann, Thorsten

    2015-03-01

    1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position. PMID:25567748

  4. P70S6 Kinase Phosphorylation: A New Site to Assess Pharmacodynamy of Sirolimus

    PubMed Central

    Wang, Jun-Yu; Fan, Hua

    2015-01-01

    Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods. Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured. Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001). Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on

  5. Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids.

    PubMed

    Ludgate, Laurie; Ning, Xiaojun; Nguyen, David H; Adams, Christina; Mentzer, Laura; Hu, Jianming

    2012-11-01

    Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

  6. Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice.

    PubMed

    Watanabe, Shigeru; Yamamori, Saori; Otsuka, Shintaro; Saito, Masanori; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

    2015-09-01

    Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of

  7. Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway.

    PubMed

    Su, Fengtao; Bhattacharya, Souparno; Abdisalaam, Salim; Mukherjee, Shibani; Yajima, Hirohiko; Yang, Yanyong; Mishra, Ritu; Srinivasan, Kalayarasan; Ghose, Subroto; Chen, David J; Yannone, Steven M; Asaithamby, Aroumougame

    2016-01-01

    Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability. PMID:26695548

  8. Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway

    PubMed Central

    Su, Fengtao; Bhattacharya, Souparno; Abdisalaam, Salim; Mukherjee, Shibani; Yajima, Hirohiko; Yang, Yanyong; Mishra, Ritu; Srinivasan, Kalayarasan; Ghose, Subroto; Chen, David J.; Yannone, Steven M.; Asaithamby, Aroumougame

    2016-01-01

    Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability. PMID:26695548

  9. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

    PubMed Central

    Nichols, M; Weih, F; Schmid, W; DeVack, C; Kowenz-Leutz, E; Luckow, B; Boshart, M; Schütz, G

    1992-01-01

    Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation. Images PMID:1354612

  10. A phosphorylation site located in the NH2-terminal domain of c-Myc increases transactivation of gene expression.

    PubMed

    Seth, A; Alvarez, E; Gupta, S; Davis, R J

    1991-12-15

    The c-myc gene encodes a sequence-specific DNA-binding protein (c-Myc) that forms leucine zipper complexes and can act as a transcription factor. Growth factor stimulation of cells causes the phosphorylation of the c-Myc transcriptional activation domain at Ser62 within a proline-rich region that is highly conserved among members of the Myc family (Alvarez, E., Northwood, I.C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). This phosphorylation site is a substrate for growth factor-regulated MAP kinases and for the cell cycle-dependent protein kinase p34cdc2. We report that serum treatment of cells results in a marked increase in the transactivation of gene expression mediated by the c-Myc transcriptional activation domain. A point mutation at the site of growth factor-stimulated phosphorylation (Ser62) decreases the serum induction of transactivation. These data indicate that the c-Myc transcriptional activation domain may be a direct target of signal transduction pathways. PMID:1748630

  11. SITES 2006 User Guide for the International Database. Second Information Technology in Education Study

    ERIC Educational Resources Information Center

    Brese, Falk, Ed.; Carstens, Ralph, Ed.

    2009-01-01

    To support and promote secondary analyses, the International Association for the Evaluation of Educational Achievement (IEA) is making the SITES 2006 international database and accompanying User Guide available to researchers, analysts, and public users. The database comprises national contexts and school- and teacher-level data from 23 education…

  12. The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization.

    PubMed

    Ando, Ryo; Shima, Hiroki; Tamahara, Toru; Sato, Yoshihiro; Watanabe-Matsui, Miki; Kato, Hiroki; Sax, Nicolas; Motohashi, Hozumi; Taguchi, Keiko; Yamamoto, Masayuki; Nio, Masaki; Maeda, Tatsuya; Ochiai, Kyoko; Muto, Akihiko; Igarashi, Kazuhiko

    2016-01-22

    The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells. PMID:26620562

  13. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    PubMed

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  14. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

    PubMed Central

    Colson, Brett A.; Thompson, Andrew R.; Espinoza-Fonseca, L. Michel; Thomas, David D.

    2016-01-01

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron–electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0–C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein’s flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein–protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  15. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.

  16. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  17. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  18. In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

    PubMed Central

    Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

    2015-01-01

    Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point

  19. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    SciTech Connect

    Sultatos, L.G. Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.

  20. Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44

    SciTech Connect

    Alvisi, Gualtiero; Marin, Oriano; Pari, Gregory; Mancini, Manuela; Avanzi, Simone; Loregian, Arianna; Jans, David A.; Ripalti, Alessandro

    2011-09-01

    The processivity factor of human cytomegalovirus DNA polymerase, phosphoprotein ppUL44, is essential for viral replication. During viral infection ppUL44 is phosphorylated by the viral kinase pUL97, but neither the target residues on ppUL44 nor the effect of phosphorylation on ppUL44's activity are known. We report here that ppUL44 is phosphorylated when transiently expressed in mammalian cells and coimmunoprecipitates with cellular kinases. Of three potential phosphorylation sites (S413, S415, S418) located upstream of ppUL44's nuclear localization signal (NLS) and one (T427) within the NLS itself, protein kinase CK2 (CK2) specifically phosphorylates S413, to trigger a cascade of phosphorylation of S418 and S415 by CK1 and CK2, respectively. Negative charge at the CK2/CK1 target serine residues facilitates optimal nuclear accumulation of ppUL44, whereas negative charge on T427, a potential cyclin-dependent 1 phosphorylation site, strongly decreases nuclear accumulation. Thus, nuclear transport of ppUL44 is finely tuned during viral infection through complex phosphorylation events.

  1. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

    PubMed

    Garver, T D; Ren, Q; Tuvia, S; Bennett, V

    1997-05-01

    This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

  2. Site-directed mutagenesis and deletion of three phosphorylation sites of calsequestrin of skeletal muscle sarcoplasmic reticulum. Effects on intracellular targeting.

    PubMed

    Nori, A; Furlan, S; Patiri, F; Cantini, M; Volpe, P

    2000-10-10

    Calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) of skeletal muscle fibers and is responsible for intraluminal Ca(2+) binding. A chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the carboxy-terminal of CS and shown to be correctly segregated to skeletal muscle jSR in vivo (A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe, 1997, Am. J. Physiol. 272, C1420-C1428), is mutagenized in order to identify domains of CS involved in targeting. Since a putative targeting mechanism of CS implies phosphorylation-dependent steps in the endoplasmic reticulum (ER) and/or Golgi complex, five CS-HA1 mutants disrupting the three phosphorylation sites of CS (Thr(189), Thr(229), and Thr(353)) were engineered by either site-directed mutagenesis or deletion: CS-HA1DeltaP1 (Thr(189) --> Ile); CS-HA1DeltaP2 (Thr(229) --> Asn); CS-HA1DeltaP1,2; in which Thr(189) and Thr(229) were changed to Ile and Asn, respectively; and CS-HA1Delta14(COOH) and CS-HA1Delta49 (COOH), in which 14 residues (Glu(354)-Asp(367)) and 49 residues (Asp(319)-Asp(367)), respectively, were deleted at the carboxy-terminal. Mutant cDNAs were transiently transfected in either HeLa cells, cultured myoblasts of rat skeletal muscle, or regenerating soleus muscle fibers of adult rats. Each CS-HA1 mutant was identified by Western blot as a single polypeptide of the predicted molecular weight. The intracellular localization of CS-HA1 mutants was studied by immunofluorescence using specific antibodies against either CS or HA1. CS-HA1 mutants colocalized with ER markers, e.g., calreticulin, and partially overlapped with Golgi complex markers, e.g., alpha-mannosidase II, in HeLa cells and myotubes. CS-HA1 mutants were expressed and retained in ER and ER/SR of HeLa cells and myotubes, respectively, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo is not affected by

  3. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  4. Preliminary Safety Analysis of the Gorleben Site: Geological Database - 13300

    SciTech Connect

    Weber, Jan Richard; Mrugalla, Sabine; Dresbach, Christian; Hammer, Joerg

    2013-07-01

    The Gorleben salt dome is 4 km wide and nearly 15 km long. It is composed of different salt rock types of the Zechstein (Upper Permian) series and extends to the Zechstein basis in a depth of more than 3 km. In the course of the salt dome formation the salt was moved several kilometers. During the uplift of the salt the initially plane-bedded strata of the Zechstein series were extensively folded. In this process anhydrite as a competent layer was broken to isolated blocks. In the core of the salt dome the Hauptsalz, which is characterized by a particularly high creeping capacity, forms a homogeneous halite body with a volume of several cubic kilometres. The Hauptsalz contains gaseous and liquid hydrocarbons in separated zones of decimeter to meter dimensions. The overall hydrocarbon content is far below 0.01 %. At the flanks the salt dome consists of salt rocks with lower creeping capacities. Brine reservoirs with fluid volumes in the range of liters to hundreds of cubic meters exist in certain regions of this part of the salt dome. The water content of the Hauptsalz is below 0.02 %. Interconnected pores do not exist in the salt rock outside of fluid bearing or fractured areas, i.e. the salt rock is impermeable. The exploration of the Gorleben site as a potential site for a HLW-repository started in 1979 and is still in progress. To date no scientific findings contest the suitability of the site for a safe HLW-repository. (authors)

  5. A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation.

    PubMed

    Havukainen, Heli; Underhaug, Jarl; Wolschin, Florian; Amdam, Gro; Halskau, Øyvind

    2012-06-01

    Vitellogenin (Vg) is an egg-yolk precursor protein in most oviparous species. In honeybee (Apis mellifera), the protein (AmVg) also affects social behavior and life-span plasticity. Despite its manifold functions, the AmVg molecule remains poorly understood. The subject of our structure-oriented AmVg study is its polyserine tract - a little-investigated repetitive protein segment mostly found in insects. We previously reported that AmVg is tissue specifically cleaved in the vicinity of this tract. Here, we show that, despite its potential for an open, disordered structure, AmVg is unexpectedly resistant to trypsin/chymotrypsin digestion at the tract. Our findings suggest that multiple phosphorylation plays a role in this resilience. Sequence variation is highly pronounced at the polyserine region in insect Vgs. We demonstrate that sequence differences in this region can lead to structural variation, as NMR and circular dichroism (CD) evidence assign different conformational propensities to polyserine peptides from the honeybee and the jewel wasp Nasonia vitripennis; the former is extended and disordered and the latter more compact and helical. CD analysis of the polyserine region of bumblebee Bombus ignitus and wasp Pimpla nipponica supports a random coil structure in these species. The spectroscopic results strengthen our model of the AmVg polyserine tract as a flexible domain linker shielded by phosphorylation. PMID:22573762

  6. A fragmented alignment method detects a putative phosphorylation site and a putative BRC repeat in the Drosophila melanogaster BRCA2 protein.

    PubMed

    Chakraborty, Sandeep

    2013-01-01

    Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html.

  7. The M3 Phosphorylation Site Is Required for Trafficking and Biological Roles of PIN-FORMED1, 2, and 7 in Arabidopsis

    PubMed Central

    Ki, Daeeun; Sasayama, Daisuke; Cho, Hyung-Taeg

    2016-01-01

    Asymmetrically localized PIN-FORMED (PIN) auxin efflux carriers play key roles in regulating directional intercellular auxin movement, generating local auxin gradients, and diverse auxin-mediated growth and development. The polar localization of PINs is controlled by phosphorylation in the central hydrophilic loop (HL) of PINs. Although the M3 phosphorylation site, including phosphorylatable 5 Ser/Thr residues, is conserved among long HL-PINs, its native role has only been characterized in PIN3. In this study, we examined the role of M3 phosphorylation site of PIN1, PIN2, and PIN7 in intracellular trafficking, phosphorylation, and biological functions of those PINs in their native expressing tissues. Phosphorylation-defective mutations of the phosphorylatable residues in the M3 site of PIN1-HL led to alteration in subcellular polarity of PIN1 and caused defects in PIN1-mediated biological functions such as cotyledon development, phyllotaxy of vegetative leaves, and development of reproductive organs. The M3 mutations of PIN7 interfered with its polar recycling in the root columella cell in response to gravity stimulus and partially disrupted root gravitropism. On the other hand, the M3 site of PIN2 was shown to be necessary for its targeting to the plasma membrane. In vitro phosphorylation assay showed that the M3 phosphorylation residues of PIN1 are the partial targets by PINOID kinase. Our data suggest that the M3 phosphorylation site is functionally conserved among long HL-PINs by playing roles for their subcellular trafficking and auxin-mediated developmental processes. PMID:27733863

  8. Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage

    PubMed Central

    Zolner, Angela E.; Abdou, Ismail; Ye, Ruiqiong; Mani, Rajam S.; Fanta, Mesfin; Yu, Yaping; Douglas, Pauline; Tahbaz, Nasser; Fang, Shujuan; Dobbs, Tracey; Wang, Chen; Morrice, Nick; Hendzel, Michael J.; Lees-Miller, Susan P.

    2011-01-01

    Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5′-DNA kinase/3′-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs. PMID:21824916

  9. Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage.

    PubMed

    Zolner, Angela E; Abdou, Ismail; Ye, Ruiqiong; Mani, Rajam S; Fanta, Mesfin; Yu, Yaping; Douglas, Pauline; Tahbaz, Nasser; Fang, Shujuan; Dobbs, Tracey; Wang, Chen; Morrice, Nick; Hendzel, Michael J; Weinfeld, Michael; Lees-Miller, Susan P

    2011-11-01

    Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

  10. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    SciTech Connect

    Loots, G; Ovcharenko, I

    2006-08-08

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes entitled ECRbase that is constructed from a collection of pairwise vertebrate genome alignments produced by the ECR Browser database. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a collection of promoters in all vertebrate genomes presented in the database. The database also contains a collection of annotated transcription factor binding sites (TFBS) in all ECRs and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and two pufferfish genomes. It is freely accessible at http://ECRbase.dcode.org.

  11. Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    PubMed Central

    Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

    2011-01-01

    Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

  12. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    DOE Data Explorer

    Loots, Gabriela G. [LLNL; Ovcharenko, I. [LLNL

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. This database of evolutionary conserved regions (ECRs) in vertebrate genomes features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a comprehensive collection of promoters in all vertebrate genomes generated using multiple sources of gene annotation. The database also contains a collection of annotated transcription factor binding sites (TFBSs) in evolutionary conserved and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and fugu genomes. (taken from paper in Journal: Bioinformatics, November 7, 2006, pp. 122-124

  13. Identification of the chicken MARCKS phosphorylation site specific for differentiating neurons as Ser 25 using a monoclonal antibody and mass spectrometry.

    PubMed

    Zolessi, Flavio R; Durán, Rosario; Engström, Ulla; Cerveñansky, Carlos; Hellman, Ulf; Arruti, Cristina

    2004-01-01

    MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.

  14. Identification of the chicken MARCKS phosphorylation site specific for differentiating neurons as Ser 25 using a monoclonal antibody and mass spectrometry.

    PubMed

    Zolessi, Flavio R; Durán, Rosario; Engström, Ulla; Cerveñansky, Carlos; Hellman, Ulf; Arruti, Cristina

    2004-01-01

    MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development. PMID:14998167

  15. The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State

    PubMed Central

    Kim, Sang Y.; Bender, Kyle W.; Walker, Berkley J.; Zielinski, Raymond E.; Spalding, Martin H.; Ort, Donald R.; Huber, Steven C.

    2016-01-01

    Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions. PMID:27064346

  16. Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

    PubMed

    Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

    2014-12-01

    As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples. PMID:25403019

  17. Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

    PubMed

    Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

    2014-12-01

    As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

  18. Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli.

    PubMed

    Kato, M; Aiba, H; Tate, S; Nishimura, Y; Mizuno, T

    1989-06-01

    The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.

  19. A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ

    PubMed Central

    Gong, Jianli; Holewinski, Ronald J.; Van Eyk, Jennifer E.; Steinberg, Susan F.

    2016-01-01

    Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain).We show that S130D and T141E substitutions co-operate to increase PKCδ’s basal lipid-independent activity and that Ser130/Thr141 di-phosphorylation influences PKCδ’s substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser357, an ATP-positioning G-loop site that limits PKCδ’s threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study shows that S130D and T141E substitutions increase PKCδ’s threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser357. A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ’s maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser130/Thr141 phosphorylations relieve auto-inhibitory constraints that limit PKCδ’s activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity. PMID:26546672

  20. Site Specific Phosphorylation of Cytochrome c Oxidase Subunits I, IVi1 and Vb in Rabbit Hearts Subjected to Ischemia/Reperfusion

    PubMed Central

    Fang, Ji-Kang; Prabu, Subbuswamy K.; Sepuri, Naresh B.; Raza, Haider; Anandatheerthavarada, Hindupur K.; Galati, Domenico; Spear, Joseph; Avadhani, Narayan G.

    2007-01-01

    We have mapped the sites of ischemia/reperfusion induced phosphorylation of cytochrome c oxidase (CcO) subunits in rabbit hearts by using a combination of Blue Native gel/Tricine gel electrophoresis and nano-LC-MS/MS approaches. We used precursor ion scanning combined with neutral loss scanning and found that mature CcO subunit I was phosphorylated at tandem Ser115/Ser116 positions, subunit IVi1 at Thr52 and subunit Vb at Ser40. These sites are highly conserved in mammalian species. Molecular modeling suggests that phosphorylation sites of subunit I face the inter membrane space while those of subunits IVi1 and Vb face the matrix side. PMID:17349628

  1. Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

    PubMed

    Honarmand-Jahromy, Sahar; Siavoshi, Farideh; Malekzadeh, Reza; Sattari, Taher Nejad; Latifi-Navid, Saeid

    2015-12-01

    Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran. PMID:26408373

  2. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    PubMed Central

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  3. Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

    PubMed

    Honarmand-Jahromy, Sahar; Siavoshi, Farideh; Malekzadeh, Reza; Sattari, Taher Nejad; Latifi-Navid, Saeid

    2015-12-01

    Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran.

  4. Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.

    PubMed Central

    Sakata, J; Uyeda, K

    1990-01-01

    We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities. Images PMID:2164212

  5. Role of the amino-terminal domain in regulating interactions of annexin I with membranes: effects of amino-terminal truncation and mutagenesis of the phosphorylation sites.

    PubMed

    Wang, W; Creutz, C E

    1994-01-11

    Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8286349

  6. Differential roles of PDK1- and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9.

    PubMed

    Roelants, Françoise M; Torrance, Pamela D; Thorner, Jeremy

    2004-10-01

    Saccharomyces cerevisiae Pkh1 and Pkh2 (orthologues of mammalian protein kinase, PDK1) are functionally redundant. These kinases activate three AGC family kinases involved in the maintenance of cell wall integrity: Ypk1 and Ypk2, two closely related, functionally redundant enzymes (orthologues of mammalian protein kinase SGK), and Pkc1 (orthologue of mammalian protein kinase PRK2). Pkh1 and Pkh2 activate Ypk1, Ypk2 and Pkc1 by phosphorylating a Thr in a conserved sequence motif (PDK1 site) within the activation loop of these proteins. A fourth protein kinase involved in growth control and stress response, Sch9 (orthologue of mammalian protein kinase c-Akt/PKB), also carries the conserved activation loop motif. Like other AGC family kinases, Ypk1, Ypk2, Pkc1 and Sch9 also carry a second conserved sequence motif situated in a region C-terminal to the catalytic domain, called the hydrophobic motif (PDK2 site). Currently, there is still controversy surrounding the identity of the enzyme responsible for phosphorylating this second site and the necessity for phosphorylation at this site for in vivo function. Here, genetic and biochemical methods have been used to investigate the physiological consequences of phosphorylation at the PDK1 and PDK2 sites of Ypk1, Pkc1 and Sch9. It was found that phosphorylation at the PDK1 site in the activation loop is indispensable for the essential functions of all three kinases in vivo, whereas phosphorylation at the PDK2 motif plays a non-essential and much more subtle role in modulating the ability of these kinases to regulate the downstream processes in which they participate. PMID:15470109

  7. Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

    PubMed

    Ferrari, S; Bannwarth, W; Morley, S J; Totty, N F; Thomas, G

    1992-08-01

    Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

  8. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    PubMed

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  9. Mutation of light-dependent phosphorylation sites of the Drosophila transient receptor potential-like (TRPL) ion channel affects its subcellular localization and stability.

    PubMed

    Cerny, Alexander C; Oberacker, Tina; Pfannstiel, Jens; Weigold, Sebastian; Will, Carina; Huber, Armin

    2013-05-31

    The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

  10. Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.

    PubMed

    Hammes, S R; Shapiro, M J; Coughlin, S R

    1999-07-20

    The thrombin receptor PAR1 becomes rapidly phosphorylated upon activation by either thrombin or exogenous SFLLRN agonist peptide. Substitution of alanine for all serine and threonine residues in the receptor's cytoplasmic carboxyl-terminal tail ablated phosphorylation and yielded a receptor defective in both shutoff and agonist-triggered internalization. These observations suggested that activation-dependent phosphorylation of PAR1's cytoplasmic tail is required for both shutoff and agonist-triggered internalization. To identify the phosphorylation site(s) that are necessary for these functions, we generated three mutant receptors in which alanine was substituted for serine and threonine residues in the amino-terminal, middle, and carboxyl-terminal thirds of PAR1's cytoplasmic tail. When stably expressed in fibroblasts, all three mutated receptors were rapidly phosphorylated in response to agonist, while a mutant in which all serines and threonines in the cytoplasmic tail were converted to alanines was not. This result suggests that phosphorylation can occur at multiple sites in PAR1's cytoplasmic tail. Alanine substitutions in the N-terminal and C-terminal portions of the tail had no effect on either receptor shutoff or agonist-triggered internalization. By contrast, alanine substitutions in the "middle" serine cluster between Ser(391) and Ser(406) yielded a receptor with considerably slower shutoff of signaling after thrombin activation than the wild type. Surprisingly, this same mutant was indistinguishable from the wild type in agonist-triggered internalization and degradation. Overexpression of G protein-coupled receptor kinase 2 (GRK2) and GRK3 "suppressed" the shutoff defect of the S --> A (391-406) mutant, consistent with this defect being due to altered receptor phosphorylation. These results suggest that specific phosphorylation sites are required for rapid receptor shutoff, but phosphorylation at multiple alternative sites is sufficient for agonist

  11. TFBSshape: a motif database for DNA shape features of transcription factor binding sites

    PubMed Central

    Yang, Lin; Zhou, Tianyin; Dror, Iris; Mathelier, Anthony; Wasserman, Wyeth W.; Gordân, Raluca; Rohs, Remo

    2014-01-01

    Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein–DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone. PMID:24214955

  12. Structures of KaiC Circadian Clock Mutant Proteins: A New Phosphorylation Site at T426 and Mechanisms of Kinase, ATPase and Phosphatase

    SciTech Connect

    Pattanayek, Rekha; Mori, Tetsuya; Xu, Yao; Pattanayek, Sabuj; Johnson, Carl H.; Egli, Martin

    2010-09-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS {yields} pTS {yields} pTpS {yields} TpS {yields} TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC. The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP {gamma}-phosphate. T432 is phosphorylated first because it lies consistently closer to P{gamma}. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation. We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.

  13. Structures of KaiC Circadian Clock Mutant Proteins: A New Phosphorylation Site at T426 and Mechanisms of Kinase, ATPase and Phosphatase

    PubMed Central

    Pattanayek, Rekha; Mori, Tetsuya; Xu, Yao; Pattanayek, Sabuj; Johnson, Carl H.; Egli, Martin

    2009-01-01

    Background The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS→pTS→pTpS→TpS→TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC. Methodology and Principal Findings The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP γ-phosphate. T432 is phosphorylated first because it lies consistently closer to Pγ. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation. Conclusions and Significance We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities. PMID:19956664

  14. Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

    SciTech Connect

    Tachikawa, E.; Tank, A.W.; Weiner, D.H.; Mosimann, W.F.; Yanagihara, N.; Weiner, N.

    1986-03-01

    The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin are independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.

  15. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

    PubMed

    Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

    2015-12-01

    Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

  16. MinChem: A Prototype Petrologic Database for Hanford Site Sediments

    SciTech Connect

    Mackley, Rob D.; Last, George V.; Serkowski, John A.; Middleton, Lisa A.; Cantrell, Kirk J.

    2010-09-01

    A prototype petrologic database (MinChem) has been under continual development for several years. MinChem contains petrologic, mineralogical, and bulk-rock geochemical data for Hanford Site sediments collected over multiple decades. The database is in relational form and consists of a series of related tables modeled after the Hanford Environmental Information System HEIS (BHI 2002) structures. The HEIS-compatible tables were created in anticipation of eventual migration into HEIS, or some future form of HEIS (e.g. HEIS-GEO). There are currently a total of 13,129 results in MinChem from 521 samples collected at 381 different sampling sites. These data come from 19 different original source documents published and unpublished (e.g. letter reports) between 1976 and 2009. The data in MinChem consist of results from analytical methods such as optical and electron microscopy, x-ray diffraction, x-ray fluorescence, and electron probe microanalysis.

  17. ARM Quick-looks Database for North Slope Alaska (NSA) sites

    DOE Data Explorer

    Stamnes, Knut [NSA Site Scientist

    From these pages one can monitor parts of the data acquisition process and access daily data visualizations from the different instruments. These data visualizations are produced in near real time automatically and are called Quick-Looks (QLs). The quick-looks contains unofficial data of unknown quality. Once data is released one can obtain the full data-set from any instrument available, and along with that, a statement about the data quality from the ARM archive. The database provides Quick-looks for the Barrow ACRF site (NSA C1), the Atqasuk ACRF site (NSA C2), or the SHEBA ice campaign of 1997 and 1998. As of 12-17-08, the database had more than 528,000 quick-looks available as data figures and data plots. No password is required for Quick-look access. (Specialized Interface)

  18. PlantDHS: a database for DNase I hypersensitive sites in plants

    PubMed Central

    Zhang, Tao; Marand, Alexandre P.; Jiang, Jiming

    2016-01-01

    Gene expression is regulated by orchestrated binding of regulatory proteins to promoters and other cis-regulatory DNA elements (CREs). Several plant databases have been developed for mapping promoters or DNA motifs associated with promoters. However, there is a lack of databases that allow investigation for all CREs. Here we present PlantDHS (http://plantdhs.org), a plant DNase I hypersensitive site (DHS) database that integrates histone modification, RNA sequencing, nucleosome positioning/occupancy, transcription factor binding sites, and genomic sequence within an easily navigated user interface. DHSs are indicative of all CREs, including promoters, enhancers, silencers, insulators and transcription factor binding sites; all of which play immense roles in global gene expression regulation. PlantDHS provides a platform to predict all CREs associated with individual genes from three model plant species, including Arabidopsis thaliana, Brachypodium distachyon and rice (Oryza sativa). PlantDHS is especially valuable in the detection of distant CREs that are located away from promoters. PMID:26400163

  19. STarMirDB: A database of microRNA binding sites.

    PubMed

    Rennie, William; Kanoria, Shaveta; Liu, Chaochun; Mallick, Bibekanand; Long, Dang; Wolenc, Adam; Carmack, C Steven; Lu, Jun; Ding, Ye

    2016-06-01

    microRNAs (miRNAs) are an abundant class of small endogenous non-coding RNAs (ncRNAs) of ∼22 nucleotides (nts) in length. These small regulatory molecules are involved in diverse developmental, physiological and pathological processes. miRNAs target mRNAs (mRNAs) for translational repression and/or mRNA degradation. Predictions of miRNA binding sites facilitate experimental validation of miRNA targets. Models developed with data from CLIP studies have been used for predictions of miRNA binding sites in the whole transcriptomes of human, mouse and worm. The prediction results have been assembled into STarMirDB, a new database of miRNA binding sites available at http://sfold.wadsworth.org/starmirDB.php . STarMirDB can be searched by miRNAs or mRNAs separately or in combination. The search results are categorized into seed and seedless sites in 3' UTR, CDS and 5' UTR. For each predicted site, STarMirDB provides a comprehensive list of sequence, thermodynamic and target structural features that are known to influence miRNA: target interaction. A high resolution PDF diagram of the conformation of the miRNA:target hybrid is also available for visualization and publication. The results of a database search are available through both an interactive viewer and downloadable text files. PMID:27144897

  20. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins.

    PubMed

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J; Camiña, Jesús P

    2016-03-03

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.

  1. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins

    PubMed Central

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F.; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J.; Camiña, Jesús P.

    2016-01-01

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser362, Ser363 and Thr366 residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr350 and Ser349 are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

  2. Intracytoplasmic phosphorylation sites of Tac antigen (p55) are not essential for the conformation, function, and regulation of the human interleukin 2 receptor.

    PubMed Central

    Hatakeyama, M; Minamoto, S; Taniguchi, T

    1986-01-01

    Tac antigen, the receptor for human interleukin 2 (IL-2), contains in its intracytoplasmic region a serine residue (Ser-247) that is seemingly the predominant site of protein kinase C-mediated phosphorylation. A number of studies on growth factor receptors have suggested the importance of phosphorylation in receptor structure, function, and regulation. In this study, we generated site-directed mutations in the Tac antigen cDNA to generate mutant receptors in which Ser-247 or Thr-250, a probable site of minor phosphorylation, was replaced with another amino acid that is not accessible to phosphorylation. Study of the expression of these mutant genes in a T-lymphoid cell line has provided no evidence as to the essential role of the above-mentioned residues in determining the degree of receptor affinity, its ability for signal transduction, and phorbol ester-mediated regulation of the receptor. Our results strongly suggest the existence of an IL-2 receptor "complex" in which the Tac antigen is associated with another molecule(s) that is involved in receptor structure, function, and regulation. PMID:3099287

  3. Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence

    PubMed Central

    Plafker, Scott M.; Woods, Amina S.; Gibson, Wade

    1999-01-01

    The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications. PMID:10516011

  4. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    PubMed

    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.

  5. Phosphorylation of arylsulphatase A occurs through multiple interactions with the UDP-N-acetylglucosamine-1-phosphotransferase proximal and distal to its retrieval site by the KDEL receptor.

    PubMed Central

    Dittmer, F; von Figura, K

    1999-01-01

    Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-phosphate receptors, was found to be site specific. ASA residing within the secretory route of these cells contains about one third of the incorporated [2-3H]mannose in phosphorylated oligosaccharides. Oligosaccharides carrying two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addition of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence time of ASA within the secretory route 6-fold, but does not result in more efficient phosphorylation. In contrast, more than 90% of the [2-3H]mannose incorporated into secreted ASA (with or without a KDEL retention signal) is present in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located within the secretory route proximal and distal to the site where ASA is retrieved by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a single oligosaccharide. Of several drugs known to inhibit transit of ASA through the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation. PMID:10359658

  6. Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.

    PubMed

    Maierhofer, Tobias; Diekmann, Marion; Offenborn, Jan Niklas; Lind, Christof; Bauer, Hubert; Hashimoto, Kenji; S Al-Rasheid, Khaled A; Luan, Sheng; Kudla, Jörg; Geiger, Dietmar; Hedrich, Rainer

    2014-09-09

    Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

  7. MeRNA: a Database of Metal Ion Binding Sites in RNAStructures

    SciTech Connect

    Stefan, Liliana R.; Zhang, Rui; Levitan, Aaron G.; Hendrix, DonnaF.; Brenner, Steven E.; Holbrook, Stephen R.

    2005-10-05

    Metal ions are essential for the folding of RNA into stable tertiary structures and for the catalytic activity of some RNA enzymes. To aid in the study of the roles of metal ions in RNA structural biology, we have created MeRNA (Metals in RNA), a comprehensive compilation of all metal binding sites identified in RNA three-dimensional structures available from the Protein Data Bank (PDB) and Nucleic Acid Database (NDB). Currently, our database contains information relating to binding of 9764 metal ions corresponding to 23 distinct elements; in 256 RNA structures. The metal ion locations were confirmed and ligands characterized using original literature references. MeRNA includes eight manually identified metal-ion binding motifs, which are described in the literature. MeRNA is searchable by PDB identifier, metal ion, method of structure determination, resolution and R-values for X-ray structure, and distance from metal to any RNA atom or to water. New structures with their respective binding motifs will be added to the database as they become available. The MeRNA database will further our understanding of the roles of metal ions in RNA folding and catalysis and have applications in structural and functional analysis, RNA design and engineering.

  8. A database of information on technologies for hazardous waste site remediation

    SciTech Connect

    Holter, G.M.; White, M.K.; Byrant, J.L.

    1992-04-01

    A personal-computer-based database and user interface has been developed for retrieving and reviewing information on technologies applicable to the environmental remediation of hazardous waste sites. This system and its information represent a useful source of technology information for people preparing, reviewing, and approving site remediation plans or evaluating remediation technologies. The system includes a variety of information for approximately 90 distinct remedial action technologies. A general text description of each technology is provided, together with basic engineering or design parameters and flowcharts. Information on applying a given technology includes the applicability of the technology to specific contaminants, associated technologies that may be required in conjunction to provide for complete remediation of a site, technical limitations and constraints on the use of the technology, and identification of information or site data needed to deploy the technology at a particular site. US federal regulatory information relating to each technology is also provided. In addition, the system identifies sources for more detailed information for these technologies (i.e., references and specific sites where these technologies have been used). Technologies to be considered can be selected from the complete list of technologies for which information is included, or can be chosen from a shorter list of technologies matching a set of user-specific remediation objectives. The technology information is compiled from a wide variety of sources. The system is designed to support the assessment of remedial alternatives at US sites, but should be readily adaptable to other environmental remediation situations throughout the world.

  9. Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability.

    PubMed Central

    Pevzner, V; Kraft, R; Kostka, S; Lipp, M

    2000-01-01

    We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down-regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli. PMID:10727398

  10. Phosphorylation at serine 52 and 635 does not alter the transport properties of glucosinolate transporter AtGTR1

    PubMed Central

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Halkier, Barbara Ann; Nour-Eldin, Hussam Hassan

    2016-01-01

    Little is known about how plants regulate transporters of defense compounds. In A. thaliana, glucosinolates are transported between tissues by NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2). Mining of the PhosPhat4.0 database showed two cytosol exposed phosphorylation sites for AtGTR1 and one membrane-buried phosphorylation site for AtGTR2. In this study, we investigate whether mutation of the two potential regulatory sites of AtGTR1 affected transport of glucosinolates in Xenopus oocytes. Characterization of AtGTR1 phosphorylation mutants showed that phosphorylation of AtGTR1 - at the two reported phosphorylation sites - is not directly involved in regulating AtGTR1 transport activity. We hypothesize a role for AtGTR1-phosphorylation in regulating protein-protein interactions. PMID:26340317

  11. The MANAGE database: nutrient load and site characteristic updates and runoff concentration data.

    PubMed

    Harmel, Daren; Qian, Song; Reckhow, Ken; Casebolt, Pamela

    2008-01-01

    The "Measured Annual Nutrient loads from AGricultural Environments" (MANAGE) database was developed to be a readily accessible, easily queried database of site characteristic and field-scale nutrient export data. The original version of MANAGE, which drew heavily from an early 1980s compilation of nutrient export data, created an electronic database with nutrient load data and corresponding site characteristics from 40 studies on agricultural (cultivated and pasture/range) land uses. In the current update, N and P load data from 15 additional studies of agricultural runoff were included along with N and P concentration data for all 55 studies. The database now contains 1677 watershed years of data for various agricultural land uses (703 for pasture/rangeland; 333 for corn; 291 for various crop rotations; 177 for wheat/oats; and 4-33 yr for barley, citrus, vegetables, sorghum, soybeans, cotton, fallow, and peanuts). Across all land uses, annual runoff loads averaged 14.2 kg ha(-1) for total N and 2.2 kg ha(-1) for total P. On average, these losses represented 10 to 25% of applied fertilizer N and 4 to 9% of applied fertilizer P. Although such statistics produce interesting generalities across a wide range of land use, management, and climatic conditions, regional crop-specific analyses should be conducted to guide regulatory and programmatic decisions. With this update, MANAGE contains data from a vast majority of published peer-reviewed N and P export studies on homogeneous agricultural land uses in the USA under natural rainfall-runoff conditions and thus provides necessary data for modeling and decision-making related to agricultural runoff. The current version can be downloaded at http://www.ars.usda.gov/spa/manage-nutrient.

  12. Phosphorylation Sites in the Hook Domain of CaVβ Subunits Differentially Modulate CaV1.2 Channel Function

    PubMed Central

    Brunet, Sylvain; Emrick, Michelle A.; Sadilek, Martin; Scheuer, Todd; Catterall, William A.

    2015-01-01

    Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. CaV1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of CaVβ subunits in vivo and test the impact of mutations of those sites on CaV1.2 channel function in vitro. Using the CaV1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser193 and Thr205 in the HOOK domain of CaVβ1a subunits were both phosphorylated in vivo. Ser193 is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr205 is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple CaVβ subunit isoforms, including the principal CaVβ subunit of cardiac CaV1.2 channels, CaVβ2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in CaVβ2b and analyzed their effects on CaV1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation CaVβ2bS152E decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation CaVβ2bS152A had opposite effects. There were no differences in peak CaV1.2 currents or voltage dependence between the phosphomimetic mutation CaVβ2bT164D and the phosphoinhibitory mutation CaVβ2bT164A. However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation CaVβ2bT164D. This effect was subunit

  13. The C-terminal tail inhibitory phosphorylation sites of PTEN regulate its intrinsic catalytic activity and the kinetics of its binding to phosphatidylinositol-4,5-bisphosphate.

    PubMed

    Chia, Yeong-Chit Joel; Catimel, Bruno; Lio, Daisy Sio Seng; Ang, Ching-Seng; Peng, Benjamin; Wu, Hong; Zhu, Hong-Jian; Cheng, Heung-Chin

    2015-12-01

    Dephosphorylation of four major C-terminal tail sites and occupancy of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]-binding site of PTEN cooperate to activate its phospholipid phosphatase activity and facilitate its recruitment to plasma membrane. Our investigation of the mechanism by which phosphorylation of these C-terminal sites controls the PI(4,5)P2-binding affinity and catalytic activity of PTEN resulted in the following findings. First, dephosphorylation of all four sites leads to full activation; and phosphorylation of any one site significantly reduces the intrinsic catalytic activity of PTEN. These findings suggest that coordinated inhibition of the upstream protein kinases and activation of the protein phosphatases targeting the four sites are needed to fully activate PTEN phosphatase activity. Second, PI(4,5)P2 cannot activate the phosphopeptide phosphatase activity of PTEN, suggesting that PI(4,5)P2 can only activate the phospholipid phosphatase activity but not the phosphoprotein phosphatase activity of PTEN. Third, dephosphorylation of all four sites significantly decreases the affinity of PTEN for PI(4,5)P2. Since PI(4,5)P2 is a major phospholipid co-localizing with the phospholipid- and phosphoprotein-substrates in plasma membrane, we hypothesise that the reduced affinity facilitates PTEN to "hop" on the plasma membrane to dephosphorylate these substrates. PMID:26471078

  14. Monocular deprivation delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice.

    PubMed

    Fu, Tao; Su, Qing; Xi, Ping; Han, Song; Li, Junfa

    2015-03-01

    Synapsins as a family of presynaptic terminal phosphoprotein participates in neuronal development, but their role in the synaptic plasticity of visual cortex is unclear. In this study, the impact of monocular deprivation (MD) on dynamic changes of isoform-specific protein expression and site 1 phosphorylation of synapsins in visual cortex of the postnatal mice were observed by using the technique of Western blot analysis. The results showed that the total (T-) protein levels of synapsins including the isoform of Ia/b, IIa/b and IIIa were about 21-26% of adult level in visual cortex of mice at postnatal 7 days (P7), and then the T-synapsin Ia/b and IIb could quickly reach adult level at P35. However, the T-synapsin IIa and IIIa increased more slowly (71-74% at P35), and then kept increasing in the visual cortex of mice at P60. Unlike to the changes of T-synapsins, the level of phosphorylated (P-) synapsin Ia/b (not IIa/b and IIIa) at site 1 increased with development to the highest level at P21, and then decreased rapidly to a low level in visual cortex of mice at P35-60. In addition, we found that the levels of P-synapsin Ia/b increased significantly in left visual cortex of P28 and P35 (not P21 and P42) mice with 1-week MD of right eye; and no significant changes of T-synapsins were observed in both left and right sides of visual cortex in P21-42 mice with MD treatment. These results suggested that the isoform-specific protein expression and site-1 phosphorylation of synapsins might play a different role in the synaptic plasticity of visual cortex, and MD delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice.

  15. Caspase Cleavage Sites in the Human Proteome: CaspDB, a Database of Predicted Substrates

    PubMed Central

    Kumar, Sonu; van Raam, Bram J.; Salvesen, Guy S.; Cieplak, Piotr

    2014-01-01

    Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency. PMID:25330111

  16. Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion

    PubMed Central

    Liu, Yuying; Conaway, LaShardai; Rutherford Bethard, Jennifer; Al-Ayoubi, Adnan M.; Thompson Bradley, Amber; Zheng, Hui; Weed, Scott A.; Eblen, Scott T.

    2013-01-01

    Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1. PMID:23519612

  17. Regulation of the substrate preference of p190RhoGAP by protein kinase C-mediated phosphorylation of a phospholipid binding site.

    PubMed

    Lévay, Magdolna; Settleman, Jeffrey; Ligeti, Erzsébet

    2009-09-15

    The Rho family GTPases are stringently regulated through the action of a large family of GTPase activating proteins (GAPs) that stimulate their relatively weak intrinsic GTP hydrolyzing activity. The p190RhoGAPs, which include the p190A and p190B proteins, are potent and widely expressed GAPs acting on both Rho and Rac GTPases. We have observed that several acidic phospholipids inhibit the RhoGAP activity and promote the RacGAP activity of p190 proteins. In liposome binding assays we have demonstrated that binding of p190A to phospholipids is controlled by electrostatic interactions. Using mapping techniques, we determined that a small polybasic peptide stretch within p190A is a common site for both the phospholipid binding and PKC phosphorylation. Moreover, PKC-mediated phosphorylation of two amino acids (serine-1221 and threonine-1226) within this region of p190A prevents the binding and substrate specificity regulation by phospholipids. Transfection of COS-7 cells with mutant forms of p190A either unable to bind to phospholipids or unable to become phosphorylated induced distinct morphological changes. Together, these findings reveal a novel GAP regulatory mechanism in which phosphorylation indirectly alters GTPase substrate preference by affecting the interaction with acidic phospholipids. Our observations provide a potential mechanism of Rac/Rho antagonism described in several cellular functions.

  18. Identification of the site on calcineurin phosphorylated by Ca sup + /CaM-dependent kinase II: Modification of the CaM-binding domain

    SciTech Connect

    Martensen, T.M.; Kincaid, R.L. ); Martin, B.M. )

    1989-11-28

    The catalytic subunit of the Ca{sup 2+}/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca{sup 2+}/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported. Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released ({sup 32}P)phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding sites. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM.

  19. ABS: a database of Annotated regulatory Binding Sites from orthologous promoters

    PubMed Central

    Blanco, Enrique; Farré, Domènec; Albà, M. Mar; Messeguer, Xavier; Guigó, Roderic

    2006-01-01

    Information about the genomic coordinates and the sequence of experimentally identified transcription factor binding sites is found scattered under a variety of diverse formats. The availability of standard collections of such high-quality data is important to design, evaluate and improve novel computational approaches to identify binding motifs on promoter sequences from related genes. ABS () is a public database of known binding sites identified in promoters of orthologous vertebrate genes that have been manually curated from bibliography. We have annotated 650 experimental binding sites from 68 transcription factors and 100 orthologous target genes in human, mouse, rat or chicken genome sequences. Computational predictions and promoter alignment information are also provided for each entry. A simple and easy-to-use web interface facilitates data retrieval allowing different views of the information. In addition, the release 1.0 of ABS includes a customizable generator of artificial datasets based on the known sites contained in the collection and an evaluation tool to aid during the training and the assessment of motif-finding programs. PMID:16381947

  20. Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation

    PubMed Central

    McKerlie, Megan; Walker, John R.; Mitchell, Taylor R. H.; Wilson, Florence R.; Zhu, Xu-Dong

    2013-01-01

    TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. PMID:23997120

  1. Microtubule Destabilizer KIF2A Undergoes Distinct Site-Specific Phosphorylation Cascades that Differentially Affect Neuronal Morphogenesis.

    PubMed

    Ogawa, Tadayuki; Hirokawa, Nobutaka

    2015-09-22

    Neurons exhibit dynamic structural changes in response to extracellular stimuli. Microtubules (MTs) provide rapid and dramatic cytoskeletal changes within the structural framework. However, the molecular mechanisms and signaling networks underlying MT dynamics remain unknown. Here, we have applied a comprehensive and quantitative phospho-analysis of the MT destabilizer KIF2A to elucidate the regulatory mechanisms of MT dynamics within neurons in response to extracellular signals. Interestingly, we identified two different sets of KIF2A phosphorylation profiles that accelerate (A-type) and brake (B-type) the MT depolymerization activity of KIF2A. Brain-derived neurotrophic factor (BDNF) stimulates PAK1 and CDK5 kinases, which decrease the MT depolymerizing activity of KIF2A through B-type phosphorylation, resulting in enhanced outgrowth of neural processes. In contrast, lysophosphatidic acid (LPA) induces ROCK2 kinase, which suppresses neurite outgrowth from round cells via A-type phosphorylation. We propose that these two mutually exclusive forms of KIF2A phosphorylation differentially regulate neuronal morphogenesis during development. PMID:26344760

  2. Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction.

    PubMed

    Schaffer, Bethany E; Levin, Rebecca S; Hertz, Nicholas T; Maures, Travis J; Schoof, Michael L; Hollstein, Pablo E; Benayoun, Bérénice A; Banko, Max R; Shaw, Reuben J; Shokat, Kevan M; Brunet, Anne

    2015-11-01

    AMP-activated protein kinase (AMPK) is a central energy gauge that regulates metabolism and has been increasingly involved in non-metabolic processes and diseases. However, AMPK's direct substrates in non-metabolic contexts are largely unknown. To better understand the AMPK network, we use a chemical genetics screen coupled to a peptide capture approach in whole cells, resulting in identification of direct AMPK phosphorylation sites. Interestingly, the high-confidence AMPK substrates contain many proteins involved in cell motility, adhesion, and invasion. AMPK phosphorylation of the RHOA guanine nucleotide exchange factor NET1A inhibits extracellular matrix degradation, an early step in cell invasion. The identification of direct AMPK phosphorylation sites also facilitates large-scale prediction of AMPK substrates. We provide an AMPK motif matrix and a pipeline to predict additional AMPK substrates from quantitative phosphoproteomics datasets. As AMPK is emerging as a critical node in aging and pathological processes, our study identifies potential targets for therapeutic strategies. PMID:26456332

  3. Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site.

    PubMed Central

    Neuschäfer-Rube, Frank; Hermosilla, Ricardo; Rehwald, Mathias; Rönnstrand, Lars; Schülein, Ralf; Wernstedt, Christer; Püschel, Gerhard Paul

    2004-01-01

    hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary. PMID:14709160

  4. Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.

    PubMed Central

    Whiting, P; McKernan, R M; Iversen, L L

    1990-01-01

    Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes. Images PMID:1702226

  5. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    PubMed

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  6. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

    PubMed Central

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements. PMID:21087992

  7. Attributes of the Federal Energy Management Program's Federal Site Building Characteristics Database

    SciTech Connect

    Loper, Susan A.; Sandusky, William F.

    2010-12-31

    Typically, the Federal building stock is referred to as a group of about one-half million buildings throughout the United States. Additional information beyond this level is generally limited to distribution of that total by agency and maybe distribution of the total by state. However, additional characterization of the Federal building stock is required as the Federal sector seeks ways to implement efficiency projects to reduce energy and water use intensity as mandated by legislation and Executive Order. Using a Federal facility database that was assembled for use in a geographic information system tool, additional characterization of the Federal building stock is provided including information regarding the geographical distribution of sites, building counts and percentage of total by agency, distribution of sites and building totals by agency, distribution of building count and floor space by Federal building type classification by agency, and rank ordering of sites, buildings, and floor space by state. A case study is provided regarding how the building stock has changed for the Department of Energy from 2000 through 2008.

  8. APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals.

    PubMed

    You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

    2015-01-01

    Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3'-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3'-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3'-untranslated regions (3'-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish.

  9. APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

    PubMed Central

    You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

    2015-01-01

    Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish. PMID:25378337

  10. Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line.

    PubMed

    Cutillas, Pedro R; Geering, Barbara; Waterfield, Mike D; Vanhaesebroeck, Bart

    2005-08-01

    Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a non-isotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase Cgamma2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospholipase Cgamma2 phosphorylation sites. Our results illustrate the capacity of this conceptually

  11. Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits*

    PubMed Central

    Gray, Erin E.; Guglietta, Ryan; Khakh, Baljit S.; O'Dell, Thomas J.

    2014-01-01

    The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca2+ sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca2+ influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function. PMID:24706758

  12. Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis

    PubMed Central

    Archambault, Vincent; D’Avino, Pier Paolo; Deery, Michael J.; Lilley, Kathryn S.; Glover, David M.

    2008-01-01

    The conserved Polo kinase controls multiple events in mitosis and cytokinesis. Although Polo-like kinases are regulated by phosphorylation and proteolysis, control of subcellular localization plays a major role in coordinating their mitotic functions. This is achieved largely by the Polo-Box Domain, which binds prephosphorylated targets. However, it remains unclear whether and how Polo might interact with partner proteins when priming mitotic kinases are inactive. Here we show that Polo associates with microtubules in interphase and cytokinesis, through a strong interaction with the microtubule-associated protein Map205. Surprisingly, this interaction does not require priming phosphorylation of Map205, and the Polo-Box Domain of Polo is required but not sufficient for this interaction. Moreover, phosphorylation of Map205 at a CDK site relieves this interaction. Map205 can stabilize Polo and inhibit its cellular activity in vivo. In syncytial embryos, the centrosome defects observed in polo hypomorphs are enhanced by overexpression of Map205 and suppressed by its deletion. We propose that Map205-dependent targeting of Polo to microtubules provides a stable reservoir of Polo that can be rapidly mobilized by the activity of Cdk1 at mitotic entry. PMID:18832073

  13. Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice.

    PubMed

    Morgan, Daniel J; Davis, Brian J; Kearn, Chris S; Marcus, David; Cook, Alex J; Wager-Miller, Jim; Straiker, Alex; Myoga, Michael H; Karduck, Jeffrey; Leishman, Emma; Sim-Selley, Laura J; Czyzyk, Traci A; Bradshaw, Heather B; Selley, Dana E; Mackie, Ken

    2014-04-01

    For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

  14. PRECISE: a Database of Predicted and Consensus Interaction Sites in Enzymes.

    PubMed

    Sheu, Shu-Hsien; Lancia, David R; Clodfelter, Karl H; Landon, Melissa R; Vajda, Sandor

    2005-01-01

    PRECISE (Predicted and Consensus Interaction Sites in Enzymes) is a database of interactions between the amino acid residues of an enzyme and its ligands (substrate and transition state analogs, cofactors, inhibitors and products). It is available online at http://precise.bu.edu/. In the current version, all information on interactions is extracted from the enzyme-ligand complexes in the Protein Data Bank (PDB) by performing the following steps: (i) clustering homologous enzyme chains such that, in each cluster, the proteins have the same EC number and all sequences are similar; (ii) selecting a representative chain for each cluster; (iii) selecting ligand types; (iv) finding non-bonded interactions and hydrogen bonds; and (v) summing the interactions for all chains within the cluster. The output of the search is the color-coded sequence of the representative. The colors indicate the total number of interactions found at each amino acid position in all chains of the cluster. Clicking on a residue displays a detailed list of interactions for that residue. Optional filters allow restricting the output to selected chains in the cluster, to non-bonded or hydrogen bonding interactions, and to selected ligand types. The binding site information is essential for understanding and altering substrate specificity and for the design of enzyme inhibitors.

  15. A database of global reference sites to support validation of satellite surface albedo datasets (SAVS 1.0)

    NASA Astrophysics Data System (ADS)

    Loew, Alexander; Bennartz, Ralf; Fell, Frank; Lattanzio, Alessio; Doutriaux-Boucher, Marie; Schulz, Jörg

    2016-09-01

    Validating the accuracy and long-term stability of terrestrial satellite data products necessitates a network of reference sites. This paper documents a global database of more than 2000 sites globally which have been characterized in terms of their spatial heterogeneity. The work was motivated by the need for potential validation sites for geostationary surface albedo data products, but the resulting database is useful also for other applications. The database (SAVS 1.0) is publicly available through the EUMETSAT website (http://savs.eumetsat.int/, doi:10.15770/EUM_SEC_CLM_1001). Sites can be filtered according to different criteria, providing a flexible way to identify potential validation sites for further studies and a traceable approach to characterize the heterogeneity of these reference sites. The present paper describes the detailed information on the generation of the SAVS 1.0 database and its characteristics.

  16. Development of an Integrated Natural Barrier Database System for Site Evaluation of a Deep Geologic Repository in Korea - 13527

    SciTech Connect

    Jung, Haeryong; Lee, Eunyong; Jeong, YiYeong; Lee, Jeong-Hwan

    2013-07-01

    Korea Radioactive-waste Management Corporation (KRMC) established in 2009 has started a new project to collect information on long-term stability of deep geological environments on the Korean Peninsula. The information has been built up in the integrated natural barrier database system available on web (www.deepgeodisposal.kr). The database system also includes socially and economically important information, such as land use, mining area, natural conservation area, population density, and industrial complex, because some of this information is used as exclusionary criteria during the site selection process for a deep geological repository for safe and secure containment and isolation of spent nuclear fuel and other long-lived radioactive waste in Korea. Although the official site selection process has not been started yet in Korea, current integrated natural barrier database system and socio-economic database is believed that the database system will be effectively utilized to narrow down the number of sites where future investigation is most promising in the site selection process for a deep geological repository and to enhance public acceptance by providing readily-available relevant scientific information on deep geological environments in Korea. (authors)

  17. AraPPISite: a database of fine-grained protein-protein interaction site annotations for Arabidopsis thaliana.

    PubMed

    Li, Hong; Yang, Shiping; Wang, Chuan; Zhou, Yuan; Zhang, Ziding

    2016-09-01

    Knowledge about protein interaction sites provides detailed information of protein-protein interactions (PPIs). To date, nearly 20,000 of PPIs from Arabidopsis thaliana have been identified. Nevertheless, the interaction site information has been largely missed by previously published PPI databases. Here, AraPPISite, a database that presents fine-grained interaction details for A. thaliana PPIs is established. First, the experimentally determined 3D structures of 27 A. thaliana PPIs are collected from the Protein Data Bank database and the predicted 3D structures of 3023 A. thaliana PPIs are modeled by using two well-established template-based docking methods. For each experimental/predicted complex structure, AraPPISite not only provides an interactive user interface for browsing interaction sites, but also lists detailed evolutionary and physicochemical properties of these sites. Second, AraPPISite assigns domain-domain interactions or domain-motif interactions to 4286 PPIs whose 3D structures cannot be modeled. In this case, users can easily query protein interaction regions at the sequence level. AraPPISite is a free and user-friendly database, which does not require user registration or any configuration on local machines. We anticipate AraPPISite can serve as a helpful database resource for the users with less experience in structural biology or protein bioinformatics to probe the details of PPIs, and thus accelerate the studies of plant genetics and functional genomics. AraPPISite is available at http://systbio.cau.edu.cn/arappisite/index.html .

  18. AraPPISite: a database of fine-grained protein-protein interaction site annotations for Arabidopsis thaliana.

    PubMed

    Li, Hong; Yang, Shiping; Wang, Chuan; Zhou, Yuan; Zhang, Ziding

    2016-09-01

    Knowledge about protein interaction sites provides detailed information of protein-protein interactions (PPIs). To date, nearly 20,000 of PPIs from Arabidopsis thaliana have been identified. Nevertheless, the interaction site information has been largely missed by previously published PPI databases. Here, AraPPISite, a database that presents fine-grained interaction details for A. thaliana PPIs is established. First, the experimentally determined 3D structures of 27 A. thaliana PPIs are collected from the Protein Data Bank database and the predicted 3D structures of 3023 A. thaliana PPIs are modeled by using two well-established template-based docking methods. For each experimental/predicted complex structure, AraPPISite not only provides an interactive user interface for browsing interaction sites, but also lists detailed evolutionary and physicochemical properties of these sites. Second, AraPPISite assigns domain-domain interactions or domain-motif interactions to 4286 PPIs whose 3D structures cannot be modeled. In this case, users can easily query protein interaction regions at the sequence level. AraPPISite is a free and user-friendly database, which does not require user registration or any configuration on local machines. We anticipate AraPPISite can serve as a helpful database resource for the users with less experience in structural biology or protein bioinformatics to probe the details of PPIs, and thus accelerate the studies of plant genetics and functional genomics. AraPPISite is available at http://systbio.cau.edu.cn/arappisite/index.html . PMID:27338257

  19. Expanding tandem mass spectral libraries of phosphorylated peptides: advances and applications.

    PubMed

    Hu, Yingwei; Lam, Henry

    2013-12-01

    The identification of phosphorylated proteins remains a challenge in proteomics, partially due to the difficulty in assigning tandem mass (MS/MS) spectra to their originating peptide sequences with correct phosphosite localization. Because of its advantages in efficiency and sensitivity, spectral library searching is a promising alternative to conventional sequence database searching. Our work aims to construct the largest collision-induced dissociation (CID) MS/MS spectral libraries of phosphorylated peptides in human (Homo sapiens) and four model organisms (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) to date, to facilitate phosphorylated peptide identification by spectral library searching. We employed state-of-the-art search methods to published data and applied two recently published phosphorylation site localization tools (PhosphoRS and PTMProphet) to ascertain the phosphorylation sites. To further increase the coverage of this library, we predicted "semi-empirical" spectra for peptides containing known phosphorylation sites from the corresponding template unphosphorylated peptide spectra. The performance of the spectral libraries built were evaluated and found to be superior to conventional database searching in terms of sensitivity. Updated spectral libraries of phosphorylated peptides are made freely available for use with the spectral search engine SpectraST. The work flow being developed will be used to continuously update the libraries when new data become available.

  20. Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of {alpha}-synuclein

    SciTech Connect

    Sasakawa, Hiroaki |; Sakata, Eri; Yamaguchi, Yoshiki; Masuda, Masami |; Mori, Tetsuya; Kurimoto, Eiji; Iguchi, Takeshi; Hisanaga, Shin-ichi; Iwatsubo, Takeshi; Hasegawa, Masato; Kato, Koichi |

    2007-11-23

    Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of {alpha}-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-{alpha}-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of {alpha}-synuclein.

  1. Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

    PubMed

    Martin, D W; Sachs, J R

    1999-06-01

    The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

  2. P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons

    PubMed Central

    Pougnet, Johan-Till; Compans, Benjamin; Martinez, Audrey; Choquet, Daniel; Hosy, Eric; Boué-Grabot, Eric

    2016-01-01

    Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression. PMID:27624155

  3. P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons.

    PubMed

    Pougnet, Johan-Till; Compans, Benjamin; Martinez, Audrey; Choquet, Daniel; Hosy, Eric; Boué-Grabot, Eric

    2016-01-01

    Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression. PMID:27624155

  4. Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: Fluorescence correlation spectroscopy and femtosecond solvation dynamics

    NASA Astrophysics Data System (ADS)

    Chowdhury, Aritra; Mojumdar, Supratik Sen; Choudhury, Aparajita; Banerjee, Rajat; Das, Kali Pada; Sasmal, Dibyendu Kumar; Bhattacharyya, Kankan

    2012-04-01

    Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (Dt) of the αA-crystallin oligomer (mass ˜800 kDa) increases from ˜35 μm2 s-1 to ˜68 μm2 s-1. This corresponds to a decrease in hydrodynamic radius (rh) from ˜6.9 nm to ˜3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ˜80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ˜20 kDa). The steady state emission maximum and average solvation time (<τs>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.

  5. DISSS/PSDB - Personnel Security Database Modernization Project: Compilation of data gathered from DOE Operations Office`s site visits

    SciTech Connect

    Carpenter, R.; Sweeney, D.

    1995-03-15

    This document is a compilation of the information gathered from visits to the DOE Operations Offices. The purpose of these visits was to gather requirements for the modernization of the personnel security database. The initial phase of visits were to sites which had known local systems to augment CPCI. They were; Rocky Flats, Richland, Las Vegas, Savannah River, Oak Ridge, and Oakland. The second phase of site visits were to; Headquarters, Schenectady, Pittsburgh, Idaho Falls, Chicago, and Albuquerque. We also visited the NRC. At each site we reviewed the current clearance process in use at the field office. If the site had a local personnel security database (PSDB), we also reviewed the current PSDB processing. Each meeting was began with the a discussion on the purpose of the meeting and the background of the redesign effort.

  6. Identifying opportune landing sites in degraded visual environments with terrain and cultural databases

    NASA Astrophysics Data System (ADS)

    Moody, Marc; Fisher, Robert; Little, J. Kristin

    2014-06-01

    Boeing has developed a degraded visual environment navigational aid that is flying on the Boeing AH-6 light attack helicopter. The navigational aid is a two dimensional software digital map underlay generated by the Boeing™ Geospatial Embedded Mapping Software (GEMS) and fully integrated with the operational flight program. The page format on the aircraft's multi function displays (MFD) is termed the Approach page. The existing work utilizes Digital Terrain Elevation Data (DTED) and OpenGL ES 2.0 graphics capabilities to compute the pertinent graphics underlay entirely on the graphics processor unit (GPU) within the AH-6 mission computer. The next release will incorporate cultural databases containing Digital Vertical Obstructions (DVO) to warn the crew of towers, buildings, and power lines when choosing an opportune landing site. Future IRAD will include Light Detection and Ranging (LIDAR) point cloud generating sensors to provide 2D and 3D synthetic vision on the final approach to the landing zone. Collision detection with respect to terrain, cultural, and point cloud datasets may be used to further augment the crew warning system. The techniques for creating the digital map underlay leverage the GPU almost entirely, making this solution viable on most embedded mission computing systems with an OpenGL ES 2.0 capable GPU. This paper focuses on the AH-6 crew interface process for determining a landing zone and flying the aircraft to it.

  7. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  8. Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.

    PubMed

    Bavli-Kertselli, Ira; Melamed, Daniel; Bar-Ziv, Lavi; Volf, Hila; Arava, Yoav

    2015-02-01

    Cells respond to changes in their environment through mechanisms that often necessitate reprogramming of the translation machinery. The fastest and strongest of all tested responses is the translation inhibition observed following abrupt depletion of glucose from the media of yeast cells. The speed of the response suggests a post-translational modification of a key component of the translation machinery. This translation factor is as yet unknown. A cAMP-dependent protein kinase mutant yeast strain (tpk1(w)) that does not respond properly to glucose depletion and maintains translation was described previously. We hypothesized that the inability of tpk1(w) to arrest translation results from abnormal expression of key translation mediators. Genome-wide analysis of steady-state mRNA levels in tpk1(w) revealed underexpression of several candidates. Elevating the cellular levels of eukaryotic initiation factor (eIF) 5 by overexpression rescued the translational defect of tpk1(w). Restoring ribosomal dissociation by eIF5 necessitated an active GAP domain and multiple regions throughout this protein. Phosphoproteomics analysis of wild-type cells overexpressing eIF5 revealed increased phosphorylation in a novel site (Thr191) upon glucose depletion. Mutating this residue and introducing it into tpk1(w) abolished the ability of eIF5 to rescue the translational defect. Intriguingly, introducing this mutation into the wild-type strain did not hamper its translational response. We further show that Thr191 is phosphorylated in vitro by Casein Kinase II (CKII), and yeast cells with a mutated CKII have a reduced response to glucose depletion. These results implicate phosphorylation of eIF5 at Thr191 by CKII as one of the pathways for regulating translation upon glucose depletion.

  9. Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

    PubMed Central

    1995-01-01

    We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel. PMID:8786340

  10. Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

    PubMed Central

    Lu, Yali; Zhou, Xiao; Stemmer, Paul M.; Reid, Gavin E.

    2014-01-01

    An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency. PMID:21952753

  11. MetalS(3), a database-mining tool for the identification of structurally similar metal sites.

    PubMed

    Valasatava, Yana; Rosato, Antonio; Cavallaro, Gabriele; Andreini, Claudia

    2014-08-01

    We have developed a database search tool to identify metal sites having structural similarity to a query metal site structure within the MetalPDB database of minimal functional sites (MFSs) contained in metal-binding biological macromolecules. MFSs describe the local environment around the metal(s) independently of the larger context of the macromolecular structure. Such a local environment has a determinant role in tuning the chemical reactivity of the metal, ultimately contributing to the functional properties of the whole system. The database search tool, which we called MetalS(3) (Metal Sites Similarity Search), can be accessed through a Web interface at http://metalweb.cerm.unifi.it/tools/metals3/ . MetalS(3) uses a suitably adapted version of an algorithm that we previously developed to systematically compare the structure of the query metal site with each MFS in MetalPDB. For each MFS, the best superposition is kept. All these superpositions are then ranked according to the MetalS(3) scoring function and are presented to the user in tabular form. The user can interact with the output Web page to visualize the structural alignment or the sequence alignment derived from it. Options to filter the results are available. Test calculations show that the MetalS(3) output correlates well with expectations from protein homology considerations. Furthermore, we describe some usage scenarios that highlight the usefulness of MetalS(3) to obtain mechanistic and functional hints regardless of homology.

  12. THE ECOTOX DATABASE AND ECOLOGICAL SOIL SCREENING LEVEL (ECO-SSL) WEB SITES

    EPA Science Inventory

    The EPA's ECOTOX database (http://www.epa.gov/ecotox/) provides a web browser search interface for locating aquatic and terrestrial toxic effects information. Data on more than 8100 chemicals and 5700 terrestrial and aquatic species are included in the database. Information is ...

  13. Geologic structure mapping database Spent Fuel Test - Climax, Nevada Test Site

    SciTech Connect

    Yow, J.L. Jr.

    1984-12-04

    Information on over 2500 discontinuities mapped at the SFT-C is contained in the geologic structure mapping database. Over 1800 of these features include complete descriptions of their orientations. This database is now available for use by other researchers. 6 references, 3 figures, 2 tables.

  14. A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans.

    PubMed Central

    Bockmühl, D P; Ernst, J F

    2001-01-01

    Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues. PMID:11290709

  15. Evaluating, Migrating, and Consolidating Databases and Applications for Long-Term Surveillance and Maintenance Activities at the Rocky Flats Site

    SciTech Connect

    Surovchak, S.; Marutzky, S.; Thompson, B.; Miller, K.; Labonte, E.

    2006-07-01

    The U.S. Department of Energy (DOE) Office of Legacy Management (LM) is assuming responsibilities for long-term surveillance and maintenance (LTS and M) activities at the Rocky Flats Environmental Technology Site (RFETS) during fiscal year 2006. During the transition, LM is consolidating databases and applications that support these various functions into a few applications which will streamline future management and retrieval of data. This paper discussed the process of evaluating, migrating, and consolidating these databases and applications for LTS and M activities and provides lessons learned that will benefit future transitions. (authors)

  16. Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites.

    PubMed

    Lee, Tae-Seong; Karl, Rosi; Moosmang, Sven; Lenhardt, Peter; Klugbauer, Norbert; Hofmann, Franz; Kleppisch, Thomas; Welling, Andrea

    2006-09-01

    Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II. PMID:16820363

  17. Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: studies with site-specific mutants of HPr.

    PubMed Central

    Reizer, J; Sutrina, S L; Saier, M H; Stewart, G C; Peterkofsky, A; Reddy, P

    1989-01-01

    The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein-kinase-catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site-specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild-type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase-catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS-mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS-mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation. Images PMID:2507315

  18. Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2alpha under oxidative stress.

    PubMed

    MacCallum, Paul R; Jack, Samantha C; Egan, Philip A; McDermott, Benjamin T; Elliott, Richard M; Chan, Shiu-Wan

    2006-11-01

    Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2alpha phosphorylation. A phosphomimetic of eIF2alpha, which mimics the structure of the phosphorylated eIF2alpha, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2alpha. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2alpha-independent mechanism, whereas the simultaneous phosphorylation of eIF2alpha repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2alpha-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation. PMID:17030858

  19. Database of groundwater levels and hydrograph descriptions for the Nevada Test Site area, Nye County, Nevada

    USGS Publications Warehouse

    Elliott, Peggy E.; Fenelon, Joseph M.

    2010-01-01

    Water levels in the database were quality assured and analyzed. Multiple conditions were assigned to each water‑level measurement to describe the hydrologic conditions at the time of measurement. General quality, temporal variability, regional significance, and hydrologic conditions are attributed to each water-level measurement.

  20. SELEX_DB: a database on in vitro selected oligomers adapted for recognizing natural sites and for analyzing both SNPs and site-directed mutagenesis data.

    PubMed

    Ponomarenko, Julia V; Orlova, Galina V; Frolov, Anatoly S; Gelfand, Mikhail S; Ponomarenko, Mikhail P

    2002-01-01

    SELEX_DB is an online resource containing both the experimental data on in vitro selected DNA/RNA oligomers (aptamers) and the applets for recognition of these oligomers. Since in vitro experimental data are evidently system-dependent, the new release of the SELEX_DB has been supplemented by the database SYSTEM storing the experimental design. In addition, the recognition applet package, SELEX_TOOLS, applying in vitro selected data to annotation of the genome DNA, is accompanied by the cross-validation test database CROSS_TEST discriminating the sites (natural or other) related to in vitro selected sites out of random DNA. By cross-validation testing, we have unexpectedly observed that the recognition accuracy increases with the growth of homology between the training and test sets of protein binding sequences. For natural sites, the recognition accuracy was lower than that for the nearest protein homologs and higher than that for distant homologs and non-homologous proteins binding the common site. The current SELEX_DB release is available at http://wwwmgs.bionet.nsc.ru/mgs/systems/selex/. PMID:11752291

  1. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes.

    PubMed

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5'→3', 3' →5' or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm.

  2. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

    PubMed Central

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

  3. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes.

    PubMed

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5'→3', 3' →5' or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm. PMID:27515825

  4. Polo Kinase Phosphorylates Miro to Control ER-Mitochondria Contact Sites and Mitochondrial Ca(2+) Homeostasis in Neural Stem Cell Development.

    PubMed

    Lee, Seongsoo; Lee, Kyu-Sun; Huh, Sungun; Liu, Song; Lee, Do-Yeon; Hong, Seung Hyun; Yu, Kweon; Lu, Bingwei

    2016-04-18

    Mitochondria play central roles in buffering intracellular Ca²⁺ transients. While basal mitochondrial Ca²⁺ (Ca²⁺ mito) is needed to maintain organellar physiology, Ca²⁺ mito overload can lead to cell death. How Ca²⁺ mito homeostasis is regulated is not well understood. Here we show that Miro, a known component of the mitochondrial transport machinery, regulates Drosophila neural stem cell (NSC) development through Ca²⁺ mito homeostasis control, independent of its role in mitochondrial transport. Miro interacts with Ca²⁺ transporters at the ER-mitochondria contact site (ERMCS). Its inactivation causes Ca²⁺ mito depletion and metabolic impairment, whereas its overexpression results in Ca²⁺ mito overload, mitochondrial morphology change, and apoptotic response. Both conditions impaired NSC lineage progression. Ca²⁺ mito homeostasis is influenced by Polo-mediated phosphorylation of a conserved residue in Miro, which positively regulates Miro localization to, and the integrity of, ERMCS. Our results elucidate a regulatory mechanism underlying Ca²⁺ mito homeostasis and how its dysregulation may affect NSC metabolism/development and contribute to disease. PMID:27093086

  5. Polo Kinase Phosphorylates Miro to Control ER-Mitochondria Contact Sites and Mitochondrial Ca(2+) Homeostasis in Neural Stem Cell Development.

    PubMed

    Lee, Seongsoo; Lee, Kyu-Sun; Huh, Sungun; Liu, Song; Lee, Do-Yeon; Hong, Seung Hyun; Yu, Kweon; Lu, Bingwei

    2016-04-18

    Mitochondria play central roles in buffering intracellular Ca²⁺ transients. While basal mitochondrial Ca²⁺ (Ca²⁺ mito) is needed to maintain organellar physiology, Ca²⁺ mito overload can lead to cell death. How Ca²⁺ mito homeostasis is regulated is not well understood. Here we show that Miro, a known component of the mitochondrial transport machinery, regulates Drosophila neural stem cell (NSC) development through Ca²⁺ mito homeostasis control, independent of its role in mitochondrial transport. Miro interacts with Ca²⁺ transporters at the ER-mitochondria contact site (ERMCS). Its inactivation causes Ca²⁺ mito depletion and metabolic impairment, whereas its overexpression results in Ca²⁺ mito overload, mitochondrial morphology change, and apoptotic response. Both conditions impaired NSC lineage progression. Ca²⁺ mito homeostasis is influenced by Polo-mediated phosphorylation of a conserved residue in Miro, which positively regulates Miro localization to, and the integrity of, ERMCS. Our results elucidate a regulatory mechanism underlying Ca²⁺ mito homeostasis and how its dysregulation may affect NSC metabolism/development and contribute to disease.

  6. The abandoned surface mining sites in the Czech Republic: mapping and creating a database with a GIS web application

    NASA Astrophysics Data System (ADS)

    Pokorný, Richard; Tereza Peterková, Marie

    2016-05-01

    Based on the vectorization of the 55-volume book series the Quarry Inventories of the Czechoslovak Republic/Czechoslovak Socialist Republic, published in the years 1932-1961, a new comprehensive database was built comprising 9958 surface mining sites of raw materials, which were active in the first half of the 20th century. The mapped area covers 40.9 % of the territory of the Czech Republic. For the purposes of visualization, a map application, the Quarry Inventories Online, was created that enables the data visualization.

  7. 65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites

    SciTech Connect

    Zu, Youli; Shigesada, Katsuya; Hanaoka, Masao; Namba, Yuziro ); Nishida, Eisuke ); Kubota, Ichiro ); Kohno, Michiaki )

    1990-09-11

    The authors have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin. In this paper, they present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.

  8. Prototype Database and User's Guide of Saturated Zone Hydraulic Properties forthe Hanford Site

    SciTech Connect

    Thorne, Paul D.; Newcomer, Darrell R.

    2002-09-01

    Predicting the movement of contaminants in groundwater beneath the Hanford Site is important for both understanding the impacts of these contaminants and for planning effective cleanup activities. These predictions are based on knowledge of the distribution of hydraulic properties within the aquifers underlying the Hanford Site. The Characterization of Systems (CoS) Task, under the Groundwater/Vadose Integration Project, is responsible for establishing a consistent set of data, parameters, and conceptual models to support estimates contaminant migration and impact.

  9. Identification and Validation of Differential Phosphorylation Sites of the Nuclear FOXL2 Protein as Potential Novel Biomarkers for Adult-Type Granulosa Cell Tumors.

    PubMed

    Suh, Dae-Shik; Oh, Hoon Kyu; Kim, Jae-Hong; Park, Seeun; Shin, Eunkyoung; Lee, Kangseok; Kim, Yong-Hak; Bae, Jeehyeon

    2015-06-01

    Granulosa cell tumor (GCT) is a rare form of ovarian cancer classified as a sex cord-stromal tumor. The c.402C→G missense mutation in the FOXL2 gene that changes cysteine 134 to tryptophan (C134W) is found in more than 97% of adult-type GCTs, and the C134W FOXL2 mutant is hyperphosphorylated. We identified three differential phosphorylation sites, at serine 33 (S33), tyrosine 186 (Y186), and serine 238 (S238), of the C134W mutant by tandem mass spectrometry. Among these sites, antibodies were raised against the pS33 and pY186 epitopes using specific peptides, and they were tested by immunostaining tissue microarrays of archival adult-type GCT specimens, other tumors, and normal tissues. The pS33 antibody showed greater sensitivity and specificity for the detection of adult-type GCTs than that of the other phospho and nonphospho antibodies. The specificity of the pS33 antibody to the pS33 epitope was further confirmed by enriching the pS33 peptide by affinity chromatography using the immobilized antibody, followed by mass spectrometric and western blot analyses from whole cell lysates of the adult-type GCT cell line, KGN. pS33 FOXL2 immunostaining levels were significantly higher in adult-type GCTs than those in other tumors and tissues. The receiver operating characteristic curve analysis of pS33 FOXL2 showed high sensitivity (1.0) and specificity (0.76) to adult-type GCTs with a cutoff score of >30% positive cells, and the area under the curve was 0.96. This suggests the potential of pS33 FOXL2 to serve as a new biomarker for the diagnosis of adult-type GCT.

  10. RESOPS: A Database for Analyzing the Correspondence of RNA Editing Sites to Protein Three-Dimensional Structures

    PubMed Central

    Yura, Kei; Sulaiman, Sintawee; Hatta, Yosuke; Shionyu, Masafumi; Go, Mitiko

    2009-01-01

    Transcripts from mitochondrial and chloroplast DNA of land plants often undergo cytidine to uridine conversion-type RNA editing events. RESOPS is a newly built database that specializes in displaying RNA editing sites of land plant organelles on protein three-dimensional (3D) structures to help elucidate the mechanisms of RNA editing for gene expression regulation. RESOPS contains the following information: unedited and edited cDNA sequences with notes for the target nucleotides of RNA editing, conceptual translation from the edited cDNA sequence in pseudo-UniProt format, a list of proteins under the influence of RNA editing, multiple amino acid sequence alignments of edited proteins, the location of amino acid residues coded by codons under the influence of RNA editing in protein 3D structures and the statistics of biased distributions of the edited residues with respect to protein structures. Most of the data processing procedures are automated; hence, it is easy to keep abreast of updated genome and protein 3D structural data. In the RESOPS database, we clarified that the locations of residues switched by RNA editing are significantly biased to protein structural cores. The integration of different types of data in the database also help advance the understanding of RNA editing mechanisms. RESOPS is accessible at http://cib.cf.ocha.ac.jp/RNAEDITING/. PMID:19808808

  11. Infection with CagA-Positive Helicobacter Pylori Strain Containing Three EPIYA C Phosphorylation Sites is Associated with More Severe Gastric Lesions in Experimentally Infected Mongolian Gerbils (Meriones Unguiculatus)

    PubMed Central

    Junior, M. Ferreira; Batista, S.A.; Vidigal, P.V.T; Cordeiro, A.A.C.; Oliveira, F.M.S.; Prata, L.O.; Diniz, A.E.T.; Barral, C.M.; Barbuto, R.C.; Comes, A.D.; Araujo, I.D.; Queiroz, D.M.M.; Caliari, M.V.

    2015-01-01

    Infection with Helicobacter pylori strains containing high number of EPIYA-C phosphorylation sites in the CagA is associated with significant gastritis and increased risk of developing pre-malignant gastric lesions and gastric carcinoma. However, these findings have not been reproduced in animal models yet. Therefore, we investigated the effect on the gastric mucosa of Mongolian gerbil (Meriones unguiculatus) infected with CagA-positive H. pylori strains exhibiting one or three EPIYA-C phosphorilation sites. Mongolian gerbils were inoculated with H. pylori clonal isolates containing one or three EPIYA-C phosphorylation sites. Control group was composed by uninfected animals challenged with Brucella broth alone. Gastric fragments were evaluated by the modified Sydney System and digital morphometry. Clonal relatedness between the isolates was considered by the identical RAPD-PCR profiles and sequencing of five housekeeping genes, vacA i/d region and of oipA. The other virulence markers were present in both isolates (vacA s1i1d1m1, iceA2, and intact dupA). CagA of both isolates was translocated and phosphorylated in AGS cells. After 45 days of infection, there was a significant increase in the number of inflammatory cells and in the area of the lamina propria in the infected animals, notably in those infected by the CagA-positive strain with three EPIYA-C phosphorylation sites. After six months of infection, a high number of EPIYA-C phosphorylation sites was associated with progressive increase in the intensity of gastritis and in the area of the lamina propria. Atrophy, intestinal metaplasia, and dysplasia were also observed more frequently in animals infected with the CagA-positive isolate with three EPIYA-C sites. We conclude that infection with H. pylori strain carrying a high number of CagA EPIYA-C phosphorylation sites is associated with more severe gastric lesions in an animal model of H. pylori infection. PMID:26150158

  12. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    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, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

  13. An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We conducted a family-wide study to identify and characterize sites of autophosphorylation in 73 representative LRR RLKs of the 223 member LRR RLK family in Arabidopsis thaliana. His-tagged constructs of intact cytoplasmic domains (CDs) for 73 of 223 A. thaliana LRR RLKs were cloned into E. coli BL-...

  14. The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

    PubMed Central

    Goedert, M; Jakes, R; Crowther, R A; Six, J; Lübke, U; Vandermeeren, M; Cras, P; Trojanowski, J Q; Lee, V M

    1993-01-01

    Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8506352

  15. A Novel Phosphorylation Site, Serine 199, in the C-Terminus of Cardiac Troponin I Regulates Calcium Sensitivity and Susceptibility to Calpain-Induced Proteolysis

    PubMed Central

    Wijnker, Paul J.M.; Li, Yuejin; Zhang, Pingbo; Foster, D. Brian; dos Remedios, Cris; Van Eyk, Jennifer E.; Stienen, Ger J.M.; Murphy, Anne M.; van der Velden, Jolanda

    2015-01-01

    Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50=5.79±0.01) compared to 199A (pCa50=5.65±0.01) and Wt (pCa50=5.66±0.02) at ~63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9±2.5% 199D exchange compared to 199A, and saturated at 12.3±2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. PMID:25771144

  16. Digital geologic map database of the Nevada Test Site area, Nevada

    USGS Publications Warehouse

    Wahl, R.R.; Sawyer, D.A.; Minor, S.A.; Carr, M.D.; Cole, J.C.; Swadley, W.C.; Laczniak, R.J.; Warren, R.G.; Green, K.S.; Engle, C.M.

    1997-01-01

    Forty years of geologic investigations at the Nevada Test Site (NTS) have been digitized. These data include all geologic information that: (1) has been collected, and (2) can be represented on a map within the map borders at the map scale is included in the map digital coverages. The following coverages are included with this dataset: Coverage Type Description geolpoly Polygon Geologic outcrops geolflts line Fault traces geolatts Point Bedding attitudes, etc. geolcald line Caldera boundaries geollins line Interpreted lineaments geolmeta line Metamorphic gradients The above coverages are attributed with numeric values and interpreted information. The entity files documented below show the data associated with each coverage.

  17. Digital geologic map database of the Nevada Test Site area, Nevada

    SciTech Connect

    Wahl, Ronald R.; Sawyer, David A.; Minor, Scott A.; Carr, Michael D.; Cole, James C.; Swadley, W.C.; Laczniak, Randell J.; Warren, Richard G.; Green, Katryn S.; Engle, Colin M.

    1997-09-09

    Forty years of geologic investigations at the Nevada Test Site (NTS) have been digitized. These data include all geologic information that: (1) has been collected, and (2) can be represented on a map within the map borders at the map scale is included in the map digital coverages. The following coverages are included with this dataset: Coverage Type Description geolpoly Polygon Geologic outcrops geolflts line Fault traces geolatts Point Bedding attitudes, etc. geolcald line Caldera boundaries geollins line Interpreted lineaments geolmeta line Metamorphic gradients. The above coverages are attributed with numeric values and interpreted information. The entity files documented below show the data associated with each coverage.

  18. Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.

    PubMed Central

    Tavaré, J M; Denton, R M

    1988-01-01

    1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3166375

  19. FLUXNET. Database of fluxes, site characteristics, and flux-community information

    SciTech Connect

    Olson, R. J.; Holladay, S. K.; Cook, R. B.; Falge, E.; Baldocchi, D.; Gu, L.

    2004-02-28

    FLUXNET is a “network of regional networks” created by international scientists to coordinate regional and global analysis of observations from micrometeorological tower sites. The flux tower sites use eddy covariance methods to measure the exchanges of carbon dioxide (CO2), water vapor, and energy between terrestrial ecosystems and the atmosphere. FLUXNET’S goals are to aid in understanding the mechanisms controlling the exchanges of CO2, water vapor, and energy across a range of time (0.5 hours to annual periods) and space scales. FLUXNET provides an infrastructure for the synthesis and analysis of world-wide, long-term flux data compiled from various regional flux networks. Information compiled by the FLUXNET project is being used to validate remote sensing products associated with the National Aeronautics and Space Administration (NASA) Terra and Aqua satellites. FLUXNET provides access to ground information for validating estimates of net primary productivity, and energy absorption that are being generated by the Moderate Resolution Imaging Spectroradiometer (MODIS) sensors. In addition, this information is also used to develop and validate ecosystem models.

  20. Peptidase specificity from the substrate cleavage collection in the MEROPS database and a tool to measure cleavage site conservation

    PubMed Central

    Rawlings, Neil D.

    2016-01-01

    One peptidase can usually be distinguished from another biochemically by its action on proteins, peptides and synthetic substrates. Since 1996, the MEROPS database (http://merops.sanger.ac.uk) has accumulated a collection of cleavages in substrates that now amounts to 66,615 cleavages. The total number of peptidases for which at least one cleavage is known is 1700 out of a total of 2457 different peptidases. This paper describes how the cleavages are obtained from the scientific literature, how they are annotated and how cleavages in peptides and proteins are cross-referenced to entries in the UniProt protein sequence database. The specificity profiles of 556 peptidases are shown for which ten or more substrate cleavages are known. However, it has been proposed that at least 40 cleavages in disparate proteins are required for specificity analysis to be meaningful, and only 163 peptidases (6.6%) fulfil this criterion. Also described are the various displays shown on the website to aid with the understanding of peptidase specificity, which are derived from the substrate cleavage collection. These displays include a logo, distribution matrix, and tables to summarize which amino acids or groups of amino acids are acceptable (or not acceptable) in each substrate binding pocket. For each protein substrate, there is a display to show how it is processed and degraded. Also described are tools on the website to help with the assessment of the physiological relevance of cleavages in a substrate. These tools rely on the hypothesis that a cleavage site that is conserved in orthologues is likely to be physiologically relevant, and alignments of substrate protein sequences are made utilizing the UniRef50 database, in which in each entry sequences are 50% or more identical. Conservation in this case means substitutions are permitted only if the amino acid is known to occupy the same substrate binding pocket from at least one other substrate cleaved by the same peptidase. PMID

  1. Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells.

    PubMed

    Cobb, Melanie M; Austin, Daniel C; Sack, Jon T; Trimmer, James S

    2015-12-01

    The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.

  2. TESS: a geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites.

    PubMed

    Wallace, A C; Borkakoti, N; Thornton, J M

    1997-11-01

    It is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X-ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O-His-O "catalytic triad" and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions.

  3. UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

    PubMed

    Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

    2016-01-01

    The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/.

  4. SISMA (Site of Italian Strong Motion Accelerograms): a Web-Database of Ground Motion Recordings for Engineering Applications

    SciTech Connect

    Scasserra, Giuseppe; Lanzo, Giuseppe; D'Elia, Beniamino; Stewart, Jonathan P.

    2008-07-08

    The paper describes a new website called SISMA, i.e. Site of Italian Strong Motion Accelerograms, which is an Internet portal intended to provide natural records for use in engineering applications for dynamic analyses of structural and geotechnical systems. SISMA contains 247 three-component corrected motions recorded at 101 stations from 89 earthquakes that occurred in Italy in the period 1972-2002. The database of strong motion accelerograms was developed in the framework of a joint project between Sapienza University of Rome and University of California at Los Angeles (USA) and is described elsewhere. Acceleration histories and pseudo-acceleration response spectra (5% damping) are available for download from the website. Recordings can be located using simple search parameters related to seismic source and the recording station (e.g., magnitude, V{sub s30}, etc) as well as ground motion characteristics (e.g. peak ground acceleration, peak ground velocity, peak ground displacement, Arias intensity, etc.)

  5. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  6. Mitogen-Activated Protein Kinase Phosphorylation of Splicing Factor 45 (SPF45) Regulates SPF45 Alternative Splicing Site Utilization, Proliferation, and Cell Adhesion

    PubMed Central

    Al-Ayoubi, Adnan M.; Zheng, Hui; Liu, Yuying; Bai, Tao

    2012-01-01

    The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38α also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H2O2, UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation. PMID:22615491

  7. Development of a priority list of chemical mixtures occurring at 1188 hazardous waste sites, using the HazDat database.

    PubMed

    Fay, R M; Mumtaz, M M

    1996-01-01

    Under the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund) section 104 mandate, as amended by the Superfund Amendments and Reauthorization Act (SARA) of 1986 USC 9604 (i)(2), the Agency for Toxic Substances and Disease Registry (ATSDR) is to identify individual substances and combinations of substances that pose the greatest public health hazard at hazardous waste sites. This has led to certain mandated activities of the Agency, including development of toxicological profiles, identification of data gaps, and, ultimately, establishment of a research agenda. The Agency has also developed HazDat, a database that captures pertinent information from public health assessments conducted at hazardous waste sites. As a preliminary step, data from sites have been analysed to identify the combinations of chemicals found in various environmental media. The most frequently found combinations were perchloroethylene (PERC) and trichloroethylene (TCE) in water (23.5% of sites); chromium (Cr) and lead (Pb) in soil (20.5%); benzene and toluene in air (3.5%); PERC, 1,1,1-trichloroethane (1,1,1-TCA) and TCE in water (11.6%); Cr, cadmium (Cd) and Pb in soil (12.0%); and benzene, PERC and TCE in air (2.2%). The findings of this analysis can be enhanced by factoring into the algorithm paramenters such as toxicity, source contribution, and likelihood of human exposure similar to that used for the Agency's priority list of 275 single substances. Assessment of the impact of chemical mixtures on human health is a formidable task, and estimating the toxicity of such mixtures, including the role of chemical interactions, is an equally demanding challenge. Because limited experimental data exist for chemical interactions, alternative methods such as predictive approaches and in vitro techniques are needed to address the many substances and their potential combinations. PMID:9119332

  8. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome

    PubMed Central

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A.

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. PMID:25324314

  9. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome.

    PubMed

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser.

  10. Can collision-induced negative-ion fragmentations of [M-H](-) anions be used to identify phosphorylation sites in peptides?

    PubMed

    Tran, T T Nha; Wang, Tianfang; Hack, Sandra; Hoffmann, Peter; Bowie, John H

    2011-12-15

    A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.

  11. Toward Non-Ergodic and Site-Specific Probabilistic Seismic Hazard Assessment: Requirements for the Next Generation of Strong Motion Network and Databases.

    NASA Astrophysics Data System (ADS)

    Cotton, F.; Ktenidou, O. J.; Derras, B.; Roumelioti, Z.; Pierre-Yves, B.; Hollender, F.

    2014-12-01

    Ground-motion models used in engineering seismology are usually calibrated on global databases that are usually created by mixing data from different regions. These models also assume that the ground-motion variability observed in a global dataset is the same as the variability in ground motion at a single site-source combination. This assumption is referred to as the ergodic assumption. New data give a unique opportunity to remove the ergodic assumption and take into account regional source, path and site specificities. Using recent data analysis performed on the EUROSEISTEST valley (Greece) and global ground-motion datasets (Kiknet, Knet, NGA2 and the European strong-motion databases) we will show the impact of source parameters, site monitoring and site-characterisation on the uncertainty of the ground motion estimates and associated hazard curves. Our results suggest that future strong-motion networks should use higher sampling rates (to better evaluate site-specific high frequency attenuations) and record both strong and weak motions (to evaluate single-station sigma). These results also quantify the impact of a better characterisation of source parameters (depth, fault maturity, source to site distances) ans site parameters on ground-motion models. We finally will show how new networks and high-level strong-motion databases may help to built consistent ergodic PSHA at a regional scale and non-ergodic, site specific, PSHA.

  12. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

    PubMed Central

    Pelton, J. G.; Torchia, D. A.; Meadow, N. D.; Roseman, S.

    1993-01-01

    IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed. PMID:8518729

  13. A User’s Guide to the Comprehensive Water Quality Database for Groundwater in the Vicinity of the Nevada Test Site, Rev. No.: 1

    SciTech Connect

    Farnham, Irene

    2006-09-01

    This water quality database (viz.GeochemXX.mdb) has been developed as part of the Underground Test Area (UGTA) Program with the cooperation of several agencies actively participating in ongoing evaluation and characterization activities under contract to the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office (NNSA/NSO). The database has been constructed to provide up-to-date, comprehensive, and quality controlled data in a uniform format for the support of current and future projects. This database provides a valuable tool for geochemical and hydrogeologic evaluations of the Nevada Test Site (NTS) and surrounding region. Chemistry data have been compiled for groundwater within the NTS and the surrounding region. These data include major ions, organic compounds, trace elements, radionuclides, various field parameters, and environmental isotopes. Colloid data are also included in the database. The GeochemXX.mdb database is distributed on an annual basis. The extension ''XX'' within the database title is replaced by the last two digits of the release year (e.g., Geochem06 for the version released during the 2006 fiscal year). The database is distributed via compact disc (CD) and is also uploaded to the Common Data Repository (CDR) in order to make it available to all agencies with DOE intranet access. This report provides an explanation of the database configuration and summarizes the general content and utility of the individual data tables. In addition to describing the data, subsequent sections of this report provide the data user with an explanation of the quality assurance/quality control (QA/QC) protocols for this database.

  14. FT-IR analysis of phosphorylated protein

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Yoshihashi, Sachiko S.; Chihara, Kunihiro; Awazu, Kunio

    2004-09-01

    Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).

  15. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  16. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  17. Third year nursing students' understanding of how to find and evaluate information from bibliographic databases and Internet sites.

    PubMed

    Jacobsen, Hilary E; Andenæs, Randi

    2011-11-01

    The aim of this study was to increase undergraduate nursing students' knowledge of finding and evaluating information from selected bibliographic databases and Internet sites. A quasi-experimental design was adopted. The 2004 autumn cohort (n=480) was divided into two approximately equal groups at the beginning of their studies. One group was subjected to a greater number of assignments requiring them to find and evaluate bibliographic and Internet-based information. The assignments were spread throughout the curriculum. Questionnaires were used to collect data. The low response rate makes generalizing the findings difficult. Only small differences were demonstrated between the knowledge of the revised assignment group and that of the other students. Both groups had a poor understanding of the use of important search and evaluation techniques. The results indicate that strategies proven in one context are not necessarily as effective in a new context and that more research is needed into which learning activities best enhance the development of information literacy skills during undergraduate nursing education.

  18. Database of Ground-Water Levels in the Vicinity of Rainier Mesa, Nevada Test Site, Nye County, Nevada 1957-2005.

    SciTech Connect

    Joseph M. Fenelon

    2006-08-15

    More than 1,200 water-level measurements from 1957 to 2005 in the Rainier Mesa area of the Nevada Test Site were quality assured and analyzed. Water levels were measured from 50 discrete intervals within 18 boreholes and from 4 tunnel sites. An interpretive database was constructed that describes water-level conditions for each water level measured in the Rainier Mesa area. Multiple attributes were assigned to each water-level measurement in the database to describe the hydrologic conditions at the time of measurement. General quality, temporal variability, regional significance, and hydrologic conditions are attributed for each water-level measurement. The database also includes hydrograph narratives that describe the water-level history of each well.

  19. Database of Ground-Water Levels in the Vicinity of Rainier Mesa, Nevada Test Site, Nye County, Nevada, 1957-2005

    USGS Publications Warehouse

    Fenelon, Joseph M.

    2006-01-01

    More than 1,200 water-level measurements from 1957 to 2005 in the Rainier Mesa area of the Nevada Test Site were quality assured and analyzed. Water levels were measured from 50 discrete intervals within 18 boreholes and from 4 tunnel sites. An interpretive database was constructed that describes water-level conditions for each water level measured in the Rainier Mesa area. Multiple attributes were assigned to each water-level measurement in the database to describe the hydrologic conditions at the time of measurement. General quality, temporal variability, regional significance, and hydrologic conditions are attributed for each water-level measurement. The database also includes hydrograph narratives that describe the water-level history of each well.

  20. Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms.

    PubMed

    Szöőr, Árpád; Ujlaky-Nagy, László; Tóth, Gábor; Szöllősi, János; Vereb, György

    2016-02-01

    Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently co-labeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of Tyr716 specific for the Ras/MAPK pathway and Tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, Tyr771 and Tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for Ras-GAP, which inactivates the MAPK pathway, and Tyr1021 feeds into the phospholipase C-gamma/PKC pathway. Coherent with this, MAPK phosphorylation, Ki-67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is

  1. Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms.

    PubMed

    Szöőr, Árpád; Ujlaky-Nagy, László; Tóth, Gábor; Szöllősi, János; Vereb, György

    2016-02-01

    Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently co-labeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of Tyr716 specific for the Ras/MAPK pathway and Tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, Tyr771 and Tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for Ras-GAP, which inactivates the MAPK pathway, and Tyr1021 feeds into the phospholipase C-gamma/PKC pathway. Coherent with this, MAPK phosphorylation, Ki-67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is

  2. 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.

    PubMed

    Boldyreff, B; Meggio, F; Pinna, L A; Issinger, O G

    1994-02-18

    Various beta-mutants were investigated either as subunits or as substrates for casein kinase 2 (CK-2), in the absence of presence of polylysine. A total of 21 beta-mutants were characterized for their susceptibility to autophosphorylation, by combining them in equimolar amounts with the recombinant alpha-subunit. Six mutants, i.e. beta A5,6, beta A59-61,63,64, beta A55,57, beta 55-57, beta delta 171-215, and beta delta 150-215 exhibited a > 70% reduction in autophosphorylation. This strongly suggests that in addition to amino acid residues 5,6, distant amino acid residues within the sequence 55-64 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 the autophosphorylation of beta wt (86% inhibition) inducing a parallel increase of the alpha-subunit autophosphorylation. The autophosphorylation of all mutants, with the exception of beta A55-57 and beta A59-61,63,64, is also inhibited by polylysine (>64%). The alpha-subunit autophosphorylation is increased with all 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 of mutants beta delta 171-215 and beta delta 150-215, whose phosphorylation is conversely stimulated by polylysine. The decreased phosphorylation efficiency of those mutants that are

  3. Gene expression levels of Casein kinase 1 (CK1) isoforms are correlated to adiponectin levels in adipose tissue of morbid obese patients and site-specific phosphorylation mediated by CK1 influences multimerization of adiponectin.

    PubMed

    Xu, Pengfei; Fischer-Posovszky, Pamela; Bischof, Joachim; Radermacher, Peter; Wabitsch, Martin; Henne-Bruns, Doris; Wolf, Anna-Maria; Hillenbrand, Andreas; Knippschild, Uwe

    2015-05-01

    White adipose tissue has now been recognized as an important endocrine organ secreting bioactive molecules termed adipocytokines. In obesity, anti-inflammatory adipocytokines like adiponectin are decreased while pro-inflammatory factors are over-produced. These changes contribute to the development of insulin resistance and obesity-associated diseases. Since members of the casein kinase 1 (CK1) family are involved in the regulation of various signaling pathways we ask here whether they are able to modulate the functions of adiponectin. We show that CK1δ and ε are expressed in adipose tissue and that the expression of CK1 isoforms correlates with that of adiponectin. Furthermore, adiponectin co-immunoprecipitates with CK1δ and CK1ε and is phosphorylated by CK1δ at serine 174 and threonine 235, thereby influencing the formation of adiponectin oligomeric complexes. Furthermore, inhibition of CK1δ in human adipocytes by IC261 leads to an increase in basal and insulin-stimulated glucose uptake. In summary, our data indicate that site-specific phosphorylation of adiponectin, especially at sites targeted by CK1δ in vitro, provides an additional regulatory mechanism for modulating adiponectin complex formation and function. PMID:25724478

  4. Drinking Water Treatability Database (Database)

    EPA Science Inventory

    The drinking Water Treatability Database (TDB) will provide data taken from the literature on the control of contaminants in drinking water, and will be housed on an interactive, publicly-available USEPA web site. It can be used for identifying effective treatment processes, rec...

  5. The Factor VIII Mutation Database on the World Wide Web: the haemophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0).

    PubMed Central

    Kemball-Cook, G; Tuddenham, E G

    1997-01-01

    The HAMSTeRS WWW site was set up in 1996 in order to facilitate easy access to, and aid understanding of, the causes of haemophilia A at the molecular level; previously, the first and second text editions of the database have been published in Nucleic Acids Research. This report describes the facilities originally available at the site and the recent additions which we have made to increase its usefulness to clinicians, the molecular genetics community and structural biologists interested in factor VIII. The database (version 3.0) has been completely updated with easy submission of point mutations, deletions and insertions via e-mail of custom-designed forms. The searching of point mutations in the database has been made simpler and more robust, with a concomitantly expanded real-time bioinformatic analysis of the database. A methods section devoted to mutation detection has been added, highlighting issues such as choice of technique and PCR primer sequences. Finally, a FVIII structure section gives access to 3D VRML (Virtual Reality Modelling Language) files for any user-definable residue in a FVIII A domain homology model based on the crystal structure of human caeruloplasmin, together with secondary structural data and a sound+video animation of the model. It is intended that the general availability of this model will assist both in interpretation of causative mutations and selection of candidate residues forin vitromutagenesis. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk. PMID:9016520

  6. The Factor VIII Mutation Database on the World Wide Web: the haemophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0).

    PubMed

    Kemball-Cook, G; Tuddenham, E G

    1997-01-01

    The HAMSTeRS WWW site was set up in 1996 in order to facilitate easy access to, and aid understanding of, the causes of haemophilia A at the molecular level; previously, the first and second text editions of the database have been published in Nucleic Acids Research. This report describes the facilities originally available at the site and the recent additions which we have made to increase its usefulness to clinicians, the molecular genetics community and structural biologists interested in factor VIII. The database (version 3.0) has been completely updated with easy submission of point mutations, deletions and insertions via e-mail of custom-designed forms. The searching of point mutations in the database has been made simpler and more robust, with a concomitantly expanded real-time bioinformatic analysis of the database. A methods section devoted to mutation detection has been added, highlighting issues such as choice of technique and PCR primer sequences. Finally, a FVIII structure section gives access to 3D VRML (Virtual Reality Modelling Language) files for any user-definable residue in a FVIII A domain homology model based on the crystal structure of human caeruloplasmin, together with secondary structural data and a sound+video animation of the model. It is intended that the general availability of this model will assist both in interpretation of causative mutations and selection of candidate residues forin vitromutagenesis. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk.

  7. Relationships between histone phosphorylation and cell proliferation

    SciTech Connect

    Gurley, L.R.; D'Anna, J.A.; Halleck, M.S.; Barham, S.S.; Walters, R.A.; Jett, J.H.; Tobey, R.A.

    1980-01-01

    From studies with various Peromyscus cell lines, correlations were made which led to the proposal that H2A phosphorylation is most active in constitutive heterochromatin. Recent studies on the two H2A variants found in these cells have revealed that the high level of H2A phosphorylation associated with heterochromatin is not the result of an increase in H2A phosphorylation rate or an increase in the number of phosphorylation sites, but rather, is due to an increase in the proportion of one of the H2A variants which is more highly phosphorylated than the other. If H2A phosphorylation is necessary for the constitutive heterochromatin state, it is reasonable that the cell would accomplish the generation of this structure by permanently installing a more highly phosphorylated H2A in the heterochromatin nucleosome rather than by trying to modulate the phosphorylation rate in such a condensed structure. The proposal that histone phosphorylation is involved with the condensed structures of chromatin is based primarily on correlations between histone phosphorylation measurements and cellular phenomena. One proof that this concept is correct ultimately rests in the ability to demonstrate these correlations in isolated chromosomes and chromatin fractions. This demonstration is presently limited by the excessive dephosphorylation of histones which occurs during the isolation of chromosomes and chromatin fractions. Thus, the demonstration of an effective inhibitor of histone dephosphorylation which is compatible with the isolation of nuclear structures and chromatin fractions having native morphologies is essential for future studies on the biological function of histone phosphorylation. (ERB)

  8. Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin [alpha]1

    SciTech Connect

    Tong, Yufeng; Tempel, Wolfram; Wang, Hui; Yamada, Kaori; Shen, Limin; Senisterra, Guillermo A.; MacKenzie, Farrell; Chishti, Athar H.; Park, Hee-Won

    2011-11-07

    Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin {alpha}1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P2). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

  9. The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

    PubMed

    Ferranti, P; Scaloni, A; Caira, S; Chianese, L; Malorni, A; Addeo, F

    1998-11-01

    The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.

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

    PubMed

    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

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

    PubMed

    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

  12. Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes.

    PubMed

    Kim, Kwangsoo; Kim, Hye-Jung; Oh, Deok-Kun; Cha, Sun-Shin; Rhee, Sangkee

    2006-09-01

    D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.

  13. Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding*

    PubMed Central

    Moritz, Amy E.; Foster, James D.; Gorentla, Balachandra K.; Mazei-Robison, Michelle S.; Yang, Jae-Won; Sitte, Harald H.; Blakely, Randy D.; Vaughan, Roxanne A.

    2013-01-01

    As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states. PMID:23161550

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

    PubMed

    Yates, Darran M; Manser, Catherine; De Vos, Kurt J; Shaw, Christopher E; McLoughlin, Declan M; Miller, Christopher C J

    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.

  15. THE NASA AMES PAH IR SPECTROSCOPIC DATABASE VERSION 2.00: UPDATED CONTENT, WEB SITE, AND ON(OFF)LINE TOOLS

    SciTech Connect

    Boersma, C.; Mattioda, A. L.; Allamandola, L. J.; Bauschlicher, C. W. Jr.; Ricca, A.; Cami, J.; Peeters, E.; De Armas, F. Sánchez; Saborido, G. Puerta; Hudgins, D. M.

    2014-03-01

    A significantly updated version of the NASA Ames PAH IR Spectroscopic Database, the first major revision since its release in 2010, is presented. The current version, version 2.00, contains 700 computational and 75 experimental spectra compared, respectively, with 583 and 60 in the initial release. The spectra span the 2.5-4000 μm (4000-2.5 cm{sup -1}) range. New tools are available on the site that allow one to analyze spectra in the database and compare them with imported astronomical spectra as well as a suite of IDL object classes (a collection of programs utilizing IDL's object-oriented programming capabilities) that permit offline analysis called the AmesPAHdbIDLSuite. Most noteworthy among the additions are the extension of the computational spectroscopic database to include a number of significantly larger polycyclic aromatic hydrocarbons (PAHs), the ability to visualize the molecular atomic motions corresponding to each vibrational mode, and a new tool that allows one to perform a non-negative least-squares fit of an imported astronomical spectrum with PAH spectra in the computational database. Finally, a methodology is described in the Appendix, and implemented using the AmesPAHdbIDLSuite, that allows the user to enforce charge balance during the fitting procedure.

  16. Dr.VIS v2.0: an updated database of human disease-related viral integration sites in the era of high-throughput deep sequencing.

    PubMed

    Yang, Xiaobo; Li, Ming; Liu, Qi; Zhang, Yabing; Qian, Junyan; Wan, Xueshuai; Wang, Anqiang; Zhang, Haohai; Zhu, Chengpei; Lu, Xin; Mao, Yilei; Sang, Xinting; Zhao, Haitao; Zhao, Yi; Zhang, Xiaoyan

    2015-01-01

    Dr.VIS is a database of human disease-related viral integration sites (VIS). The number of VIS has grown rapidly since Dr.VIS was first released in 2011, and there is growing recognition of the important role that viral integration plays in the development of malignancies. The updated database version, Dr.VIS v2.0 (http://www.bioinfo.org/drvis or bminfor.tongji.edu.cn/drvis_v2), represents 25 diseases, covers 3340 integration sites of eight oncogenic viruses in human chromosomes and provides more accurate information about VIS from high-throughput deep sequencing results obtained mainly after 2012. Data of VISes for three newly identified oncogenic viruses for 14 related diseases have been added to this 2015 update, which has a 5-fold increase of VISes compared to Dr.VIS v1.0. Dr.VIS v2.0 has 2244 precise integration sites, 867 integration regions and 551 junction sequences. A total of 2295 integration sites are located near 1730 involved genes. Of the VISes, 1153 are detected in the exons or introns of genes, with 294 located up to 5 kb and a further 112 located up to 10 kb away. As viral integration may alter chromosome stability and gene expression levels, characterizing VISes will contribute toward the discovery of novel oncogenes, tumor suppressor genes and tumor-associated pathways. PMID:25355513

  17. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    PubMed

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins.

  18. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    PubMed

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins. PMID:27522420

  19. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    PubMed

    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.

  20. LymPHOS 2.0: an update of a phosphosite database of primary human T cells.

    PubMed

    Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

    2015-01-01

    LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org.

  1. LymPHOS 2.0: an update of a phosphosite database of primary human T cells

    PubMed Central

    Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

    2015-01-01

    LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org. PMID:26708986

  2. The Chemical Biology of Protein Phosphorylation

    PubMed Central

    Tarrant, Mary Katherine; Cole, Philip A.

    2011-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

  3. The chemical biology of protein phosphorylation.

    PubMed

    Tarrant, Mary Katherine; Cole, Philip A

    2009-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain.

  4. dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice-Site SNVs.

    PubMed

    Liu, Xiaoming; Wu, Chunlei; Li, Chang; Boerwinkle, Eric

    2016-03-01

    The purpose of the dbNSFP is to provide a one-stop resource for functional predictions and annotations for human nonsynonymous single-nucleotide variants (nsSNVs) and splice-site variants (ssSNVs), and to facilitate the steps of filtering and prioritizing SNVs from a large list of SNVs discovered in an exome-sequencing study. A list of all potential nsSNVs and ssSNVs based on the human reference sequence were created and functional predictions and annotations were curated and compiled for each SNV. Here, we report a recent major update of the database to version 3.0. The SNV list has been rebuilt based on GENCODE 22 and currently the database includes 82,832,027 nsSNVs and ssSNVs. An attached database dbscSNV, which compiled all potential human SNVs within splicing consensus regions and their deleteriousness predictions, add another 15,030,459 potentially functional SNVs. Eleven prediction scores (MetaSVM, MetaLR, CADD, VEST3, PROVEAN, 4× fitCons, fathmm-MKL, and DANN) and allele frequencies from the UK10K cohorts and the Exome Aggregation Consortium (ExAC), among others, have been added. The original seven prediction scores in v2.0 (SIFT, 2× Polyphen2, LRT, MutationTaster, MutationAssessor, and FATHMM) as well as many SNV and gene functional annotations have been updated. dbNSFP v3.0 is freely available at http://sites.google.com/site/jpopgen/dbNSFP. PMID:26555599

  5. dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice-Site SNVs.

    PubMed

    Liu, Xiaoming; Wu, Chunlei; Li, Chang; Boerwinkle, Eric

    2016-03-01

    The purpose of the dbNSFP is to provide a one-stop resource for functional predictions and annotations for human nonsynonymous single-nucleotide variants (nsSNVs) and splice-site variants (ssSNVs), and to facilitate the steps of filtering and prioritizing SNVs from a large list of SNVs discovered in an exome-sequencing study. A list of all potential nsSNVs and ssSNVs based on the human reference sequence were created and functional predictions and annotations were curated and compiled for each SNV. Here, we report a recent major update of the database to version 3.0. The SNV list has been rebuilt based on GENCODE 22 and currently the database includes 82,832,027 nsSNVs and ssSNVs. An attached database dbscSNV, which compiled all potential human SNVs within splicing consensus regions and their deleteriousness predictions, add another 15,030,459 potentially functional SNVs. Eleven prediction scores (MetaSVM, MetaLR, CADD, VEST3, PROVEAN, 4× fitCons, fathmm-MKL, and DANN) and allele frequencies from the UK10K cohorts and the Exome Aggregation Consortium (ExAC), among others, have been added. The original seven prediction scores in v2.0 (SIFT, 2× Polyphen2, LRT, MutationTaster, MutationAssessor, and FATHMM) as well as many SNV and gene functional annotations have been updated. dbNSFP v3.0 is freely available at http://sites.google.com/site/jpopgen/dbNSFP.

  6. A systems model of phosphorylation for inflammatory signaling events.

    PubMed

    Sadreev, Ildar I; Chen, Michael Z Q; Welsh, Gavin I; Umezawa, Yoshinori; Kotov, Nikolay V; Valeyev, Najl V

    2014-01-01

    Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits.

  7. The Global Terrestrial Network for Permafrost Database: metadata statistics and prospective analysis on future permafrost temperature and active layer depth monitoring site distribution

    NASA Astrophysics Data System (ADS)

    Biskaborn, B. K.; Lanckman, J.-P.; Lantuit, H.; Elger, K.; Streletskiy, D. A.; Cable, W. L.; Romanovsky, V. E.

    2015-03-01

    The Global Terrestrial Network for Permafrost (GTN-P) provides the first dynamic database associated with the Thermal State of Permafrost (TSP) and the Circumpolar Active Layer Monitoring (CALM) programs, which extensively collect permafrost temperature and active layer thickness data from Arctic, Antarctic and Mountain permafrost regions. The purpose of the database is to establish an "early warning system" for the consequences of climate change in permafrost regions and to provide standardized thermal permafrost data to global models. In this paper we perform statistical analysis of the GTN-P metadata aiming to identify the spatial gaps in the GTN-P site distribution in relation to climate-effective environmental parameters. We describe the concept and structure of the Data Management System in regard to user operability, data transfer and data policy. We outline data sources and data processing including quality control strategies. Assessment of the metadata and data quality reveals 63% metadata completeness at active layer sites and 50% metadata completeness for boreholes. Voronoi Tessellation Analysis on the spatial sample distribution of boreholes and active layer measurement sites quantifies the distribution inhomogeneity and provides potential locations of additional permafrost research sites to improve the representativeness of thermal monitoring across areas underlain by permafrost. The depth distribution of the boreholes reveals that 73% are shallower than 25 m and 27% are deeper, reaching a maximum of 1 km depth. Comparison of the GTN-P site distribution with permafrost zones, soil organic carbon contents and vegetation types exhibits different local to regional monitoring situations on maps. Preferential slope orientation at the sites most likely causes a bias in the temperature monitoring and should be taken into account when using the data for global models. The distribution of GTN-P sites within zones of projected temperature change show a high

  8. Global Database of GSSR Mars Delay-Doppler Radar Observations: Analysis for Landing Site Characterization and Rover Trafficability

    NASA Technical Reports Server (NTRS)

    Haldemann, A. F. C.; Jurgens, R. F.; Slade, M. A.; Thompson, T. W.

    1999-01-01

    Earth-based radar data remain an important part of the information set used to select and certify spacecraft landing sites on Mars. Constraints on robotic landings on Mars include: terrain elevation, radar reflectivity, regional and local slopes, rock distribution and coverage, and surface roughness, all of which are addressed by radar data. Indeed, the usefulness of radar data for Mars exploration has been demonstrated in the past. Radar data were critical in assessing the Viking Lander 1 site, and more recently, the Mars Pathfinder landing site.

  9. A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

    PubMed

    Panda, Swarupa; Vedagiri, Dhiviya; Viveka, Thangaraj Soundara; Harshan, Krishnan Harinivas

    2014-09-01

    We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

  10. Preliminary Safety Analysis of the Gorleben Site: Safety Concept and Application to Scenario Development Based on a Site-Specific Features, Events and Processes (FEP) Database - 13304

    SciTech Connect

    Moenig, Joerg; Beuth, Thomas; Wolf, Jens; Lommerzheim, Andre; Mrugalla, Sabine

    2013-07-01

    Based upon the German safety criteria, released in 2010 by the Federal Ministry of the Environment (BMU), a safety concept and a safety assessment concept for the disposal of heat-generating high-level waste have both been developed in the framework of the preliminary safety case for the Gorleben site (Project VSG). The main objective of the disposal is to contain the radioactive waste inside a defined rock zone, which is called containment-providing rock zone. The radionuclides shall remain essentially at the emplacement site, and at the most, a small defined quantity of material shall be able to leave this rock zone. This shall be accomplished by the geological barrier and a technical barrier system, which is required to seal the inevitable penetration of the geological barrier by the construction of the mine. The safe containment has to be demonstrated for probable and less probable evolutions of the site, while evolutions with very low probability (less than 1 % over the demonstration period of 1 million years) need not to be considered. Owing to the uncertainty in predicting the real evolution of the site, plausible scenarios have been derived in a systematic manner. Therefore, a comprehensive site-specific features, events and processes (FEP) data base for the Gorleben site has been developed. The safety concept was directly taken into account, e.g. by identification of FEP with direct influence on the barriers that provide the containment. No effort was spared to identify the interactions of the FEP, their probabilities of occurrence, and their characteristics (values). The information stored in the data base provided the basis for the development of scenarios. The scenario development methodology is based on FEP related to an impairment of the functionality of a subset of barriers, called initial barriers. By taking these FEP into account in their probable characteristics the reference scenario is derived. Thus, the reference scenario describes a

  11. Mitotic phosphorylation of histone H3 threonine 80

    PubMed Central

    Hammond, Sharra L; Byrum, Stephanie D; Namjoshi, Sarita; Graves, Hillary K; Dennehey, Briana K; Tackett, Alan J; Tyler, Jessica K

    2014-01-01

    The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells. PMID:24275038

  12. Mitotic phosphorylation of histone H3 threonine 80.

    PubMed

    Hammond, Sharra L; Byrum, Stephanie D; Namjoshi, Sarita; Graves, Hillary K; Dennehey, Briana K; Tackett, Alan J; Tyler, Jessica K

    2014-01-01

    The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells.

  13. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    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.

  14. Doubling down on phosphorylation as a variable peptide modification.

    PubMed

    Cooper, Bret

    2016-09-01

    Some mass spectrometrists believe that searching for variable PTMs like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false-positive matching. The basis for this is the premise that the algorithm compares a spectrum to both a nonphosphorylated peptide candidate and a phosphorylated candidate, which is double the number of candidates compared to a search with no possible phosphorylation. Hence, if the search space doubles, false-positive matching could increase accordingly as the algorithm considers more candidates to which false matches could be made. In this study, it is shown that the search for variable phosphoserine and phosphothreonine modifications does not always double the search space or unduly impinge upon the FDR. A breakdown of how one popular database-search algorithm deals with variable phosphorylation is presented.

  15. Systematic Discovery of In Vivo Phosphorylation Networks

    PubMed Central

    Linding, Rune; Jensen, Lars Juhl; Ostheimer, Gerard J.; van Vugt, Marcel A.T.M.; Jørgensen, Claus; Miron, Ioana M.; Diella, Francesca; Colwill, Karen; Taylor, Lorne; Elder, Kelly; Metalnikov, Pavel; Nguyen, Vivian; Pasculescu, Adrian; Jin, Jing; Park, Jin Gyoon; Samson, Leona D.; Woodgett, James R.; Russell, Robert B.; Bork, Peer; Yaffe, Michael B.; Pawson, Tony

    2009-01-01

    Summary Protein kinases control cellular decision processes by phosphorylating specific substrates. Proteome-wide mapping has identified thousands of in vivo phosphorylation sites. However, systematically resolving which kinase targets each site is presently infeasible, due to the limited specificity of consensus motifs and the potential influence of contextual factors, such as protein scaffolds, localisation and expression, on cellular substrate specificity. We have therefore developed a computational method, NetworKIN, that augments motifs with context for kinases and phosphoproteins. This can pinpoint individual kinases responsible for specific in vivo phosphorylation events and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. We show that context provides 60–80% of the computational capability to assign in vivo substrate specificity. Applying this approach to a DNA damage signalling network, we extend its cell-cycle regulation by showing that 53BP1 is a CDK1 substrate, show that Rad50 is phosphorylated by ATM kinase under genotoxic stress, and suggest novel roles of ATM in apoptosis. Finally, we present a scalable strategy to validate our predictions and use it to support the prediction that BCLAF1 is a GSK3 substrate. PMID:17570479

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

    PubMed

    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

  17. IDENTIFICATION OF IN VITRO AUTOPHOSPHORYLATION SITES AND THE EFFECTS OF PHOSPHORYLATION ON THE ARABIDOPSIS CRINKLY4 (ACR4) RECEPTOR-LIKE KINASE INTRACELLULAR DOMAIN: INSIGHTS INTO CONFORMATION, OLIGOMERIZATION AND ACTIVITY

    PubMed Central

    Meyer, Matthew R.; Lichti, Cheryl F.; Townsend, R. Reid; Rao, A. Gururaj

    2011-01-01

    Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that consists of an extracellular domain and an intracellular domain (ICD) with serine/threonine kinase activity. While genetic and cell biology experiments have demonstrated that ACR4 is important in cell fate specification and overall development of the plant, little is known about the biochemical properties of the kinase domain and the mechanisms that underlie the overall function of the receptor. To complement in planta studies on the function of ACR4, we have expressed the ICD in Escherichia coli as a soluble C-terminal fusion to the N-utilization substance A (NusA) protein, purified the recombinant protein and characterized the enzymatic and conformational properties. The protein autophosphorylates via an intramolecular mechanism, prefers Mn2+ over Mg2+ as the divalent cation and displays typical Michaelis-Menten kinetics with respect to ATP with an apparent Km of 6.67 ± 2.07 μM and Vmax of 1.83 ± 0.18 nmol/min/mg. Autophosphorylation is accompanied by a conformational change as demonstrated by circular dichroism, fluorescence spectroscopy and limited proteolysis with trypsin. Analysis by nano-liquid chromatography mass spectrometry (nano-LC-MS) revealed 16 confirmed sites of phosphorylation at Ser and Thr residues. Sedimentation velocity and gel-filtration experiments indicate that the ICD has a propensity to oligomerize and that this property is lost upon autophosphorylation. PMID:21294549

  18. Isolation of 3-phosphohistidine from phosphorylated pyruvate, phosphate dikinase.

    PubMed Central

    Spronk, A M; Yoshida, H; Wood, H G

    1976-01-01

    Pyruvate, phosphate dikinase (EC 2-7-9-1) catalyzes formation of phosphoenolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate. A pyrophosphoryl and phosphoryl form of the enzyme is involved in this transfer. The [32P]phosphoryl form of pyruvate, phosphate dikinase was prepared with enzyme isolated from Bacteroides symbiosus. The [32P]phosphoryl enzyme was found to have properties corresponding to a phosphoramidate linkage and this was confirmed by isolation of 3-[32P]phosphohistidine from alkaline hydrolysates of the enzyme. The histidyl residue is considered to be the pyrophosphoryl- and phosphoryl-carrier between the three substrate sites of this enzyme. PMID:12506

  19. Global Database of GSSR Mars Delay-Doppler Radar Obsevations: Analysis for Landing Site Characterization and Rover Trafficability

    NASA Technical Reports Server (NTRS)

    Haldemann, A. F. C.; Jurgens, R. F.; Slade, M. A.; Thompson, T. W.; Rojas, F.

    2000-01-01

    Earth-based radar data remain an important part of the information set used to select and certify spacecraft landing sites on Mars. Constraints on robotic landings on Mars include: terrain elevation, radar reflectivity, regional and local slopes, rock distribution and coverage, and surface roughness, all of which are addressed by radar data. Indeed, the usefulness of radar data for Mars exploration has been demonstrated in the past. Radar data were critical in assessing the Viking Lander, and more recently, the Mars Pathfinder landing.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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/

  2. Cascadia Tsunami Deposit Database

    USGS Publications Warehouse

    Peters, Robert; Jaffe, Bruce; Gelfenbaum, Guy; Peterson, Curt

    2003-01-01

    The Cascadia Tsunami Deposit Database contains data on the location and sedimentological properties of tsunami deposits found along the Cascadia margin. Data have been compiled from 52 studies, documenting 59 sites from northern California to Vancouver Island, British Columbia that contain known or potential tsunami deposits. Bibliographical references are provided for all sites included in the database. Cascadia tsunami deposits are usually seen as anomalous sand layers in coastal marsh or lake sediments. The studies cited in the database use numerous criteria based on sedimentary characteristics to distinguish tsunami deposits from sand layers deposited by other processes, such as river flooding and storm surges. Several studies cited in the database contain evidence for more than one tsunami at a site. Data categories include age, thickness, layering, grainsize, and other sedimentological characteristics of Cascadia tsunami deposits. The database documents the variability observed in tsunami deposits found along the Cascadia margin.

  3. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  4. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

  5. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

  6. The NCBI Taxonomy database.

    PubMed

    Federhen, Scott

    2012-01-01

    The NCBI Taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy) is the standard nomenclature and classification repository for the International Nucleotide Sequence Database Collaboration (INSDC), comprising the GenBank, ENA (EMBL) and DDBJ databases. It includes organism names and taxonomic lineages for each of the sequences represented in the INSDC's nucleotide and protein sequence databases. The taxonomy database is manually curated by a small group of scientists at the NCBI who use the current taxonomic literature to maintain a phylogenetic taxonomy for the source organisms represented in the sequence databases. The taxonomy database is a central organizing hub for many of the resources at the NCBI, and provides a means for clustering elements within other domains of NCBI web site, for internal linking between domains of the Entrez system and for linking out to taxon-specific external resources on the web. Our primary purpose is to index the domain of sequences as conveniently as possible for our user community.

  7. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  8. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

  9. Oxidative phosphorylation revisited.

    PubMed

    Nath, Sunil; Villadsen, John

    2015-03-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of

  10. Unlimited multistability in multisite phosphorylation systems.

    PubMed

    Thomson, Matthew; Gunawardena, Jeremy

    2009-07-01

    Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n sites, to eukaryotes, with examples having n >/= 150 sites. Multisite phosphorylation has many roles and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2(n)) of phospho-forms and individual phospho-forms may have distinct biological effects. The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 x 2(n) differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification 'codes' such as the histone code. Whereas simulations struggle with exponentially increasing molecular complexity

  11. Requirements Management Database

    2009-08-13

    This application is a simplified and customized version of the RBA and CTS databases to capture federal, site, and facility requirements, link to actions that must be performed to maintain compliance with their contractual and other requirements.

  12. VIEWCACHE: An incremental database access method for autonomous interoperable databases

    NASA Technical Reports Server (NTRS)

    Roussopoulos, Nick; Sellis, Timoleon

    1991-01-01

    The objective is to illustrate the concept of incremental access to distributed databases. An experimental database management system, ADMS, which has been developed at the University of Maryland, in College Park, uses VIEWCACHE, a database access method based on incremental search. VIEWCACHE is a pointer-based access method that provides a uniform interface for accessing distributed databases and catalogues. The compactness of the pointer structures formed during database browsing and the incremental access method allow the user to search and do inter-database cross-referencing with no actual data movement between database sites. Once the search is complete, the set of collected pointers pointing to the desired data are dereferenced.

  13. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  14. Androgen Receptor Exon 1 Mutation Causes Androgen Insensitivity by Creating Phosphorylation Site and Inhibiting Melanoma Antigen-A11 Activation of NH2- and Carboxyl-terminal Interaction-dependent Transactivation*

    PubMed Central

    Lagarde, William H.; Blackwelder, Amanda J.; Minges, John T.; Hnat, Andrew T.; French, Frank S.; Wilson, Elizabeth M.

    2012-01-01

    Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH2- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH2-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif 433WHTLF437 required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH2- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH2- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero. PMID:22334658

  15. A Computerized Data-Base System for Land-Use and Land-Cover Data Collected at Ground-Water Sampling Sites in the Pilot National Water Quality Assessment Program

    USGS Publications Warehouse

    Scott, Jonathon C.

    1989-01-01

    Data-base software has been developed for the management of land-use and land-cover data collected by the U.S. Geological Survey as part of a pilot program to test and refine concepts for a National Water-Quality Assessment Program. This report describes the purpose, use, and design of the land-use and land-cover data-base software. The software provides capabilities for interactive storage and retrieval of land-use and land-cover data collected at ground-water sampling sites. Users of the software can add, update, and delete land-use and land-cover data. The software also provides capabilities to group, print, and summarize the data. The land-use and land-cover data-base software supports multiple data-base systems so that data can be accessed by persons in different offices. Data-base systems are organized in a tiered structure. Each data-base system contains all the data stored in the data-base systems located in the lower tiers of the structure. Data can be readily transmitted from lower tiers to high tiers of the structure. Therefore, the data-base system at the highest tier of the structure contains land-use and land-cover data for the entire pilot program.

  16. Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

    SciTech Connect

    Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

    1986-03-05

    The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with ..gamma..-/sup 32/P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity.

  17. The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

    PubMed Central

    2002-01-01

    Background Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. Results We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. Conclusions This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site. PMID:11869452

  18. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    PubMed

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868487

  19. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    PubMed

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc.

  20. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  1. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  2. Doubling down on peptide phosphorylation as a variable mass modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some mass spectrometrists believe that searching for variable post-translational modifications like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false positive rates. The basis for this is the premise that the al...

  3. Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants.

    PubMed

    Kimura, Taeko; Hosokawa, Tomohisa; Taoka, Masato; Tsutsumi, Koji; Ando, Kanae; Ishiguro, Koichi; Hosokawa, Masato; Hasegawa, Masato; Hisanaga, Shin-Ichi

    2016-01-01

    Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation in vitro by Cdk5, in cultured cells and in mouse brain. Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau. PMID:27641626

  4. Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants

    PubMed Central

    Kimura, Taeko; Hosokawa, Tomohisa; Taoka, Masato; Tsutsumi, Koji; Ando, Kanae; Ishiguro, Koichi; Hosokawa, Masato; Hasegawa, Masato; Hisanaga, Shin-ichi

    2016-01-01

    Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation in vitro by Cdk5, in cultured cells and in mouse brain. Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau. PMID:27641626

  5. Biofuel Database

    National Institute of Standards and Technology Data Gateway

    Biofuel Database (Web, free access)   This database brings together structural, biological, and thermodynamic data for enzymes that are either in current use or are being considered for use in the production of biofuels.

  6. Database Administrator

    ERIC Educational Resources Information Center

    Moore, Pam

    2010-01-01

    The Internet and electronic commerce (e-commerce) generate lots of data. Data must be stored, organized, and managed. Database administrators, or DBAs, work with database software to find ways to do this. They identify user needs, set up computer databases, and test systems. They ensure that systems perform as they should and add people to the…

  7. Database-assisted promoter analysis.

    PubMed

    Hehl, R; Wingender, E

    2001-06-01

    The analysis of regulatory sequences is greatly facilitated by database-assisted bioinformatic approaches. The TRANSFAC database contains information on transcription factors and their origins, functional properties and sequence-specific binding activities. Software tools enable us to screen the database with a given DNA sequence for interacting transcription factors. If a regulatory function is already attributed to this sequence then the database-assisted identification of binding sites for proteins or protein classes and subsequent experimental verification might establish functionally relevant sites within this sequence. The binding transcription factors and interacting factors might already be present in the database.

  8. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

  9. Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells.

    PubMed

    Thomas, Yann; Cirillo, Luca; Panbianco, Costanza; Martino, Lisa; Tavernier, Nicolas; Schwager, Françoise; Van Hove, Lucie; Joly, Nicolas; Santamaria, Anna; Pintard, Lionel; Gotta, Monica

    2016-04-19

    The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.

  10. Nitration of JAK-2 at the 1007Y-1008Y activation epitope impedes phosphorylation at this site: defining a GH, AKT/protein kinase B and nitric oxide synthase axis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Generalized liver protein tyrosine nitration (3’-nitrotyrosine, 3’-NT) increases in vivo after GH injection with immunohistocellular patterns strikingly similar to those we observed for a specific nitration of JAK2 at its 1007Y-1008Y regulatory phosphorylation epitope following proinflammatory chall...

  11. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.

    PubMed

    Huguenin-Dezot, Nicolas; De Cesare, Virginia; Peltier, Julien; Knebel, Axel; Kristaryianto, Yosua Adi; Rogerson, Daniel T; Kulathu, Yogesh; Trost, Matthias; Chin, Jason W

    2016-07-26

    Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages. PMID:27425610

  12. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  13. Phosphorylation of yeast hexokinases.

    PubMed

    Vojtek, A B; Fraenkel, D G

    1990-06-20

    We show by the use of 32P-labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP-dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcy1) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpk1w1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase-dependent high-affinity glucose uptake.

  14. The Molecular Biology Database Collection: 2008 update

    PubMed Central

    Galperin, Michael Y.

    2008-01-01

    The Nucleic Acids Research online Molecular Biology Database Collection is a public repository that lists more than 1000 databases described in this and previous Nucleic Acids Research annual database issues, as well as a selection of molecular biology databases described in other journals. All databases included in this Collection are freely available to the public. The 2008 update includes 1078 databases, 110 more than the previous one. The links to more than 80 databases have been updated and 25 obsolete databases have been removed from the list. The complete database list and summaries are available online at the Nucleic Acids Research web site, http://nar.oxfordjournals.org/. PMID:18025043

  15. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    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.

  16. Multisite phosphorylation of spinach leaf sucrose-phosphate synthase

    SciTech Connect

    Huber, J.L.; Huber, S.C. )

    1990-05-01

    Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

  17. National Ambient Radiation Database

    SciTech Connect

    Dziuban, J.; Sears, R.

    2003-02-25

    The U.S. Environmental Protection Agency (EPA) recently developed a searchable database and website for the Environmental Radiation Ambient Monitoring System (ERAMS) data. This site contains nationwide radiation monitoring data for air particulates, precipitation, drinking water, surface water and pasteurized milk. This site provides location-specific as well as national information on environmental radioactivity across several media. It provides high quality data for assessing public exposure and environmental impacts resulting from nuclear emergencies and provides baseline data during routine conditions. The database and website are accessible at www.epa.gov/enviro/. This site contains (1) a query for the general public which is easy to use--limits the amount of information provided, but includes the ability to graph the data with risk benchmarks and (2) a query for a more technical user which allows access to all of the data in the database, (3) background information on ER AMS.

  18. Global phosphoproteomic analysis of Daphnia pulex reveals evolutionary conservation of Ser/Thr/Tyr phosphorylation.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Yun, Ki Na; Kim, Jin Young; Lee, Sangkyu

    2014-03-01

    Reversible protein phosphorylations of serine, threonine, and tyrosine are critical processes in organisms ranging from prokaryotes to eukaryotes. Water fleas (Daphnids) have been used widely in ecologic and ecotoxicological studies, with more than 80% of ecotoxicological publications over the last 10 years involving planktonic genera, including Daphnia. However, the substrate proteins and the functions of phosphorylation in Daphnia remain largely unknown. Here, we report the first global screening of phosphoproteins and their sites of phosphorylation in D. pulex. We identified 103 phosphorylation sites in 91 Daphnia proteins by phosphopeptide enrichment using titanium dioxide isolation technology and an online two-dimensional liquid chromatography (2D-LC) system supported by high accuracy mass spectrometry. The identified Serine/threonine/tyrosine phosphorylation sites showed enrichment in the unstructured regions. Using Gene Ontology analysis, phosphorylated proteins were identified mainly as membrane proteins with essential biological roles such as protein binding, catalytic activity and nucleotide binding. BLASTP searching identified 21 phosphorylated sites in 20 D. pulex proteins that were evolutionally conserved between D. pulex and human. Here, we report the phosphorylation in Daphnia proteins and the predicted biological and functional roles of these phosphorylations. D. pulex might provide a promising model for examining the role of phosphorylation in biological functions.

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

    SciTech Connect

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

    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.

  20. Database Manager

    ERIC Educational Resources Information Center

    Martin, Andrew

    2010-01-01

    It is normal practice today for organizations to store large quantities of records of related information as computer-based files or databases. Purposeful information is retrieved by performing queries on the data sets. The purpose of DATABASE MANAGER is to communicate to students the method by which the computer performs these queries. This…

  1. Maize databases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter is a succinct overview of maize data held in the species-specific database MaizeGDB (the Maize Genomics and Genetics Database), and selected multi-species data repositories, such as Gramene/Ensembl Plants, Phytozome, UniProt and the National Center for Biotechnology Information (NCBI), ...

  2. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage.

    PubMed

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-03-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells. PMID:26666690

  3. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

    PubMed Central

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo

    2015-01-01

    ABSTRACT The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. IMPORTANCE It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. PMID:26608325

  4. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    PubMed Central

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells. PMID:26666690

  5. Proline-directed phosphorylation of the dopamine transporter N-terminal domain

    PubMed Central

    Gorentla, Balachandra K.; Moritz, Amy E.; Foster, James D.; Vaughan, Roxanne A.

    2009-01-01

    Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses. PMID:19146407

  6. Bak apoptotic function is not directly regulated by phosphorylation.

    PubMed

    Tran, V H; Bartolo, R; Westphal, D; Alsop, A; Dewson, G; Kluck, R M

    2013-01-01

    During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation. PMID:23303126

  7. Construction of protein phosphorylation networks by data mining, text mining and ontology integration: analysis of the spindle checkpoint.

    PubMed

    Ross, Karen E; Arighi, Cecilia N; Ren, Jia; Huang, Hongzhan; Wu, Cathy H

    2013-01-01

    Knowledge representation of the role of phosphorylation is essential for the meaningful understanding of many biological processes. However, such a representation is challenging because proteins can exist in numerous phosphorylated forms with each one having its own characteristic protein-protein interactions (PPIs), functions and subcellular localization. In this article, we evaluate the current state of phosphorylation event curation and then present a bioinformatics framework for the annotation and representation of phosphorylated proteins and construction of phosphorylation networks that addresses some of the gaps in current curation efforts. The integrated approach involves (i) text mining guided by RLIMS-P, a tool that identifies phosphorylation-related information in scientific literature; (ii) data mining from curated PPI databases; (iii) protein form and complex representation using the Protein Ontology (PRO); (iv) functional annotation using the Gene Ontology (GO); and (v) network visualization and analysis with Cytoscape. We use this framework to study the spindle checkpoint, the process that monitors the assembly of the mitotic spindle and blocks cell cycle progression at metaphase until all chromosomes have made bipolar spindle attachments. The phosphorylation networks we construct, centered on the human checkpoint kinase BUB1B (BubR1) and its yeast counterpart MAD3, offer a unique view of the spindle checkpoint that emphasizes biologically relevant phosphorylated forms, phosphorylation-state-specific PPIs and kinase-substrate relationships. Our approach for constructing protein phosphorylation networks can be applied to any biological process that is affected by phosphorylation. Database URL: http://www.yeastgenome.org/

  8. Construction of protein phosphorylation networks by data mining, text mining and ontology integration: analysis of the spindle checkpoint

    PubMed Central

    Ross, Karen E.; Arighi, Cecilia N.; Ren, Jia; Huang, Hongzhan; Wu, Cathy H.

    2013-01-01

    Knowledge representation of the role of phosphorylation is essential for the meaningful understanding of many biological processes. However, such a representation is challenging because proteins can exist in numerous phosphorylated forms with each one having its own characteristic protein–protein interactions (PPIs), functions and subcellular localization. In this article, we evaluate the current state of phosphorylation event curation and then present a bioinformatics framework for the annotation and representation of phosphorylated proteins and construction of phosphorylation networks that addresses some of the gaps in current curation efforts. The integrated approach involves (i) text mining guided by RLIMS-P, a tool that identifies phosphorylation-related information in scientific literature; (ii) data mining from curated PPI databases; (iii) protein form and complex representation using the Protein Ontology (PRO); (iv) functional annotation using the Gene Ontology (GO); and (v) network visualization and analysis with Cytoscape. We use this framework to study the spindle checkpoint, the process that monitors the assembly of the mitotic spindle and blocks cell cycle progression at metaphase until all chromosomes have made bipolar spindle attachments. The phosphorylation networks we construct, centered on the human checkpoint kinase BUB1B (BubR1) and its yeast counterpart MAD3, offer a unique view of the spindle checkpoint that emphasizes biologically relevant phosphorylated forms, phosphorylation-state–specific PPIs and kinase–substrate relationships. Our approach for constructing protein phosphorylation networks can be applied to any biological process that is affected by phosphorylation. Database URL: http://www.yeastgenome.org/ PMID:23749465

  9. Genome databases

    SciTech Connect

    Courteau, J.

    1991-10-11

    Since the Genome Project began several years ago, a plethora of databases have been developed or are in the works. They range from the massive Genome Data Base at Johns Hopkins University, the central repository of all gene mapping information, to small databases focusing on single chromosomes or organisms. Some are publicly available, others are essentially private electronic lab notebooks. Still others limit access to a consortium of researchers working on, say, a single human chromosome. An increasing number incorporate sophisticated search and analytical software, while others operate as little more than data lists. In consultation with numerous experts in the field, a list has been compiled of some key genome-related databases. The list was not limited to map and sequence databases but also included the tools investigators use to interpret and elucidate genetic data, such as protein sequence and protein structure databases. Because a major goal of the Genome Project is to map and sequence the genomes of several experimental animals, including E. coli, yeast, fruit fly, nematode, and mouse, the available databases for those organisms are listed as well. The author also includes several databases that are still under development - including some ambitious efforts that go beyond data compilation to create what are being called electronic research communities, enabling many users, rather than just one or a few curators, to add or edit the data and tag it as raw or confirmed.

  10. Hawaii bibliographic database

    USGS Publications Warehouse

    Wright, T.L.; Takahashi, T.J.

    1998-01-01

    The Hawaii bibliographic database has been created to contain all of the literature, from 1779 to the present, pertinent to the volcanological history of the Hawaiian-Emperor volcanic chain. References are entered in a PC- and Macintosh-compatible EndNote Plus bibliographic database with keywords and abstracts or (if no abstract) with annotations as to content. Keywords emphasize location, discipline, process, identification of new chemical data or age determinations, and type of publication. The database is updated approximately three times a year and is available to upload from an ftp site. The bibliography contained 8460 references at the time this paper was submitted for publication. Use of the database greatly enhances the power and completeness of library searches for anyone interested in Hawaiian volcanism.

  11. Phosphorylation of a neuronal-specific beta-tubulin isotype

    SciTech Connect

    Diaz-Nido, J.; Serrano, L.; Lopez-Otin, C.; Vandekerckhove, J.; Avila, J. )

    1990-08-15

    Adult rats were intracraneally injected with ({sup 32}P) phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.

  12. Data on the peptide mapping and MS identification for phosphorylated peptide.

    PubMed

    Wang, Hui; Tu, Zong-Cai; Liu, Guang-Xian; Zhang, Lu; Chen, Yuan

    2016-09-01

    This article contains peptides mapping, mass spectrometry and processed data related to the research "Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment" [1]. Fourier transform ion cyclotron mass spectrometry (FTICR MS) was used to investigate the specific phosphorylation sites and the degree of phosphorylation (DSP) at each site. Specifically, phosphorylated peptides were monitored through mass shift on the FTICR MS spectrum. DSP was evaluated through the relative abundance levels of the FTICR MS spectrometry. From these data, the calculation method of DSP was exemplified. PMID:27274527

  13. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

    PubMed

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

    2015-08-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin

  14. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

    PubMed Central

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

    2015-01-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

  15. Regulation of peroxisome dynamics by phosphorylation.

    PubMed

    Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W; Warscheid, Bettina

    2016-05-01

    Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies.

  16. AthaMap web tools for database-assisted identification of combinatorial cis-regulatory elements and the display of highly conserved transcription factor binding sites in Arabidopsis thaliana.

    PubMed

    Steffens, Nils Ole; Galuschka, Claudia; Schindler, Martin; Bülow, Lorenz; Hehl, Reinhard

    2005-07-01

    The AthaMap database generates a map of cis-regulatory elements for the Arabidopsis thaliana genome. AthaMap contains more than 7.4 x 10(6) putative binding sites for 36 transcription factors (TFs) from 16 different TF families. A newly implemented functionality allows the display of subsets of higher conserved transcription factor binding sites (TFBSs). Furthermore, a web tool was developed that permits a user-defined search for co-localizing cis-regulatory elements. The user can specify individually the level of conservation for each TFBS and a spacer range between them. This web tool was employed for the identification of co-localizing sites of known interacting TFs and TFs containing two DNA-binding domains. More than 1.8 x 10(5) combinatorial elements were annotated in the AthaMap database. These elements can also be used to identify more complex co-localizing elements consisting of up to four TFBSs. The AthaMap database and the connected web tools are a valuable resource for the analysis and the prediction of gene expression regulation at http://www.athamap.de. PMID:15980498

  17. Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels

    PubMed Central

    Cerda, Oscar; Trimmer, James S.

    2012-01-01

    Phosphorylation is the most common and abundant posttranslational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies that reveal the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events. PMID:20600597

  18. Estrogen receptor alpha phosphorylation and its functional impact in human breast cancer.

    PubMed

    Anbalagan, Muralidharan; Rowan, Brian G

    2015-12-15

    Estrogen receptor α (ERα) is a member of the nuclear receptor superfamily of transcription factors that regulates cell proliferation, differentiation and homeostasis in various tissues. Sustained exposure to estrogen/estradiol (E2) increases the risk of breast, endometrial and ovarian cancers. ERα function is also regulated by phosphorylation through various kinase signaling pathways that will impact various ERα functions including chromatin interaction, coregulator recruitment and gene expression, as well impact breast tumor growth/morphology and breast cancer patient response to endocrine therapy. However, many of the previously characterized ERα phosphorylation sites do not fully explain the impact of receptor phosphorylation on ERα function. This review discusses work from our laboratory toward understanding a role of ERα site-specific phosphorylation in ERα function and breast cancer. The key findings discussed in this review are: (1) the effect of site specific ERα phosphorylation on temporal recruitment of ERα and unique coactivator complexes to specific genes; (2) the impact of stable disruption of ERα S118 and S167 phosphorylation in breast cancer cells on eliciting unique gene expression profiles that culminate in significant effects on breast cancer growth/morphology/migration/invasion; (3) the Src kinase signaling pathway that impacts ERα phosphorylation to alter ERα function; and (4) circadian disruption by light exposure at night leading to elevated ERK1/2 and Src kinase and phosphorylation of ERα, concomitant with tamoxifen resistance in breast tumor models. Results from these studies demonstrate that even changes to single ERα phosphorylation sites can have a profound impact on ERα function in breast cancer. Future work will extend beyond single site phosphorylation analysis toward identification of specific patterns/profiles of ERα phosphorylation under different physiological/pharmacological conditions to understand how common

  19. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  20. Phosphorylation Signals in Striatal Medium Spiny Neurons.

    PubMed

    Nagai, Taku; Yoshimoto, Junichiro; Kannon, Takayuki; Kuroda, Keisuke; Kaibuchi, Kozo

    2016-10-01

    Dopamine signaling in the brain is a complex phenomenon that strongly contributes to emotional behaviors. Medium spiny neurons (MSNs) play a major role in dopamine signaling through dopamine D1 receptors (D1Rs) or dopamine D2 receptors (D2Rs) in the striatum. cAMP/protein kinase A (PKA) regulates phosphorylation signals downstream of D1Rs, which affects the excitability of MSNs, leading to reward-associated emotional expression and memory formation. A combination of phosphoproteomic approaches and the curated KANPHOS database can be used to elucidate the physiological and pathophysiological functions of dopamine signaling and other monoamines. Emerging evidence from these techniques suggests that the Rap1 pathway plays a crucial role in the excitability of MSNs, leading to the expression of emotional behaviors. PMID:27546785

  1. Phosphorylation Signals in Striatal Medium Spiny Neurons.

    PubMed

    Nagai, Taku; Yoshimoto, Junichiro; Kannon, Takayuki; Kuroda, Keisuke; Kaibuchi, Kozo

    2016-10-01

    Dopamine signaling in the brain is a complex phenomenon that strongly contributes to emotional behaviors. Medium spiny neurons (MSNs) play a major role in dopamine signaling through dopamine D1 receptors (D1Rs) or dopamine D2 receptors (D2Rs) in the striatum. cAMP/protein kinase A (PKA) regulates phosphorylation signals downstream of D1Rs, which affects the excitability of MSNs, leading to reward-associated emotional expression and memory formation. A combination of phosphoproteomic approaches and the curated KANPHOS database can be used to elucidate the physiological and pathophysiological functions of dopamine signaling and other monoamines. Emerging evidence from these techniques suggests that the Rap1 pathway plays a crucial role in the excitability of MSNs, leading to the expression of emotional behaviors.

  2. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    SciTech Connect

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

  3. Role of phosphorylation in the mammalian circadian clock.

    PubMed

    Vanselow, K; Kramer, A

    2007-01-01

    Circadian clocks regulate a wide variety of processes ranging from gene expression to behavior. At the molecular level, circadian rhythms are thought to be produced by a set of clock genes and proteins interconnected to form transcriptional-translational feedback loops. Rhythmic gene expression was formerly regarded as the major drive for rhythms in clock protein abundance, but recent findings underline the crucial importance of posttranslational mechanisms for both the generation and dynamics of circadian rhythms. In particular, the reversible phosphorylation of PER proteins-essential components within the negative feedback loop in Drosophila and mammals-seems to have a key role for the correct timing of nuclear repression. To understand how PER protein phosphorylation regulates the dynamics of the circadian oscillator, we have mapped endogenous phosphorylation sites in mPER2. Detailed investigation of the functional role of one particular phosphorylation site (Ser-659, which is mutated in the familial advanced sleep phase syndrome [FASPS]) led us propose a model of functionally different phosphorylation sites in PER2. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster. PMID:18419274

  4. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis.

    PubMed

    Jones, Danielle M; Murray, Christian M; Ketelaar, KassaDee J; Thomas, Joseph J; Villalobos, Jose A; Wallace, Ian S

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  5. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  6. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

    PubMed Central

    Jones, Danielle M.; Murray, Christian M.; Ketelaar, KassaDee J.; Thomas, Joseph J.; Villalobos, Jose A.; Wallace, Ian S.

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  7. National Geo-Database for Biofuel Simulations and Regional Analysis of Biorefinery Siting Based on Cellulosic Feedstock Grown on Marginal Lands

    SciTech Connect

    Izaurralde, Roberto C.; Zhang, Xuesong; Sahajpal, Ritvik; Manowitz, David H.

    2012-04-01

    The goal of this project undertaken by GLBRC (Great Lakes Bioenergy Research Center) Area 4 (Sustainability) modelers is to develop a national capability to model feedstock supply, ethanol production, and biogeochemical impacts of cellulosic biofuels. The results of this project contribute to sustainability goals of the GLBRC; i.e. to contribute to developing a sustainable bioenergy economy: one that is profitable to farmers and refiners, acceptable to society, and environmentally sound. A sustainable bioenergy economy will also contribute, in a fundamental way, to meeting national objectives on energy security and climate mitigation. The specific objectives of this study are to: (1) develop a spatially explicit national geodatabase for conducting biofuel simulation studies and (4) locate possible sites for the establishment of cellulosic ethanol biorefineries. To address the first objective, we developed SENGBEM (Spatially Explicit National Geodatabase for Biofuel and Environmental Modeling), a 60-m resolution geodatabase of the conterminous USA containing data on: (1) climate, (2) soils, (3) topography, (4) hydrography, (5) land cover/ land use (LCLU), and (6) ancillary data (e.g., road networks, federal and state lands, national and state parks, etc.). A unique feature of SENGBEM is its 2008-2010 crop rotation data, a crucially important component for simulating productivity and biogeochemical cycles as well as land-use changes associated with biofuel cropping. ARRA support for this project and to the PNNL Joint Global Change Research Institute enabled us to create an advanced computing infrastructure to execute millions of simulations, conduct post-processing calculations, store input and output data, and visualize results. These computing resources included two components installed at the Research Data Center of the University of Maryland. The first resource was 'deltac': an 8-core Linux server, dedicated to county-level and state-level simulations and Postgre

  8. Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System

    PubMed Central

    2016-01-01

    Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking–MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry–MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation. PMID:27504491

  9. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    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.

  10. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    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. PMID:26375013

  11. NPM phosphorylation stimulates Cdk1, overrides G2/M checkpoint and increases leukemic blasts in mice.

    PubMed

    Du, Wei; Zhou, Yun; Pike, Suzette; Pang, Qishen

    2010-02-01

    An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CDK) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Simultaneous inactivation of two CDK phosphorylation sites at Ser10 and Ser70 (NPM-AA) induced G(2)/M cell cycle arrest, phosphorylation of Cdk1 at Tyr15 (Cdc2(Tyr15)) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1(Tyr15) and Cdc25C sequestration was suppressed by expression of a phosphomimetic NPM mutant created on the same CDK sites (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 was required for proper interaction between Cdk1 and Cdc25C. Moreover, NPM-EE directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G(2)/M arrest and increased leukemia blasts in a NOD/SCID xenograft model. Thus, these findings reveal a novel function of NPM on regulation of cell cycle progression, in which phosphorylation of NPM controls cell cycle progression at G(2)/M transition through modulation of Cdk1 and Cdc25C activities.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27586301

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

    PubMed

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

    2016-01-01

    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 IICB(Glc), 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. PMID:27586301

  15. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

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

    2016-01-01

    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. PMID:27088325

  16. Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes.

    PubMed

    Vener, Alexander V

    2007-06-01

    Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO(2) availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes. PMID:17184728

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

    PubMed

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

    2016-01-01

    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. PMID:27088325

  18. Formation and Dissociation of Phosphorylated Peptide Radical Cations

    NASA Astrophysics Data System (ADS)

    Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-Kuen; Siu, Chi-Kit; Chu, Ivan K.

    2012-12-01

    In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N, N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the p M·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model— N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

  19. Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase

    SciTech Connect

    Lobjois, Valerie; Froment, Carine; Braud, Emmanuelle; Grimal, Fanny; Burlet-Schiltz, Odile; Ducommun, Bernard; Bouche, Jean-Pierre

    2011-06-24

    Highlights: {yields} Phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro. {yields} Sequential phosphorylation of CDC25B is analyzed using {sup 16}O and {sup 18}O ATP. {yields} Thirteen sites phosphorylated by PLK1 have been identified. -- Abstract: CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with {sup 16}O and {sup 18}O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.

  20. FGF23 is endogenously phosphorylated in bone cells.

    PubMed

    Lindberg, Iris; Pang, Hong Weng; Stains, Joseph P; Clark, David; Yang, Austin J; Bonewald, Lynda; Li, Kevin Z

    2015-03-01

    Levels of serum phosphate are controlled by the peptide hormone FGF23, secreted from bone osteocytes. Elevated levels of circulating FGF23 are a key factor in several hypophosphatemic disorders and play a role in chronic kidney disease. Posttranslational processing of FGF23 includes multi-site O-glycosylation, which reduces intracellular cleavage by proprotein convertases. The FGF23 protein also contains four serine phosphorylation consensus sequences (S-X-D/E); in this work, we asked whether FGF23 is a substrate for secretory phosphorylation. Both HEK cells as well as IDG-SW3 cells, an osteocyte model, incorporated radiolabeled orthophosphate into intact FGF23, as well as into the 14-kDa carboxy-terminal-but not the 17-kDa N-terminal-fragment. Sequential serine-to-alanine site-directed mutagenesis of four kinase consensus sites showed that labeling occurred on three serines within the carboxy-terminal fragment, Ser180 (adjacent to the cleavage site), Ser207, and Ser212. Liquid chromatography-coupled mass spectroscopy indicated the presence of phosphate at Ser212 in recombinant R&D mouse FGF23(R179Q) , confirming labeling results. A phosphopeptide-specific antibody was raised against phospho-Ser212 and exhibited immunoreactivity in osteocytes present in mouse long bone, providing further evidence that FGF23 is naturally phosphorylated in bone. Bone SIBLING proteins are serine-phosphorylated by the ubiquitous Golgi secretory kinase FAM20C. Cotransfection of HEK and MC3T3 cells with FGF23 and active, but not inactive, FAM20C kinase increased the storage and release of FGF23 in radiolabeling experiments, indicating potential effects of phosphorylation on FGF23 stability. Collectively, these data point to an important role for phosphorylation of FGF23 in bone.

  1. Mechanism of APC/CCDC20 activation by mitotic phosphorylation

    PubMed Central

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2016-01-01

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  2. Solubility Database

    National Institute of Standards and Technology Data Gateway

    SRD 106 IUPAC-NIST Solubility Database (Web, free access)   These solubilities are compiled from 18 volumes (Click here for List) of the International Union for Pure and Applied Chemistry(IUPAC)-NIST Solubility Data Series. The database includes liquid-liquid, solid-liquid, and gas-liquid systems. Typical solvents and solutes include water, seawater, heavy water, inorganic compounds, and a variety of organic compounds such as hydrocarbons, halogenated hydrocarbons, alcohols, acids, esters and nitrogen compounds. There are over 67,500 solubility measurements and over 1800 references.

  3. Discovery of a Previously Unrecognized Ribonuclease from Escherichia coli That Hydrolyzes 5'-Phosphorylated Fragments of RNA.

    PubMed

    Ghodge, Swapnil V; Raushel, Frank M

    2015-05-12

    TrpH or YciV (locus tag b1266) from Escherichia coli is annotated as a protein of unknown function that belongs to the polymerase and histidinol phosphatase (PHP) family of proteins in the UniProt and NCBI databases. Enzymes from the PHP family have been shown to hydrolyze organophosphoesters using divalent metal ion cofactors at the active site. We found that TrpH is capable of hydrolyzing the 3'-phosphate from 3',5'-bis-phosphonucleotides. The enzyme will also sequentially hydrolyze 5'-phosphomononucleotides from 5'-phosphorylated RNA and DNA oligonucleotides, with no specificity toward the identity of the nucleotide base. The enzyme will not hydrolyze RNA or DNA oligonucleotides that are unphosphorylated at the 5'-end of the substrate, but it makes no difference whether the 3'-end of the oligonucleotide is phosphorylated. These results are consistent with the sequential hydrolysis of 5'-phosphorylated mononucleotides from oligonucleotides in the 5' → 3' direction. The catalytic efficiencies for hydrolysis of 3',5'-pAp, p(Ap)A, p(Ap)4A, and p(dAp)4dA were determined to be 1.8 × 10(5), 9.0 × 10(4), 4.6 × 10(4), and 2.9 × 10(3) M(-1) s(-1), respectively. TrpH was found to be more efficient at hydrolyzing RNA oligonucleotides than DNA oligonucleotides. This enzyme can also hydrolyze annealed DNA duplexes, albeit at a catalytic efficiency approximately 10-fold lower than that of the corresponding single-stranded oligonucleotides. TrpH is the first enzyme from E. coli that has been found to possess 5' → 3' exoribonuclease activity. We propose to name this enzyme RNase AM. PMID:25871919

  4. Phosphorylation mechanisms in chemical evolution

    NASA Astrophysics Data System (ADS)

    Schoffstall, Allen M.; Laing, Euton M.

    1985-06-01

    An objective of this work is to elucidate the mechanism of phosphorylation of nucleosides in amide solvents and in urea. A second objective is to assess the importance of phosphorylation and dephosphorylation of nucleotide derivatives in amide environments. Although the most complex amide studied here was N-methylacetamide, inferences are made on the importance of dephosphorylation for nucleotides in oligopeptide environments. Phosphorylations in amide solvents and in urea are suggested to proceed through monomeric metaphosphate, which was first postulated as a reaction intermediate thirty years ago (Butcher and Westheimer, 1955). Phosphorylation of nucleosides and nucleotides and dephosphorylation of nucleotide derivatives have been studied in formamide, N-methylformamide, urea and N-methylacetamide. Hydrated forms of 5'-ADP and 5'ATP are unstable in hot amide solvents and in urea. They decompose to a mixture of adenosine and its phosphorylated derivatives. The rate of decomposition is much slower in N-methylacetamide than in formamide or urea. Experiments designed to prepare oligonucleotides in the presence of oligopeptides have been reported (White, 1983). According to the present study, it is not unreasonable to expect that nucleotide derivatives can be condensed with nucleosides to form oligonucleotides in a peptide environment. However, nucleotide monomers such as 5'-ATP, 5'-ADP or 5'AMP will suffer isomerization or decomposition during condensation use of activated phosphate derivatives is preferable. Monomeric metaphosphate has not been isolated or characterized in amide solvents. It is proposed here as a reaction intermediate, probably in a complexed form with the amide.

  5. Glycogen phosphorylation and Lafora disease.

    PubMed

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease.

  6. Confident site localization using a simulated phosphopeptide spectral library.

    PubMed

    Suni, Veronika; Imanishi, Susumu Y; Maiolica, Alessio; Aebersold, Ruedi; Corthals, Garry L

    2015-05-01

    We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.

  7. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  8. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  9. Phosphorylated Mesoporous Carbon as a Solid Acid Catalyst

    SciTech Connect

    Dai, Sheng; Mayes, Richard T; Fulvio, Pasquale F; Ma, Zhen

    2011-01-01

    Mesoporous carbon catalyst supports are attractive due to their wide chemical stability while potentially increasing masstransport through and providing a path for larger molecules to access catalytic sites. Herein we report the synthesis of a 10 phosphorylated mesoporous carbon solid-acid catalyst characterized by NH3-TPD and isopropanol dehydration.

  10. Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function.

    PubMed

    Rajanala, Kalpana; Sarkar, Anshuk; Jhingan, Gagan Deep; Priyadarshini, Raina; Jalan, Manisha; Sengupta, Sagar; Nandicoori, Vinay Kumar

    2014-08-15

    A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.

  11. Nucleoside phosphorylation by phosphate minerals.

    PubMed

    Costanzo, Giovanna; Saladino, Raffaele; Crestini, Claudia; Ciciriello, Fabiana; Di Mauro, Ernesto

    2007-06-01

    In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.

  12. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo.

    PubMed

    Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas J T; Jensen, Ole Nørregaard; Højlund, Kurt

    2014-05-01

    There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.

  13. Cell-cycle-dependent phosphorylation of the nuclear pore Nup107–160 subcomplex

    PubMed Central

    Glavy, Joseph S.; Krutchinsky, Andrew N.; Cristea, Ileana M.; Berke, Ian C.; Boehmer, Thomas; Blobel, Günter; Chait, Brian T.

    2007-01-01

    The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107–160 subcomplex, a core building block of the NPC. Using in vivo 32P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS2, and MS3), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS2 analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G1 cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107–160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107–160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins. PMID:17360435

  14. Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.

    PubMed

    Wooldridge, Anne A; MacDonald, Justin A; Erdodi, Ferenc; Ma, Chaoyu; Borman, Meredith A; Hartshorne, David J; Haystead, Timothy A J

    2004-08-13

    Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

  15. Shuttle Hypervelocity Impact Database

    NASA Technical Reports Server (NTRS)

    Hyde, James L.; Christiansen, Eric L.; Lear, Dana M.

    2011-01-01

    With three missions outstanding, the Shuttle Hypervelocity Impact Database has nearly 3000 entries. The data is divided into tables for crew module windows, payload bay door radiators and thermal protection system regions, with window impacts compromising just over half the records. In general, the database provides dimensions of hypervelocity impact damage, a component level location (i.e., window number or radiator panel number) and the orbiter mission when the impact occurred. Additional detail on the type of particle that produced the damage site is provided when sampling data and definitive analysis results are available. Details and insights on the contents of the database including examples of descriptive statistics will be provided. Post flight impact damage inspection and sampling techniques that were employed during the different observation campaigns will also be discussed. Potential enhancements to the database structure and availability of the data for other researchers will be addressed in the Future Work section. A related database of returned surfaces from the International Space Station will also be introduced.

  16. Shuttle Hypervelocity Impact Database

    NASA Technical Reports Server (NTRS)

    Hyde, James I.; Christiansen, Eric I.; Lear, Dana M.

    2011-01-01

    With three flights remaining on the manifest, the shuttle impact hypervelocity database has over 2800 entries. The data is currently divided into tables for crew module windows, payload bay door radiators and thermal protection system regions, with window impacts compromising just over half the records. In general, the database provides dimensions of hypervelocity impact damage, a component level location (i.e., window number or radiator panel number) and the orbiter mission when the impact occurred. Additional detail on the type of particle that produced the damage site is provided when sampling data and definitive analysis results are available. The paper will provide details and insights on the contents of the database including examples of descriptive statistics using the impact data. A discussion of post flight impact damage inspection and sampling techniques that were employed during the different observation campaigns will be presented. Future work to be discussed will be possible enhancements to the database structure and availability of the data for other researchers. A related database of ISS returned surfaces that are under development will also be introduced.

  17. DAPPLE 2: a Tool for the Homology-Based Prediction of Post-Translational Modification Sites.

    PubMed

    Trost, Brett; Maleki, Farhad; Kusalik, Anthony; Napper, Scott

    2016-08-01

    The post-translational modification of proteins is critical for regulating their function. Although many post-translational modification sites have been experimentally determined, particularly in certain model organisms, experimental knowledge of these sites is severely lacking for many species. Thus, it is important to be able to predict sites of post-translational modification in such species. Previously, we described DAPPLE, a tool that facilitates the homology-based prediction of one particular post-translational modification, phosphorylation, in an organism of interest using known phosphorylation sites from other organisms. Here, we describe DAPPLE 2, which expands and improves upon DAPPLE in three major ways. First, it predicts sites for many post-translational modifications (20 different types) using data from several sources (15 online databases). Second, it has the ability to make predictions approximately 2-7 times faster than DAPPLE depending on the database size and the organism of interest. Third, it simplifies and accelerates the process of selecting predicted sites of interest by categorizing them based on gene ontology terms, keywords, and signaling pathways. We show that DAPPLE 2 can successfully predict known human post-translational modification sites using, as input, known sites from species that are either closely (e.g., mouse) or distantly (e.g., yeast) related to humans. DAPPLE 2 can be accessed at http://saphire.usask.ca/saphire/dapple2 .

  18. DAPPLE 2: a Tool for the Homology-Based Prediction of Post-Translational Modification Sites.

    PubMed

    Trost, Brett; Maleki, Farhad; Kusalik, Anthony; Napper, Scott

    2016-08-01

    The post-translational modification of proteins is critical for regulating their function. Although many post-translational modification sites have been experimentally determined, particularly in certain model organisms, experimental knowledge of these sites is severely lacking for many species. Thus, it is important to be able to predict sites of post-translational modification in such species. Previously, we described DAPPLE, a tool that facilitates the homology-based prediction of one particular post-translational modification, phosphorylation, in an organism of interest using known phosphorylation sites from other organisms. Here, we describe DAPPLE 2, which expands and improves upon DAPPLE in three major ways. First, it predicts sites for many post-translational modifications (20 different types) using data from several sources (15 online databases). Second, it has the ability to make predictions approximately 2-7 times faster than DAPPLE depending on the database size and the organism of interest. Third, it simplifies and accelerates the process of selecting predicted sites of interest by categorizing them based on gene ontology terms, keywords, and signaling pathways. We show that DAPPLE 2 can successfully predict known human post-translational modification sites using, as input, known sites from species that are either closely (e.g., mouse) or distantly (e.g., yeast) related to humans. DAPPLE 2 can be accessed at http://saphire.usask.ca/saphire/dapple2 . PMID:27367363

  19. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    NASA Astrophysics Data System (ADS)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  20. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates.

    PubMed

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  1. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    PubMed Central

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx. PMID:24681538

  2. PKC{delta}-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    SciTech Connect

    Greene, Michael W. . E-mail: michael.greene@bassett.org; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKC{delta} on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKC{delta}-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKC{delta} catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  3. Tyrosine Phosphorylation of SGEF Regulates RhoG Activity and Cell Migration

    PubMed Central

    Okuyama, Yusuke; Umeda, Kentaro; Negishi, Manabu; Katoh, Hironori

    2016-01-01

    SGEF and Ephexin4 are members of the Ephexin subfamily of RhoGEFs that specifically activate the small GTPase RhoG. It is reported that Ephexin1 and Ephexin5, two well-characterized Ephexin subfamily RhoGEFs, are tyrosine-phosphorylated by Src, and that their phosphorylation affect their activities and functions. In this study, we show that SGEF, but not Ephexin4, is tyrosine-phosphorylated by Src. Tyrosine phosphorylation of SGEF suppresses its interaction with RhoG, the elevation of RhoG activity, and SGEF-mediated promotion of cell migration. We identified tyrosine 530 (Y530), which is located within the Dbl homology domain, as a major phosphorylation site of SGEF by Src, and Y530F mutation blocked the inhibitory effect of Src on SGEF. Taken together, these results suggest that the activity of SGEF is negatively regulated by tyrosine phosphorylation of the DH domain. PMID:27437949

  4. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

    PubMed Central

    Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

    1992-01-01

    Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

  5. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    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.

  6. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  7. dimerization and DNA binding alter phosphorylation of Fos and Jun

    SciTech Connect

    Abate, C.; Baker, S.J.; Curran, T. ); Lees-Miller, S.P.; Anderson, C.W. ); Marshak, D.R. )

    1993-07-15

    Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcription factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here the authors show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34[sup cdc2] (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation. 44 refs., 4 figs.

  8. Phosphorylation of Izumo1 and its role in male infertility

    PubMed Central

    Young, Samantha AM; Aitken, John; Baker, Mark A

    2015-01-01

    Izumo1 is a testis-specific gene product, whose function is essential for sperm-egg fusion. Throughout its lifespan, Izumo1 is posttranslationally modified, being both N-linked glycosylated on its extracellular domain and phosphorylated on the intracellular C-terminal tail. Within the caput regions of the rat epididymis, two phosphorylation events have been documented. However, as sperm pass through the epididymis, this cytoplasmic portion of Izumo1 has been shown to contain up to seven phosphorylation sites. Remarkably, in the rat, in correlation with these events, Izumo1 undergoes sub-cellular re-location, moving from the head/tail regions of the spermatozoa, to a predominantly equatorial segment location once they have reached the caudal end of the epididymis. PMID:25994654

  9. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    SciTech Connect

    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.

  10. Phosphorylation of lamins determine their structural properties and signaling functions.

    PubMed

    Torvaldson, Elin; Kochin, Vitaly; Eriksson, John E

    2015-01-01

    Lamin A/C is part of the nuclear lamina, a meshwork of intermediate filaments underlying the inner nuclear membrane. The lamin network is anchoring a complex set of structural and linker proteins and is either directly or through partner proteins also associated or interacting with a number of signaling protein and transcription factors. During mitosis the nuclear lamina is dissociated by well established phosphorylation- dependent mechanisms. A-type lamins are, however, also phosphorylated during interphase. A recent study identified 20 interphase phosphorylation sites on lamin A/C and explored their functions related to lamin dynamics; movements, localization and solubility. Here we discuss these findings in the light of lamin functions in health and disease.

  11. Interaction of diphtheria toxin with phosphorylated molecules.

    PubMed Central

    Proia, R L; Hart, D A; Eidels, L

    1979-01-01

    The binding of diphtheria toxin to 125I-labeled cell surface glycoproteins from hamster thymocytes was shown to be inhibited by nucleotides. The relative effectiveness of the nucleotides (at 5 mM) was found to be thymidine triphosphate greater than adenosine triphosphate greater than guanosine triphosphate greater than uridine triphosphate greater than cytidine triphosphate. When adenine-containing compounds were used, the relative effectiveness was determined to be adenosine tetraphosphate greater than adenosine triphosphate greater than adenosine diphosphate greater than adenosine monophosphate. In addition, tetrapolyphosphate, tripolyphosphate, inositol hexaphosphate (phytic acid), and the highly phosphorylated proteins casein and phosvitin were also shown to be potent inhibitors of the binding of diphtheria toxin to 125I-labeled cell surface glycoproteins. Diphtheria toxin was shown to bind directly to 125I-casein; this binding was also inhibited by the highly phosphorylated compounds and was decreased by pretreatment of the 125I-casein with alkaline phosphatase. These results suggest that diphtheria toxin binds to regions of high phosphate density and raise the possibility that the site on the cell surface glycoproteins to which diphtheria toxin binds might be polyanionic in nature. PMID:528059

  12. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity

    PubMed Central

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor

    2016-01-01

    Summary Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non‐structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post‐translational events yet. Here, we show that the newly identified phospho‐site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double‐stranded RNA and TRIM25 as well as complex formation with RIG‐I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon‐antagonistic activity are needed. PMID:26687707

  13. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

    PubMed

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

    2016-06-01

    Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.

  14. Enhanced binding of RNAP II CTD phosphatase FCP1 to RAP74 following CK2 phosphorylation.

    PubMed

    Abbott, Karen L; Renfrow, Matthew B; Chalmers, Michael J; Nguyen, Bao D; Marshall, Alan G; Legault, Pascale; Omichinski, James G

    2005-03-01

    FCP1 (TFIIF-associated CTD phosphatase) is the first identified CTD-specific phosphatase required to recycle RNA polymerase II (RNAP II). FCP1 activity has been shown to be regulated by the general transcription factors TFIIF (RAP74) and TFIIB, protein kinase CK2 (CK2), and the HIV-1 transcriptional activator Tat. Phosphorylation of FCP1 by CK2 stimulates FCP1 phosphatase activity and enhances binding of RAP74 to FCP1. We have examined consensus CK2 phosphorylation sites (acidic residue n + 3 to serine or threonine residue) located immediately adjacent to both RAP74-binding sites of FCP1. We demonstrate that both of these consensus CK2 sites can be phosphorylated in vitro and that phosphorylation at either CK2 site results in enhanced binding of RAP74 to FCP1. The CK2 site adjacent to the RAP74-binding site in the central domain of FCP1 is phosphorylated at a single threonine site (T584). The CK2 site adjacent to the RAP74-binding site in the carboxyl-terminal domain can be phosphorylated at three successive serine residues (S942-S944), with phosphorylations at S942 and S944 both contributing to enhanced binding to RAP74. With the use of tandem Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR), we demonstrate that the phosphorylation of S942-S944 occurs in a semiordered fashion with the initial phosphorylation occurring at either S942 or S944 followed by a second phosphorylation to yield the S942/S944 diphosphorylated species. Using nuclear magnetic resonance (NMR) spectroscopy, we identify and map chemical shift changes onto the solution structure of the carboxyl-terminal domain of RAP74 (RAP74(436)(-)(517)) on complexation of RAP74(436)(-)(517) with phosphorylated FCP1 peptides. These results provide new functional and structural information on the role of phosphorylation in the recognition of acidic-rich activation domains involved in transcriptional regulation, and bring insights into how CK2 and TFIIF regulate FCP1 function. PMID:15723518

  15. novPTMenzy: a database for enzymes involved in novel post-translational modifications.

    PubMed

    Khater, Shradha; Mohanty, Debasisa

    2015-01-01

    With the recent discoveries of novel post-translational modifications (PTMs) which play important roles in signaling and biosynthetic pathways, identification of such PTM catalyzing enzymes by genome mining has been an area of major interest. Unlike well-known PTMs like phosphorylation, glycosylation, SUMOylation, no bioinformatics resources are available for enzymes associated with novel and unusual PTMs. Therefore, we have developed the novPTMenzy database which catalogs information on the sequence, structure, active site and genomic neighborhood of experimentally characterized enzymes involved in five novel PTMs, namely AMPylation, Eliminylation, Sulfation, Hydroxylation and Deamidation. Based on a comprehensive analysis of the sequence and structural features of these known PTM catalyzing enzymes, we have created Hidden Markov Model profiles for the identification of similar PTM catalyzing enzymatic domains in genomic sequences. We have also created predictive rules for grouping them into functional subfamilies and deciphering their mechanistic details by structure-based analysis of their active site pockets. These analytical modules have been made available as user friendly search interfaces of novPTMenzy database. It also has a specialized analysis interface for some PTMs like AMPylation and Eliminylation. The novPTMenzy database is a unique resource that can aid in discovery of unusual PTM catalyzing enzymes in newly sequenced genomes. Database URL: http://www.nii.ac.in/novptmenzy.html

  16. Phosphorylation stoichiometry determination in plant photosynthetic membranes.

    PubMed

    Ingelsson, Björn; Fristedt, Rikard; Turkina, Maria V

    2015-01-01

    This chapter describes different strategies for the study of phosphorylation dynamics and stoichiometry in photosynthetic membranes. Detailed procedures for the detection, large-scale identification, and quantification of phosphorylated proteins optimized for plant thylakoid proteins are given. PMID:25930698

  17. Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

    PubMed Central

    Wahl, Matthew I.; Fluckiger, Anne-Catherine; Kato, Roberta M.; Park, Hyunsun; Witte, Owen N.; Rawlings, David J.

    1997-01-01

    Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation. PMID:9326643

  18. Characteristics and comprehensiveness of adult HIV care and treatment programmes in Asia-Pacific, sub-Saharan Africa and the Americas: results of a site assessment conducted by the International epidemiologic Databases to Evaluate AIDS (IeDEA) Collaboration

    PubMed Central

    Duda, Stephany N; Farr, Amanda M; Lindegren, Mary Lou; Blevins, Meridith; Wester, C William; Wools-Kaloustian, Kara; Ekouevi, Didier K; Egger, Matthias; Hemingway-Foday, Jennifer; Cooper, David A; Moore, Richard D; McGowan, Catherine C; Nash, Denis

    2014-01-01

    Introduction HIV care and treatment programmes worldwide are transforming as they push to deliver universal access to essential prevention, care and treatment services to persons living with HIV and their communities. The characteristics and capacity of these HIV programmes affect patient outcomes and quality of care. Despite the importance of ensuring optimal outcomes, few studies have addressed the capacity of HIV programmes to deliver comprehensive care. We sought to describe such capacity in HIV programmes in seven regions worldwide. Methods Staff from 128 sites in 41 countries participating in the International epidemiologic Databases to Evaluate AIDS completed a site survey from 2009 to 2010, including sites in the Asia-Pacific region (n=20), Latin America and the Caribbean (n=7), North America (n=7), Central Africa (n=12), East Africa (n=51), Southern Africa (n=16) and West Africa (n=15). We computed a measure of the comprehensiveness of care based on seven World Health Organization-recommended essential HIV services. Results Most sites reported serving urban (61%; region range (rr): 33–100%) and both adult and paediatric populations (77%; rr: 29–96%). Only 45% of HIV clinics that reported treating children had paediatricians on staff. As for the seven essential services, survey respondents reported that CD4+ cell count testing was available to all but one site, while tuberculosis (TB) screening and community outreach services were available in 80 and 72%, respectively. The remaining four essential services – nutritional support (82%), combination antiretroviral therapy adherence support (88%), prevention of mother-to-child transmission (PMTCT) (94%) and other prevention and clinical management services (97%) – were uniformly available. Approximately half (46%) of sites reported offering all seven services. Newer sites and sites in settings with low rankings on the UN Human Development Index (HDI), especially those in the President's Emergency Plan

  19. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.; Morrison, Lynda A. . E-mail: morrisla@slu.edu

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

  20. Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

    PubMed

    McDonough, Patrick M; Maciejewski-Lenoir, Dominique; Hartig, Sean M; Hanna, Rita A; Whittaker, Ross; Heisel, Andrew; Nicoll, James B; Buehrer, Benjamin M; Christensen, Kurt; Mancini, Maureen G; Mancini, Michael A; Edwards, Dean P; Price, Jeffrey H

    2013-01-01

    Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

  1. Regulation of uridine diphosphate-glucuronosyltransferase 1A3 activity by protein phosphorylation.

    PubMed

    Xiao, Yongsheng; Yao, Yan; Jiang, Huidi; Lu, Chuan; Zeng, Su; Yu, Lushan

    2015-11-01

    Protein phosphorylation is a vital post-translational modification. This study investigated the effect of phosphorylation on human uridine diphosphate (UDP)-glucuronosyltransferase 1A3 (UGT1A3) activity. Curcumin and calphostin C suppressed the activity and phosphorylation of recombinant UGT1A3 expressed in Sf9 cells. These results indicate that UGT1A3 undergoes phosphorylation, which is required for its catalytic activity. Calphostin C is a highly specific protein kinase C (PKC) inhibitor, so three predicted PKC phosphorylation sites in UGT1A3 were examined. Site-directed mutation analysis at residues 28, 43 and 436 (from serine to glycine) was conducted. Compared with the wild-type, the S43G-mutant showed significantly decreased UGT1A3 catalytic activity. Furthermore, the UGT1A3 activity of wild-type and S43G-mutant was down-regulated by calphostin C, whereas the calphostin C inhibitory effect was much weaker on the S43G-mutant than the wild-type. In conclusion, phosphorylation plays an important role in UGT1A3 activity, and the serine at site 43 in UGT1A3 is most likely a phosphorylation site. PMID:26094731

  2. An Internet enabled impact limiter material database

    SciTech Connect

    Wix, S.; Kanipe, F.; McMurtry, W.

    1998-09-01

    This paper presents a detailed explanation of the construction of an interest enabled database, also known as a database driven web site. The data contained in the internet enabled database are impact limiter material and seal properties. The technique used in constructing the internet enabled database presented in this paper are applicable when information that is changing in content needs to be disseminated to a wide audience.

  3. Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

    SciTech Connect

    Kuga, Takahisa; Nozaki, Naohito; Matsushita, Kazuyuki; Nomura, Fumio; Tomonaga, Takeshi

    2010-08-15

    Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G{sub 2}/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G{sub 1} phase, whereas Ser387 was phosphorylated discontinuously in prophase and G{sub 1} phase. Ser401 phosphorylation was enhanced in the G{sub 1}/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G{sub 1}-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.

  4. Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

    PubMed Central

    Zheng, Weinan; Li, Jing; Wang, Shanshan; Cao, Shuaishuai; Jiang, Jingwen; Chen, Can; Ding, Chan; Qin, Chuan; Ye, Xin; Gao, George F.

    2015-01-01

    ABSTRACT The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCE It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the

  5. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

  6. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  7. High Performance Buildings Database

    DOE Data Explorer

    The High Performance Buildings Database is a shared resource for the building industry, a unique central repository of in-depth information and data on high-performance, green building projects across the United States and abroad. The database includes information on the energy use, environmental performance, design process, finances, and other aspects of each project. Members of the design and construction teams are listed, as are sources for additional information. In total, up to twelve screens of detailed information are provided for each project profile. Projects range in size from small single-family homes or tenant fit-outs within buildings to large commercial and institutional buildings and even entire campuses. The database is a data repository as well. A series of Web-based data-entry templates allows anyone to enter information about a building project into the database. Once a project has been submitted, each of the partner organizations can review the entry and choose whether or not to publish that particular project on its own Web site.

  8. LQTS gene LOVD database.

    PubMed

    Zhang, Tao; Moss, Arthur; Cong, Peikuan; Pan, Min; Chang, Bingxi; Zheng, Liangrong; Fang, Quan; Zareba, Wojciech; Robinson, Jennifer; Lin, Changsong; Li, Zhongxiang; Wei, Junfang; Zeng, Qiang; Qi, Ming

    2010-11-01

    The Long QT Syndrome (LQTS) is a group of genetically heterogeneous disorders that predisposes young individuals to ventricular arrhythmias and sudden death. LQTS is mainly caused by mutations in genes encoding subunits of cardiac ion channels (KCNQ1, KCNH2,SCN5A, KCNE1, and KCNE2). Many other genes involved in LQTS have been described recently(KCNJ2, AKAP9, ANK2, CACNA1C, SCNA4B, SNTA1, and CAV3). We created an online database(http://www.genomed.org/LOVD/introduction.html) that provides information on variants in LQTS-associated genes. As of February 2010, the database contains 1738 unique variants in 12 genes. A total of 950 variants are considered pathogenic, 265 are possible pathogenic, 131 are unknown/unclassified, and 292 have no known pathogenicity. In addition to these mutations collected from published literature, we also submitted information on gene variants, including one possible novel pathogenic mutation in the KCNH2 splice site found in ten Chinese families with documented arrhythmias. The remote user is able to search the data and is encouraged to submit new mutations into the database. The LQTS database will become a powerful tool for both researchers and clinicians. PMID:20809527

  9. LQTS gene LOVD database.

    PubMed

    Zhang, Tao; Moss, Arthur; Cong, Peikuan; Pan, Min; Chang, Bingxi; Zheng, Liangrong; Fang, Quan; Zareba, Wojciech; Robinson, Jennifer; Lin, Changsong; Li, Zhongxiang; Wei, Junfang; Zeng, Qiang; Qi, Ming

    2010-11-01

    The Long QT Syndrome (LQTS) is a group of genetically heterogeneous disorders that predisposes young individuals to ventricular arrhythmias and sudden death. LQTS is mainly caused by mutations in genes encoding subunits of cardiac ion channels (KCNQ1, KCNH2,SCN5A, KCNE1, and KCNE2). Many other genes involved in LQTS have been described recently(KCNJ2, AKAP9, ANK2, CACNA1C, SCNA4B, SNTA1, and CAV3). We created an online database(http://www.genomed.org/LOVD/introduction.html) that provides information on variants in LQTS-associated genes. As of February 2010, the database contains 1738 unique variants in 12 genes. A total of 950 variants are considered pathogenic, 265 are possible pathogenic, 131 are unknown/unclassified, and 292 have no known pathogenicity. In addition to these mutations collected from published literature, we also submitted information on gene variants, including one possible novel pathogenic mutation in the KCNH2 splice site found in ten Chinese families with documented arrhythmias. The remote user is able to search the data and is encouraged to submit new mutations into the database. The LQTS database will become a powerful tool for both researchers and clinicians.

  10. Tyrosine phosphorylation and bacterial virulence

    PubMed Central

    Whitmore, Sarah E; Lamont, Richard J

    2012-01-01

    Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2. PMID:22388693

  11. Stable Synaptic Retention of Serine-880-Phosphorylated GluR2 in Hippocampal Neurons

    PubMed Central

    States, Bradley A.; Khatri, Latika; Ziff, Edward B.

    2008-01-01

    Phosphorylation of S880 within the GluR2 C-terminus has been reported to promote endocytosis of AMPA receptors (AMPARs) by preventing GluR2 interaction with the putative synaptic anchoring proteins GRIP and ABP. It is not yet established however, whether S880 phosphorylation induces removal of AMPARs from synaptic sites, and the trafficking of phosphorylated GluR2 subunits with surface and endocytosed GluR2 has not been directly compared within the same intact neurons. Here we show that phosphorylation of GluR2 subunits by PKC activated with phorbol esters is compartmentally restricted to receptors located at the cell surface. Endogenous AMPARs containing S880-phosphorylated GluR2 remained highly synaptic and colocalized with post-synaptic markers to the same extent as AMPARs which did not contain S880-phosphorylated GluR2. Moreover, following S880 phosphorylation, exogenous GluR2 homomers were found specifically at the cell surface and did not cotraffic with the internalized endosomal GluR2 population. We also show that GluR2 is endogenously phosphorylated by a constitutively active kinase pharmacologically related to PKC, and this phosphorylation is opposed by the protein phosphatase PP1. Our results demonstrate a population of hippocampal AMPARs which do not require interaction with GRIP/ABP for synaptic anchorage. PMID:18417360

  12. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

    PubMed

    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-01

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  13. Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Ziková, Alice; Smětáková, Magdalena; Bezoušková, Silvie

    2011-03-01

    The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.

  14. Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation*

    PubMed Central

    Pavlovic, Zvezdan; Zhu, Lin; Pereira, Leanne; Singh, Ratnesh Kumar; Cornell, Rosemary B.; Bakovic, Marica

    2014-01-01

    CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation. PMID:24519946

  15. Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer.

    PubMed

    Ha, S; Iqbal, N J; Mita, P; Ruoff, R; Gerald, W L; Lepor, H; Taneja, S S; Lee, P; Melamed, J; Garabedian, M J; Logan, S K

    2013-08-22

    Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

  16. Stackfile Database

    NASA Technical Reports Server (NTRS)

    deVarvalho, Robert; Desai, Shailen D.; Haines, Bruce J.; Kruizinga, Gerhard L.; Gilmer, Christopher

    2013-01-01

    This software provides storage retrieval and analysis functionality for managing satellite altimetry data. It improves the efficiency and analysis capabilities of existing database software with improved flexibility and documentation. It offers flexibility in the type of data that can be stored. There is efficient retrieval either across the spatial domain or the time domain. Built-in analysis tools are provided for frequently performed altimetry tasks. This software package is used for storing and manipulating satellite measurement data. It was developed with a focus on handling the requirements of repeat-track altimetry missions such as Topex and Jason. It was, however, designed to work with a wide variety of satellite measurement data [e.g., Gravity Recovery And Climate Experiment -- GRACE). The software consists of several command-line tools for importing, retrieving, and analyzing satellite measurement data.

  17. SENTRA, a database of signal transduction proteins.

    SciTech Connect

    D'Souza, M.; Romine, M. F.; Maltsev, N.; Mathematics and Computer Science; PNNL

    2000-01-01

    SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

  18. A framework for multivariate data-based at-site flood frequency analysis: Essentiality of the conjugal application of parametric and nonparametric approaches

    NASA Astrophysics Data System (ADS)

    Vittal, H.; Singh, Jitendra; Kumar, Pankaj; Karmakar, Subhankar

    2015-06-01

    In watershed management, flood frequency analysis (FFA) is performed to quantify the risk of flooding at different spatial locations and also to provide guidelines for determining the design periods of flood control structures. The traditional FFA was extensively performed by considering univariate scenario for both at-site and regional estimation of return periods. However, due to inherent mutual dependence of the flood variables or characteristics [i.e., peak flow (P), flood volume (V) and flood duration (D), which are random in nature], analysis has been further extended to multivariate scenario, with some restrictive assumptions. To overcome the assumption of same family of marginal density function for all flood variables, the concept of copula has been introduced. Although, the advancement from univariate to multivariate analyses drew formidable attention to the FFA research community, the basic limitation was that the analyses were performed with the implementation of only parametric family of distributions. The aim of the current study is to emphasize the importance of nonparametric approaches in the field of multivariate FFA; however, the nonparametric distribution may not always be a good-fit and capable of replacing well-implemented multivariate parametric and multivariate copula-based applications. Nevertheless, the potential of obtaining best-fit using nonparametric distributions might be improved because such distributions reproduce the sample's characteristics, resulting in more accurate estimations of the multivariate return period. Hence, the current study shows the importance of conjugating multivariate nonparametric approach with multivariate parametric and copula-based approaches, thereby results in a comprehensive framework for complete at-site FFA. Although the proposed framework is designed for at-site FFA, this approach can also be applied to regional FFA because regional estimations ideally include at-site estimations. The framework is

  19. Database Marketplace 2002: The Database Universe.

    ERIC Educational Resources Information Center

    Tenopir, Carol; Baker, Gayle; Robinson, William

    2002-01-01

    Reviews the database industry over the past year, including new companies and services, company closures, popular database formats, popular access methods, and changes in existing products and services. Lists 33 firms and their database services; 33 firms and their database products; and 61 company profiles. (LRW)

  20. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases.

    PubMed

    Tan, Chris Soon Heng; Bodenmiller, Bernd; Pasculescu, Adrian; Jovanovic, Marko; Hengartner, Michael O; Jørgensen, Claus; Bader, Gary D; Aebersold, Ruedi; Pawson, Tony; Linding, Rune

    2009-01-01

    Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast-evolving and ancient signaling events. We reveal that multiple complex diseases seem to converge within the conserved networks, suggesting that disease development might rely on common molecular networks.

  1. The GLIMS Glacier Database

    NASA Astrophysics Data System (ADS)

    Raup, B. H.; Khalsa, S. S.; Armstrong, R.

    2007-12-01

    The Global Land Ice Measurements from Space (GLIMS) project has built a geospatial and temporal database of glacier data, composed of glacier outlines and various scalar attributes. These data are being derived primarily from satellite imagery, such as from ASTER and Landsat. Each "snapshot" of a glacier is from a specific time, and the database is designed to store multiple snapshots representative of different times. We have implemented two web-based interfaces to the database; one enables exploration of the data via interactive maps (web map server), while the other allows searches based on text-field constraints. The web map server is an Open Geospatial Consortium (OGC) compliant Web Map Server (WMS) and Web Feature Server (WFS). This means that other web sites can display glacier layers from our site over the Internet, or retrieve glacier features in vector format. All components of the system are implemented using Open Source software: Linux, PostgreSQL, PostGIS (geospatial extensions to the database), MapServer (WMS and WFS), and several supporting components such as Proj.4 (a geographic projection library) and PHP. These tools are robust and provide a flexible and powerful framework for web mapping applications. As a service to the GLIMS community, the database contains metadata on all ASTER imagery acquired over glacierized terrain. Reduced-resolution of the images (browse imagery) can be viewed either as a layer in the MapServer application, or overlaid on the virtual globe within Google Earth. The interactive map application allows the user to constrain by time what data appear on the map. For example, ASTER or glacier outlines from 2002 only, or from Autumn in any year, can be displayed. The system allows users to download their selected glacier data in a choice of formats. The results of a query based on spatial selection (using a mouse) or text-field constraints can be downloaded in any of these formats: ESRI shapefiles, KML (Google Earth), Map

  2. Nucleic Acids Research annual Database Issue and the NAR online Molecular Biology Database Collection in 2009

    PubMed Central

    Galperin, Michael Y.; Cochrane, Guy R.

    2009-01-01

    The current issue of Nucleic Acids Research includes descriptions of 179 databases, of which 95 are new. These databases (along with several molecular biology databases described in other journals) have been included in the Nucleic Acids Research online Molecular Biology Database Collection, bringing the total number of databases in the collection to 1170. In this introductory comment, we briefly describe some of these new databases and review the principles guiding the selection of databases for inclusion in the Nucleic Acids Research annual Database Issue and the Nucleic Acids Research online Molecular Biology Database Collection. The complete database list and summaries are available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/). PMID:19033364

  3. JDD, Inc. Database

    NASA Technical Reports Server (NTRS)

    Miller, David A., Jr.

    2004-01-01

    information to the database. It now consists of seven different categories of data (carpet cleaning, forms, NASA Event Schedules, training certifications, wall and vent cleaning, work schedules, and miscellaneous) . I also did some field inspecting with the supervisors around the site and was present at all of the training certification courses that have been scheduled since June 2004. My future outlook for the JDD, Inc. database is to have all of company s information from future contract proposals, weekly inventory, to employee timesheets all in this same database.

  4. Influence of diffusion on the kinetics of multisite phosphorylation.

    PubMed

    Gopich, Irina V; Szabo, Attila

    2016-01-01

    When an enzyme modifies multiple sites on a substrate, the influence of the relative diffusive motion of the reactants cannot be described by simply altering the rate constants in the rate equations of chemical kinetics. We have recently shown that, even as a first approximation, new transitions between the appropriate species must also be introduced. The physical reason for this is that a kinase, after phosphorylating one site, can rebind and modify another site instead of diffusing away. The corresponding new rate constants depend on the capture or rebinding probabilities that an enzyme-substrate pair, which is formed after dissociation from one site, reacts at the other site rather than diffusing apart. Here we generalize our previous work to describe both random and sequential phosphorylation by considering inequivalent modification sites. In addition, anisotropic reactive sites (instead of uniformly reactive spheres) are explicitly treated by using localized sink and source terms in the reaction-diffusion equations for the enzyme-substrate pair distribution function. Finally, we show that our results can be rederived using a phenomenological approach based on introducing transient encounter complexes into the standard kinetic scheme and then eliminating them using the steady-state approximation.

  5. Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh

    PubMed Central

    Nakamura, Yuka; Tatsuno, Takanori; Ma, Shaofu; Tomosugi, Naohisa

    2015-01-01

    The RNA-binding protein 8A (RBM8A)–mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh. PMID:25349214

  6. Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.

    PubMed

    Delbaere, Louis T J; Sudom, Athena M; Prasad, Lata; Leduc, Yvonne; Goldie, Hughes

    2004-03-11

    Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP). The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains. Each domain has an alpha/beta topology and the overall structure represents a new protein fold. Furthermore, PCK has a unique mononucleotide-binding fold. The 1.8 A resolution structure of the complex of ATP/Mg(2+)/oxalate with PCK revealed a 20 degrees hinge-like rotation of the N- and C-terminal domains, which closed the active site cleft. The ATP was found in the unusual syn conformation as a result of binding to the enzyme. Along with the side chain of Lys254, Mg(2+) neutralizes charges on the P beta and P gamma oxygen atoms of ATP and stabilizes an extended, eclipsed conformation of the P beta and P gamma phosphoryl groups. The sterically strained high-energy conformation likely lowers the free energy of activation for phosphoryl transfer. Additionally, the gamma-phosphoryl group becomes oriented in-line with the appropriate enolate oxygen atom, which strongly supports a direct S(N)2-type displacement of this gamma-phosphoryl group by the enolate anion. In the 2.0 A resolution structure of the complex of PCK/ADP/Mg(2+)/AlF(3), the AlF(3) moiety represents the phosphoryl group being transferred during catalysis. There are three positively charged groups that interact with the fluorine atoms, which are complementary to the three negative charges that would occur for an associative transition state. PMID:15023367

  7. Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKε promotes cell transformation

    PubMed Central

    Hutti, Jessica E.; Shen, Rhine R.; Abbott, Derek W.; Zhou, Alicia Y.; Sprott, Kam M.; Asara, John M.; Hahn, William C.; Cantley, Lewis C.

    2009-01-01

    Summary The non-canonical IKK family member IKKε is essential for regulating anti-viral signaling pathways and is a recently-discovered breast cancer oncoprotein. Although several IKKε targets have been described, direct IKKε substrates necessary for regulating cell transformation have not been identified. Here, we performed a screen for putative IKKε substrates using an unbiased proteomic and bioinformatic approach. Using a positional scanning peptide library assay we determined the optimal phosphorylation motif for IKKε and used bioinformatic approaches to predict IKKε substrates. Of these potential substrates, serine 418 of the tumor suppressor CYLD was identified as a likely site of IKKε phosphorylation. We confirmed that CYLD is directly phosphorylated by IKKε, and that IKKε phosphorylates serine 418 in vivo. Phosphorylation of CYLD at serine 418 decreases its deubiquitinase activity and is necessary for IKKε-driven transformation. Together, these observations define IKKε and CYLD as an oncogene-tumor suppressor network that participates in tumorigenesis. PMID:19481526

  8. Phosphoryl transfer reaction catalyzed by membrane diacylglycerol kinase: a theoretical mechanism study.

    PubMed

    Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Li, Xichen; Chen, Guangju; Jia, Zongchao

    2015-10-14

    Diacylglycerol kinase is an integral membrane protein which catalyzes phosphoryl transfer from ATP to diacylglycerol. As the smallest kinase known, it shares no sequence homology with conventional kinases and possesses a distinct trimer structure. Thus far, its catalytic mechanism remains elusive. Using molecular dynamics and quantum mechanics calculations, we investigated the co-factor and the substrate binding and phosphoryl transfer mechanism. Based on the analysis of density functional theory calculations, we reveal that the phosphorylation reaction of diacylglycerol kinase features the same phosphoryl transfer mechanism as other kinases, despite its unique structural properties. Our results further show that the active site is relatively open and able to accommodate ligands in multiple orientations, suggesting that the optimization of binding orientations and conformational changes would occur prior to actual phosphoryl transfer.

  9. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

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

    PubMed

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

    2016-04-27

    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

  11. Multisite Phosphorylation of NuMA-Related LIN-5 Controls Mitotic Spindle Positioning in C. elegans

    PubMed Central

    Portegijs, Vincent; van Mourik, Tim; Akhmanova, Anna; Heck, Albert J. R.; van den Heuvel, Sander

    2016-01-01

    During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα–LGN–NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation. PMID:27711157

  12. K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK

    PubMed Central

    Lin, Dao-Hong; Sterling, Hyacinth; Lerea, Kenneth M.; Welling, Paul; Jin, Lianhong; Giebisch, Gerhard; Wang, Wen-Hui

    2010-01-01

    We purified Histagged ROMK1 and carried out in vitro phosphorylation assays with 32P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [32P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [32P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333–362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion. PMID:12217858

  13. Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina: characteristics of the phosphorylated proteins and their dephosphorylation

    SciTech Connect

    Not Available

    1986-01-05

    To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, the cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein was studied. cGMP or cAMP with (..gamma..-/sup 32/P)ATP in the dark enhanced the phosphorylation of two ROS proteins with M/sub r/ = 10,500 (Band 1) and 8500 (Band 2) according to sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg/sup 2 +/. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. Both /sup 32/P-phosphorylated Bands 1 and 2 were solubilized during preparation and the molecular weight of each was 19,000. Their isoelectric point was 5.2. Th